System and method to laser heat slides containing histological samples

Abstract

A unique system and method for preparing tissue samples on slides for microscopic analysis has been developed. The system, uses a laser to directly heat slides during various processing stages, including baking, dewaxing, and curing. The laser is controlled to target specific areas of the slide, such as the bottom surface of the slide or coverslip, to promote efficient heating while protecting the tissue sample. Temperature sensors and feedback control help manage the heating process. The system can include modules for dispensing solvents and removing wax, as well as features for applying and curing coverslips. This approach reduces processing time and energy use compared to traditional oven and resistive heating methods.

Description

[0001] #3371089 OS <11 9-000025

[0002] 1

[0003] SYSTEM AND METHOD TO LASER HEAT SLIDES CONTAINING HISTOLOGICAL SAMPLES

[0004] BACKGROUND

[0005] The diagnosis of histological pathologies can be a laborious and time-consuming process. For instance, the diagnosis of histological pathologies typically requires several slide preparation stages to be performed during a typical tissue staining workflow. In some cases, formalin-fixed paraffin-embedded (FFPE) tissue sections or samples are mounted on glass slides. In a common tissue staining workflow, the slides with the tissue samples need to be processed before and after stain is applied to the tissue samples. These processes can require significant processing times which can dramatically lengthen the overall workflow times. If these processes are not properly performed, the ti sue samples may be inadequately stained and otherwise damaged such that the tissue samples cannot be analyzed.

[0006] Thus, there is a need for improvement in this field.#3371089 OS <11 9-000025

[0007] 2

[0008] SUMMARY

[0009] In laboratory settings and elsewhere, preparing tissue samples for diagnosis of histological pathologies, such as by study ing tissue samples under a microscope to look for diseases like cancer, requires several processing stages to prepare the tissue for staining and examination, These processing stages often include baking the tissue slide, removing the wax (a process called dewaxing or deparaffinization), and curing the glue that holds a thin glass coverslip on top of the tissue. Traditionally, these stages are typically performed by heating the slides. The slides are commonly heated using thermal conduction or convection ovens. When thermal conduction is used, an electrical resistor is placed in thermal contact with the slide (e.g., via a hotplate). However, thermal conduction is typically slow. Monitoring and controlling the temperature of the whole slide can be difficult. Ensuring good thermal contact between the resistor and the slide can also be troublesome. Often, an insulating airgap at the interface between the heating surface and the glass slide is formed. Ovens are usually used to heat up multiple slides in batches. It was found that the use of ovens can be slow, inefficient, and expensive, because a significant amount of time and energy is required for the oven to reach the correct temperature, and additional time is needed for the slides to cool down before further bundling is possible.

[0010] Using conventional heating techniques, the temperature of the slide is estimated by indirect measurements of the hot plate or the recirculating air in the oven. These estimated indirect temperature measurements limit the amount of energy that can be added to the system due to the risk of overheating the slides. As a result, the heating process needs to proceed at a slow rate, which in turn slows processing times. Although heat transfer can be sped up by increasing the temperature gradient of the heater, the inabili ty to directly measure slide temperature prevents this from being a safe or even practical option. Moreover, the thermal inertia or mass for heating requires the use of excess energy that needs to be managed inside the processing chambers of the system. Due to the heat involved, the temperature of adjacent chambers needs to be managed, which in turn can impact processing performance.

[0011] Baking is normally one of the first stages after placing a thin section of tissue onto a slide. This baking helps to ensure that the tissue sample adheres firmly to the slide and removes any water that may be trapped under the tissue sample. Proper adhesion is usually needed to prevent the tissue sample from detaching during later processing, which could interfere with#3371089 OS <11 9-000025

[0012] 3

[0013] accurate diagnosis. The baking process helps to flatten the paraffin wax to conform to the surface of the slide and eliminate wrinkles in the tissue sample. An angled or vertical slide orientation is commonly used during baking to facilitate water drainage. Baking is commonly performed in an oven at a set temperature (e.g., approximately 60 degrees Celsius) for a set period of time (e.g., 60 minutes), which can occupy a large portion of the total processing time for slide preparation.

[0014] Following baking, dewaxing or deparaffinization is performed to remove paraffin wax from the tissue sample. Paraffin wax is used to embed and preserve tissue samples, but the wax is usually removed before staining and microscopic examination. The wax should be completely removed, because any remaining wax can prevent staining agents from reaching the tissue, which can obscure diagnostic details in the tissue sample. For example, incomplete dewaxing of slides can lead to a blocked antigen-antibody interaction by creating a phy sical barrier. Incomplete dewaxing may also result in weak, patchy, or absent staining, together with increased background and other morphological artifacts. Dewaxing is typically achieved by heating the slides. This dewaxing process can commonly require 12 to 16 minutes of processing time. One typical dewaxing process uses a wet approach (e.g., using Ezprep). The slide is heated to melt the wax, and solvents or surfactants are used to clear away the melted wax. It was found that this wet approach wasted considerable amounts of time. Significant time is required to heat a solvent mixture placed on top of the slide in the form of a puddle due to the high specific heat of water in the solvent mixture. The temperatures used during dewaxing are limited by the boiling temperature of water. The high latent heat for water vaporization limits the slide temperature to less than 100 degrees Celsius at sea level. In other techniques, organic chemicals, such as xylene, are used to directly dissolve the wax from the slide, but this chemistry can be toxic which negatively impacts the sustainability of the process. In some cases, a series of graded alcohols are used to rehydrate the tissue.

[0015] After baking and dewaxing, staining is performed to highlight different structures within the tissue sample. Once staining is complete, a co verslip is placed on top of the tissue sample on the slide to protect the sample and to provide a clear surface for microscopic examination. A mounting media, which acts as a glue, is used to attach the coverslip. The mounting media is usually cured, or hardened, to ensure that the coverslip remains securely in place.

[0016] Traditionally, curing is also performed in an oven, which adds further time to the process and#3371089 OS <11 9-000025

[0017] 4

[0018] requires the slides to remain stationary until the mounting media is fully set. Again, it was found that all of these heating and cooling processes were time consuming as well as created other complications and quality issues in the staining process.

[0019] A unique direct heating process and system has been developed to address these as well as other issues. In one form, a laser-based approach is used to directly heat the slides, which reduces the time required for the baking, dewaxing, and curing processing stages. The laser beam can be directed to specific areas of the slide, such as the glass beneath the tissue sample, the coverslip, and / or the mounting media beneath the coverslip. For the baking stage in one example, the laser heats the glass slide rapidly, which promotes tissue adhesion and water removal. For dewaxing, the laser beam in one version melts the paraffin wax, which can then be removed with a solvent such as a DOW ANOL® brand (The Dow Chemical Company, Midland, Michigan) Dipropylene Glycol Propyl Ether (DPGPE) solvent. This is to ensure that aqueous staining solutions can effectively penetrate the tissue. A further approach uses a xylene-free and alcohol -free deparaffinization technique with non-toxic, biodegradable agents together with heat to remove molten paraffin. In some cases, both the baking and dewaxing processes are performed in one stage or in a short sequence. For example, a rapid baking and dewaxing process can be performed in about 15 seconds in some cases which is considerably faster than traditional approaches. During the curing stage in one variation, the laser beam heats the mounting media, allowing the coverslip to be fixed in place in a much shorter time compared to oven-based methods.

[0020] During development of this laser-based process and sy stem, several unique processing features were developed to enhance efficiency and processing times. In one version, a carbon dioxide laser w'ith a wavelength of about 10.6 micrometers is used to generally match the absorption properties of borosilicate and soda- lime glass, which are commonly used for slides. It was also discovered that when the laser beam is applied from below' the slide, the glass absorbs the energy and heats up quickly, while the tissue sample on top of the slide is protected from direct laser exposure. In some versions, a dark marker ink or other laser absorptive material is applied to the slide and / or the coverslip to increase absorption of visible and / or ultraviolet laser wavelengths. In some cases, the mounting media is formulated or mixed to absorb the laser energy more effectively which further reduces processing time. In some cases, the laser absorptive material can include a coating and / or be created through a#3371089 OS <11 9-000025

[0021] 5

[0022] doping process to ensure a close interface for thermal transfer into the slide, coverslip., or other materials. This enhanced thermal contact can facilitate the use of other laser wavelengths outside of those produced with carbon dioxide lasers.

[0023] In some cases, the laser beam generator in the system is controlled by a computer or other type of controller, which moves the laser beam rapidly and accurately over the slide surface (or interior). This enables custom heating patterns and precise control over the amount and location of the energy delivered. The system can adjust the power, modulation, and raster speed of the laser for different processing stages and or tissue types. Safety features, such as protective enclosures and automatic shut-offs, are included to ensure safe operation in the laboratory environment.

[0024] It was discovered that reducing the size of the darkened area on the slide can further decrease the time required for curing, as the laser energy is concentrated more efficiently.

[0025] Additionally, using the laser in short bursts, rather than as a continuous beam, can enhance heating efficiency and reduce energy waste. Considering the laser heats only a localized area, the overall electricity consumption is much lower than that of an oven, which must heat a large volume of air and metal in addition to the slides.

[0026] The laser -based process can be configured to process individual slides or multiple slides at the same time, and this process can be readily integrated into automated machines that perform other sample preparation processes. This flexibility allows for faster laboratory workflows, reduced energy use. and less manual handling of slides. The result is a more efficient and controlled method for preparing tissue slides for microscopic diagnosis, supporting faster and more reliable results for pathologists.

[0027] In a further variation, an adapti ve laser baking method or technique has been developed for the histopathology workflows to significantly enhance the adhesion of Formalin-Fixed, Paraffin-Embedded (FFPE) tissue sections to microscope slides. This technique utilizes a controlled power laser source and either passive (e.g., beam expanders) or dynamic (e.g., galvanometer mirrors) optical mechanisms to direct the laser beam so as to precisely heat the slide to reduce or eliminate trapped water through evaporation. This ensures a stronger tissueslide bond and reduces wrinkling of the tissue sample. In one example, a system for#3371089 OS <11 9-000025

[0028] 6

[0029] performing this technique incorporates a touchless sensor, such as a pyranometer or thermal camera, and a controller with PID routines to accurately measure and regulate the temperature of the slide. The controller actively estimates when the slide is dry by monitoring the laser power required to maintain the set point temperature, A sudden drop in required power for the laser indicates the cessation of evaporative cooling, which in turn confirms dryness of the tissue sample and / or the slide. In one version, a beam scanning path for the laser has been developed to leverage thermocapillary convection, an effect that moves excess humidity and wax from the center of the section toward the edges. This beam scanning path further enhances adhesion and section quality of the tissue sample.

[0030] Antigen retrieval (AR) can be a crucial step in immunohistochemistry ( IHC) to restore antigenicity masked during formalin fixation and paraffin embedding (FFPE). Traditional Heat-Induced Epitope Retrieval (HIER) uses heating in retrieval buffers (e.g., citrate pH 6.0 or Tris-EDTA pH 9.0) for approximately 15 to 30 minutes. Enzyme- Induced Epitope Retrieval (EIER) uses proteolytic enzymes for 10 to 20 minutes, but EIER is less common due to potential tissue damage. Modem automated solutions, such as the BENCHMARK® ULTRA brand system (Roche Diagnostics GmbH. Mannheim, Germany), use HIER with pre-optimized protocols, typically ranging from 32 to 104 minutes.

[0031] It was found that automated AR can be time-consuming and increases the overall turnaround time (TAT) in labs. For example, the ALK assay on the BENCHMARK® ULTRA brand system requires 104 minutes of antigen retrieval. While processes that involve pressure cookers or rapid antigen retrieval (RAR) can shorten AR time, these processes remain largely manual, and the complexity of automating slide mo vement through a pressure cooker station has been a challenge.

[0032] A unique automated AR method and system have been developed to promote rapid, tempera ture-eon trolled, a nd closed-loo p heat ing via one or more lasers for an tigen retrieval i n IHC. With this AR technique, a laser beam is used to rapidly heat slides. In some cases, a carbon dioxide laser is used to heat glass slides, but other types of lasers can be used to heat other types of slides. This laser-enabled workflow delivers heat in a targeted manner through immediate absorption in the substrate of the slide, ensuring precise thermal control. This#3371089 OS <11 9-000025

[0033] 7

[0034] rapid antigen retrieval process can further drastically reduce system complexity, such as pressurized chambers used to achieve similar retrieval times.

[0035] In an alternati ve approach, instead of scanning the slide with a focused laser beam having a diameter that is much smaller than the whole slide, one or more slides are il luminated by and heated with a static and massively expanded laser beam. To facilitate this expansion, the original laser beam, which has an extremely nonuniform Gaussian profile, is converted into a uniform flat top laser beam of appropriate size. The laser optics include a beam expander to increase the beam diameter of the laser beam and a beam shaper to achieve homogeneous illumination of the slide with the laser beam, In one particular example, the beam expander includes a Galilean beam expander that includes a diverging lens followed by a converging lens. The beam shaper in this example includes a refractive flat-top beam shaper. Gaussian- to-top-hat converter, or aspheric beam homogenizer. The refractive flat-top beam shaper is commonly identified by the brand name "pShaper" for a series of beam shapers that arc sold by AdlOptica Optical Systems GmbH of Berlin, Germany. With this construction, no mo ving parts for the galvo scanners are needed, and no controller for the galvo scanners is required. Moreover, an F -theta lens is not required for assuring a linear relation between scan angle and beam position on the slide.

[0036] Another variation concerns a highly integrated adaptive baking system that enhances the laser processing methods described above by utilizing a multi -slide asynchronous architecture and spatially resolved thermal management. This system and technique further facilitates the removal of the residual water pellicle trapped between a histological tissue section and the glass slide fol lowing microtomy. This process helps to ensure consistent and robust tissue adhesion, and this process further provides a better confirmation of the removal of the water from the slide.

[0037] As alluded to before, poor tissue adhesion frequently leads to tissue folding, damage, or the complete loss of samples during staining, which directly undermines the ability of clinicians to provide diagnostic certainty for patients. This can be particularly troublesome in immunohistochemistry (IHC) protocols involving many rinsing steps. Once more, labs commonly use convection ovens, which typically require 30 to 60 minutes of heating, hot plates, or overnight air drying, all of which create significant bottlenecks and add hours to the#3371089 OS <11 9-000025

[0038] 8

[0039] diagnostic journey of the slide, These conventional methods suffer from severe shortcomings, including poor energy efficiency as they require heating of the entire apparatus and surrounding lab environment, rather than just the slide. These conventional methods also rely on indirect temperature measurements that lead to over -engineered, worst-case protocols. This lack of direct control forces a one-size-fits-all approach that risks either overbaking the tissue or underbaking the tissue, which causes detachment. It was discovered that using adaptive control to detect dryness via the cessation of evaporati ve cooling often relied on single-sensor averaging that failed to account for localized temperature gradients caused by geometric artifacts or the agglutination of water and wax. The highly integrated adaptive baking system in this variation addresses these issues by incorporating active convection mechanisms and turbulent air to replace saturated air near the slide surface and by utilizing specialized laser scanning paths to manage temperature gradients, effectively leveraging thermocapil lary convection to sweep excess moisture out of the slide and further accelerate the drying process.

[0040] In one version, the baking system includes a tilted slide holder that maintains one or more slides at about a 45-degree angle relative to horizontal. The slide holder uses gravity as a. natural mechanism to assist in the removal of excess water. Hiis slide holder is protected by a cap that provides operator safety from laser radiation. The cap further defines an internal volume where a small fan induces airflow to replace saturated air. This in turn accelerates the drying process through active convection. To manage high-throughput workflows, the system includes one or more sensors for precise slide detection and supports independent, asynchronous processing of multiple slides. Instead of using single-point sensors, the system includes a thermal camera array that monitors spatial temperature distribution across the slides without physical contact. The digitized data from this array is utilized by multiple PID control loops to modulate laser power with spatial resolution. This allows the creation of controlled thermal gradients that ensure even drying and prevent localized artifacts or water pockets.

[0041] In one form of the system, the optical path includes an RF metal tube carbon dioxide laser source and a visible red laser for testing and alignment safety. The combined beam is reflected off a steerable mirror driven by voice-coil type actuators on X and Y axes so as to allow' the laser to execute arbitrary scanning paths, such as increasing -radius concentric#3371089 OS <11 9-000025

[0042] 9

[0043] circles, ovals, or rectangles, on the bottom face of the slide to enhance heat transfer. It was unexpectedly discovered that this configuration is able to physically drive excess water and wax toward the edges of the slide via laser-induced thermocapillary convection, In essence, the laser heating system effectively sweeps the moisture away from the tissue section to enhance adhesion. The system further includes a centralized controller to manage the entire protocol. The controller analyzes real-time thermal curves to adapt baking protocols based on individual slide wetness, and the controller is able to confirm successful dryness by detecting the drop in power demand that occurs once evaporative cooling stops.

[0044] The systems and techniques as described and illustrated herein concern a number of unique and inventive aspects. Some, but by no means all, of these unique aspects are summarized below.

[0045] Aspect 1 generally concerns a system.

[0046] Aspect 2 generally concerns the system of any previous aspect including a slide.

[0047] Aspect 3 generally concerns the system of any previous aspect in which the slide is made of glass.

[0048] Aspect 4 generally concerns the system of any previous aspect in which the slide is configured to receive a coverslip.

[0049] Aspect 5 generally concerns the system of any previous aspect in which the coverslip is mounted to the slide.

[0050] Aspect 6 generally concerns the system of any previous aspect in which the coverslip is made of glass.

[0051] Aspect 7 generally concerns the system of any previous aspect in which the coverslip is a pre-glued coverslip.#3371089 OS <11 9-000025

[0052] 10

[0053] Aspect 8 generally concerns the system of any previous aspect in which the tissue sample is disposed on the slide.

[0054] Aspect 9 generally concerns the system of any previous aspect in which the coverslip covers the tissue sample on the slide.

[0055] Aspect 10 generally concerns the system of any previous aspect in which the coverslip includes glue.

[0056] Aspect 11 generally concerns the system of any previous aspect in which the slide has a sample surface.

[0057] Aspect 12 generally concerns the sy stem of any previous aspect in which the tissue sample is disposed on the sample surface.

[0058] Aspect 13 generally concerns the system of any previous aspect in which the slide has a base surface.

[0059] Aspect 14 generally concerns the system of any previous aspect in which the base surface is disposed opposite to the sample surface.

[0060] Aspect.15 generally concerns the sy stem of any previous aspect including a module.

[0061] Aspect 16 generally concerns the system of any previous aspect in which the module is configured to process the tissue sample for analysis.

[0062] Aspect 17 generally concerns the system of any previous aspect in which the module includes a station.

[0063] Aspect 18 generally concerns the system of any previous aspect in which the module includes an input module.#3371089 OS <11 9-000025

[0064] 11

[0065] Aspect 19 generally concerns the system of any previous aspect in which the input module is configured to bake the tissue sample.

[0066] Aspect 20 generally concerns the system of any previous aspect in which the input module includes a baking station.

[0067] Aspect 21 generally concerns the system of any previous aspect in which the baking station is configured to bake the tissue sample.

[0068] Aspect 22 generally concerns the system of any previous aspect in which the input module is configured to remove wax from the tissue sample.

[0069] Aspect 23 generally concerns the system of any previous aspect in which the input module includes a dewaxing station.

[0070] Aspect 24 generally concerns the system of any previous aspect in which the dewaxing station is configured to dewax the tissue sample.

[0071] Aspect 25 generally concerns the system of any previous aspect in which the module includes a stain module.

[0072] Aspect 26 generally concerns the system of any previous aspect in which the stain module is configured to stain the tissue sample.

[0073] Aspect 27 generally concerns the system of any previous aspect in which the stain module includes an antigen retrieval station.

[0074] Aspect 28 generally concerns the system of any previous aspect in which the module includes a coverslip module.

[0075] Aspect 29 generally concerns the system of any previous aspect in which the coverslip module is configured to adhere the coverslip to the slide.#3371089 OS <11 9-000025

[0076] 12

[0077] Aspect 30 generally concerns the system of any previous aspect in which the coverslip module includes a curing station.

[0078] Aspect 31 generally concerns the system of any previous aspect in which the curing station is configured to cure the coverslip.

[0079] Aspect 32 generally concerns the system of any previous aspect in which the curing station is configured to cure the glue.

[0080] Aspect 33 generally concerns the system of any previous aspect in which the module is configured to bake and dewax at the same time.

[0081] Aspect 34 generally concerns the system of any previous aspect in which the module is configured to bake, dewax, and facilitate antigen retrieval in a single integrated unit.

[0082] Aspect 35 generally concerns the system of any previous aspect in which the module is configured to heat the tissue sample to bake, dewax, and facilitate antigen retrieval at the same time.

[0083] Aspect 36 generally concerns the system of any previous aspect including a laser.

[0084] Aspect 37 generally concerns the system of any previous aspect in which the laser is configured to produce a laser beam.

[0085] Aspect 38 generally concerns the system of any previous aspect in which the laser includes a beam generator,

[0086] Aspect 39 generally concerns the system of any previous aspect in which the beam generator is configured to generate the laser beam.

[0087] Aspect 40 generally concerns the system of any previous aspect in which the laser is a carbon dioxide type laser.#3371089 OS <11 9-000025

[0088] 13

[0089] Aspect 41 generally concerns the system of any previous aspect in which the laser includes an infrared type laser.

[0090] Aspect 42 generally concerns the system of any previous aspect in which the laser beam has a wavelength of about 10.6 micrometers.

[0091] Aspect 43 generally concerns the system of any previous aspect in which the laser is a visible light laser.

[0092] Aspect 44 generally concerns the system of any previous aspect in which the laser is a blue light laser.

[0093] Aspect 45 generally concerns the system of any previous aspect in which the laser is an ultraviolet type laser.

[0094] Aspect 46 generally concerns the system of any previous aspect in which the laser includes a beam positioner.

[0095] Aspect 47 generally concerns the system of any previous aspect in which the beam positioner is configured to direct the laser beam.

[0096] Aspect 48 generally concerns the system of any previous aspect in which the beam positioner is configured to scan the laser beam.

[0097] Aspect 49 generally concerns the system of any previous aspect in which the beam positioner includes a mirror controlled by a galvanometer motor.

[0098] Aspect 50 generally concerns the system of any previous aspect in which the beam positioner is configured to scan the laser beam in two dimensions.

[0099] Aspect 51 generally concerns the system of any previous aspect in which the beam positioner includes a two-dimensional type galvanometer.#3371089 OS <11 9-000025

[0100] 14

[0101] Aspect 52 generally concerns the system of any previous aspect in which the laser includes a power control module.

[0102] Aspect 53 generally concerns the system of any previous aspect in which the power control module is configured to control power of the laser beam emitted from the beam generator.

[0103] Aspect 54 generally concerns the system of any previous aspect in which the laser beam is a continuous beam.

[0104] Aspect 55 generally concerns the system of any previous aspect in which the laser beam is a pulsed beam.

[0105] Aspect 56 generally concerns the system of any previous aspect in which the power control module is configured to turn on and off the laser beam.

[0106] Aspect 57 generally concerns the system of any previous aspect in which the power control module is operatively coupled to the beam generator.

[0107] Aspect 58 generally concerns the system of any previous aspect in which the laser includes a position control module.

[0108] Aspect 59 generally concerns the system of any previous aspect in which the position control module is operatively coupled to the beam positioner.

[0109] Aspect 60 generally concerns the system of any previous aspect in which the position control module is confi gured to control movement of the laser beam.

[0110] Aspect 61 generally concerns the system of any previous aspect in which the laser includes a communication module.

[0111] Aspect 62 generally concerns the system of any previous aspect in which the communication module is operatively coupled to the power control module.#3371089 OS <11 9-000025

[0112] 15

[0113] Aspect 63 generally concerns the system of any previous aspect in which the communication module is operatively coupled to the position control module.

[0114] Aspect 64 generally concerns the system of any previous aspect including a controller,

[0115] Aspect 65 generally concerns the system of any previous aspect in which the controller is configured to control the laser.

[0116] Aspect 66 generally concerns the system of any previous aspect in which the controller is configured to control the laser beam.

[0117] Aspect 67 generally concerns the system of any previous aspect in which the controller is operatively coupled to the communication module.

[0118] Aspect 68 generally concerns the system of any previous aspect in which the controller is configured to control power of the laser beam.

[0119] Aspect 69 generally concerns the system of any previous aspect in which the controller is configured to control scanning of the laser beam.

[0120] Aspect 70 generally concerns the system of any previous aspect in which the controller is configured to control pulsing of the laser beam.

[0121] Aspect 71 generally concerns the system of any previous aspect including a temperature sensor.

[0122] Aspect 72 generally concerns the system of any previous aspect in which the temperature sensor is operatively coupled to the controller,

[0123] Aspect 73 generally concerns the system of any previous aspect in which the temperature sensor is configured to measure temperature of the slide.#3371089 OS <11 9-000025

[0124] 16

[0125] Aspect 74 generally concerns the system of any previous aspect in which the temperature sensor is configured to measure temperature of the base surface of the slide.

[0126] Aspect 75 generally concerns the system of any previous aspect in which the temperature sensor is configured to measure temperature of the sample surface of the slide.

[0127] Aspect 76 generally concerns the system of any previous aspect in which the temperature sensor is configured to measure temperature of the coverslip.

[0128] Aspect 77 generally concerns the system of any previous aspect in which the temperature sensor is configured to measure temperature of the tissue sample.

[0129] Aspect 78 generally concerns the system of any previous aspect in which the controller is configured to control the laser using an open loop control technique.

[0130] Aspect 79 generally concerns the system of any previous aspect in which the controller is configured to control the laser using a closed loop control technique.

[0131] Aspect 80 generally concerns the system of any previous aspect in which the controller is configured to control the laser based on the temperature sensed by the temperature sensor.

[0132] Aspect 81 generally concerns the system of any previous aspect in which the laser includes a laser head.

[0133] Aspect 82 generally concerns the system of any previous aspect in which the laser includes a position adjuster.

[0134] Aspect 83 generally concerns the system of any previous aspect in which the position adjuster is configured to adjust position of the head.

[0135] Aspect 84 generally concerns the system of any previous aspect in which the laser includes a lens.#3371089 OS <11 9-000025

[0136] 17

[0137] Aspect 85 generally concerns the system of any previous aspect in which the lens is configured to focus the laser beam.

[0138] Aspect 86 generally concerns the system of any previous aspect in which the lens is configured to defocus the laser beam.

[0139] Aspect 87 generally concerns the system of any previous aspect in which the lens is configured to project the laser beam with a flat focal plane.

[0140] Aspect 88 generally concerns the system of any previous aspect in which the lens includes an F-Theta lens.

[0141] Aspect 89 generally concerns the system of any previous aspect including a mirror positioned to reflect the laser beam.

[0142] Aspect 90 generally concerns the system of any previous aspect in which the mirror is positioned to reflect the laser beam on the base surface of the slide.

[0143] Aspect 91 generally concerns the system of any previous aspect in which the laser includes a beam expander.

[0144] Aspect 92 generally concerns the system of any previous aspect in which the beam expander is configured to defocus the laser beam.

[0145] Aspect 93 generally concerns the system of any previous aspect in which the beam expander includes a tube assembly,

[0146] Aspect 94 generally concerns the system of any previous aspect in which the laser beam travels along a beam path.

[0147] Aspect 95 generally concerns the system of any previous aspect in which the beam expander is positioned along the beam path between the lens and the base surface of the slide.#3371089 OS <11 9-000025

[0148] 18

[0149] Aspect 96 generally concerns the system of any previous aspect including a slide holder.

[0150] Aspect 97 generally concerns the system of any previous aspect in which the slide holder is configured to hold the slide.

[0151] Aspect 98 generally concerns the system of any previous aspect in which the slide holder holds the slide.

[0152] Aspect 99 generally concerns the system of any previous aspect in which the slide holder is configured to hold the edges of the slide.

[0153] Aspect 100 generally concerns the system of any previous aspect in which the slide holder is configured to engage the corners of the slide.

[0154] Aspect 101 generally concerns the system of any previous aspect in which the slide holder is integrated with the beam expander.

[0155] Aspect 102 generally concerns the sy stem of any previous aspect in which the slide holder includes one or more posts.

[0156] Aspect 103 generally concerns the system of any previous aspect in which the posts are configured to support the slide.

[0157] Aspect 104 generally concerns the system of any previous aspect in which the posts support the slide.

[0158] Aspect 105 generally concerns the system of any previous aspect in which the posts are positioned to support the corners of the slide.

[0159] Aspect 106 generally concerns the system of any previous aspect in which the posts are configured to form an air gap between the holder and the slide.#3371089 OS <11 9-000025

[0160] 19

[0161] Aspect 107 generally concerns the system of any previous aspect in which the holder defines a beam aperture.

[0162] Aspect 108 generally concerns the system of any previous aspect in which the laser beam shines through the beam aperture.

[0163] Aspect 109 generally concerns the system of any previous aspect in which the beam aperture is positioned between the posts.

[0164] Aspect 110 generally concerns the system of any previous aspect in which the holder defines a drain channel.

[0165] Aspect 111 generally concerns the system of any previous aspect in which the drain channel extends around the beam aperture.

[0166] Aspect 112 generally concerns the system of any previous aspect in which the drain channel extends around at least one of the posts.

[0167] Aspect 113 generally concerns the system of any previous aspect in which the drain channel is configured to drain wax from the slide.

[0168] Aspect 114 generally concerns the system of any previous aspect including a dispenser head.

[0169] Aspect 115 generally concerns the system of any previous aspect in which the dispenser head is configured to move relative to the slide.

[0170] Aspect 116 generally concerns the system of any previous aspect in which the dispenser head includes a nozzle.

[0171] Aspect 117 generally concerns the system of any previous aspect in which the nozzle is configured to dispense a solvent.#3371089 OS <11 9-000025

[0172] 20

[0173] Aspect 118 generally concerns the system of any previous aspect in which the nozzle is configured to dispense the solvent onto the tissue sample.

[0174] Aspect 119 generally concerns the system of any previous aspect in which the nozzle is configured to dispense the solvent onto the sample surface of the slide.

[0175] Aspect 120 generally concerns the system of any previous aspect in which the nozzle is configured to form a puddle of the solvent on the slide.

[0176] Aspect 121 generally concerns the system of any previous aspect in which the solvent is configured to mix with molten wax.

[0177] Aspect 122 generally concerns the system of any previous aspect in which the dispenser head includes an air knife.

[0178] Aspect 123 generally concerns the system of any previous aspect in which the air knife is configured to blow a gas across the slide.

[0179] Aspect 124 generally concerns the system of any previous aspect in which the air knife is configured to sweep a mixture of the solvent and wax from the slide.

[0180] Aspect 125 generally concerns the system of any previous aspect in which the coverslip is at least partially covered with an absorptive material.

[0181] Aspect 126 generally concerns the system of any previous aspect in which the slide is at least partially covered with an absorptive material.

[0182] Aspect 127 generally concerns the system of any previous aspect in which the absorptive material is configured to absorb the laser beam.

[0183] Aspect 128 generally concerns the system of any previous aspect in which the absorptive material is configured to absorb visible light.#3371089 OS <11 9-000025

[0184] 21

[0185] Aspect 129 generally concerns the system of any previous aspect in which the absorptive material is configured to absorb ultraviolet light.

[0186] Aspect 130 generally concerns the system of any previous aspect in which the absorptive material includes black ink.

[0187] Aspect 131 generally concerns the system of any previous aspect in which the absorptive material is in the form of a machine-readable identifier.

[0188] Aspect 132 generally concerns the system of any previous aspect in which the absorptive material is positioned to not obstruct visibility during tissue analysis.

[0189] Aspect 133 generally concerns the system of any previous aspect in which the absorptive material includes one or more absorptive bars.

[0190] Aspect 134 generally concerns the system of any previous aspect in which the absorptive bars cover one or more edges of the coverslip.

[0191] Aspect 135 generally concerns the system of any previous aspect in which the absorptive material includes one or more dots.

[0192] Aspect 136 generally concerns the system of any previous aspect in which the dots are positioned at the corners of the coverslip.

[0193] Aspect 137 generally concerns the system of any previous aspect in which the laser beam is configured to create heat to prepare for analysis of a tissue sample on a slide.

[0194] Aspect 138 generally concerns the system of any previous aspect in which the laser beam is configured to heat the base surface of the slide.

[0195] Aspect 139 generally concents the sy stem of any previous aspect in which the laser beam is configured to heat the coverslip.#3371089 OS <11 9-000025

[0196] 22

[0197] Aspect 140 generally concerns the system of any previous aspect in which the laser beam is configured to heat the absorptive material.

[0198] Aspect 141 generally concerns the system of any previous aspect in which the laser beam is configured to heat the puddle.

[0199] Aspect 142 generally concerns the system of any previous aspect in which the laser is configured to bake the tissue sample on the slide.

[0200] Aspect 143 generally concerns the system of any previous aspect in which the laser is configured to dewax the tissue sample on the slide.

[0201] Aspect 144 generally concerns the system of any previous aspect in which the laser is configured to facilitate antigen retrieval.

[0202] Aspect 145 generally concerns the system of any previous aspect in which the laser is configured to cure the coverslip.

[0203] Aspect 146 generally concerns the system of any previous aspect in which the laser is configured to perform a flash baking technique.

[0204] Aspect 147 generally concerns the system of any previous aspect in which the laser is configured to perform a gentle baking technique.

[0205] Aspect 148 generally concerns the system of any previous aspect in which the laser is configured to scan the laser beam along a laser scan path.

[0206] Aspect 149 generally concerns the system of any previous aspect in which the laser scan path is in an outward radial direction.

[0207] Aspect 150 generally concerns the system of any previous aspect in which the laser scan path has an outbound rectangular hatch pattern.#3371089 OS <11 9-000025

[0208] 23

[0209] Aspect 151 generally concerns the system of any previous aspect in which the laser scan path leverages thermocapillary convection.

[0210] Aspect 152 generally concerns the system of any previous aspect in which the laser scan path generates thermal gradients to sweep excess humidity from the center of the slide to the edges of the slide.

[0211] Aspect 153 generally concerns the system of any previous aspect in which the laser scan path generates thermal gradients to sweep wax from the center of the slide to the edges of the slide.

[0212] Aspect 154 generally concerns the system of any previous aspect in which the controller is configured to perform closed loop temperature control.

[0213] Aspect 155 generally concerns the system of any previous aspect in which the controller includes a proportional-integral-derivative (PID) controller.

[0214] Aspect 156 generally concerns the system of any previous aspect in which the PID controller is operatively coupled to the temperature sensor.

[0215] Aspect 157 generally concerns the system of any previous aspect in which the PID controller is configured to regulate power of the laser.

[0216] Aspect 158 generally concerns the system of any previous aspect in which the controller is configured to regulate the power of the laser via a pulse-width modulation (PWM) signal.

[0217] Aspect 159 generally concerns the system of any previous aspect in which the PID controller is configured to receive temperature feedback from the temperature sensor.

[0218] Aspect 160 generally concerns the system of any previous aspect in which the PID controller is configured to regulate power of the laser to achieve a temperature set point.#3371089 OS <11 9-000025

[0219] 24

[0220] Aspect 161 generally concerns the system of any previous aspect in which the controller is configured to detect dryness of the slide based on a threshold reduction in the power of the laser required to maintain the temperature set point.

[0221] Aspect 162 generally concerns the system of any previous aspect in which the controller is configured to detect cessation of evaporative cooling based on a sudden drop in the power of the laser required to maintain the temperature set point.

[0222] Aspect 163 generally concerns the system of any previous aspect including a liquid puddle covering the tissue sample on the slide.

[0223] Aspect 164 generally concerns the system of any previous aspect in which the puddle includes a cell conditioning reagent and a liquid coverslip (LCS),

[0224] Aspect 165 generally concerns the system of any previous aspect in which the cell conditioning reagent is configured to promote antigen retrieval.

[0225] Aspect 166 generally concerns the system of any previous aspect in which the cell conditioning reagent includes a high-temperature cell conditioning liquid.

[0226] Aspect 167 generally concerns the system of any previous aspect in which the cell conditioning reagent includes an ethylene glycol cell conditioning reagent.

[0227] Aspect 168 generally concerns the system of any previous aspect in which the cell conditioning reagent includes a mixture of Tris-EDTA with ethylene glycol (EG),

[0228] Aspect 169 generally concerns the system of any previous aspect in which the cell conditioning reagent is composed of at least 60% ethylene glycol (EG).

[0229] Aspect 170 generally concerns the system of any previous aspect in which the cell conditioning reagent has a boiling point of at least about 110 degrees Celsius.#3371089 OS <11 9-000025

[0230] 25

[0231] Aspect 171 generally concerns the system of any previous aspect in which the cell conditioning reagent covers the tissue sample on the slide.

[0232] Aspect 172 generally concerns the system of any previous aspect in which the liquid coverslip covers the cell conditioning reagent.

[0233] Aspect 173 generally concerns the system of any previous aspect in which the liquid coverslip forms a seal that maintains an isolated microenvironment for the cell conditioning reagent.

[0234] Aspect 174 generally concerns the system of any previous aspect in which the liquid coverslip is configured to reduce evaporation of the cell conditioning reagent during heating with the laser beam.

[0235] Aspect 175 generally concerns the system of any previous aspect in which the liquid coverslip includes mineral oil.

[0236] Aspect 176 generally concerns the system of any previous aspect including an optical system configured to expand and flatten the laser beam.

[0237] Aspect 177 generally concerns the system of any previous aspect in which the optical system is configured to provide passive heating.

[0238] Aspect 178 generally concerns the system of any previous aspect in which the optical system is disposed between the laser and the slide.

[0239] Aspect 179 generally concerns the system of any previous aspect in which the optical system includes a beam expander,

[0240] Aspect 180 generally concerns the system of any previous aspect in which the beam expander is configured to expand the laser beam.#3371089 OS <11 9-000025

[0241] 26

[0242] Aspect 181 generally concerns the system of any previous aspect in which the beam expander includes a Galilean beam expander.

[0243] Aspect 182 generally concerns the system of any previous aspect in which the optical system includes a field stop.

[0244] Aspect 183 generally concerns the system of any previous aspect in which the field stop is configured to clip the laser beam expanded by the beam expander,

[0245] Aspect 184 generally concerns the system of any previous aspect in which the field stop is configured to shape the laser beam from the beam expander to have a circular shape.

[0246] Aspect 185 generally concerns the system of any previous aspect in which the field stop includes a pillow-shaped field aperture.

[0247] Aspect 186 generally concerns the system of any previous aspect in which the field stop is configured to shape the laser beam from the beam expander to have a barrel shape.

[0248] Aspect 187 generally concerns the system of any previous aspect in which the optical system includes a beam shaper.

[0249] Aspect 188 generally concerns the system of any previous aspect in which the beam shaper is configured to redistribute the intensity profile of the laser beam.

[0250] Aspect 189 generally concerns the system of any previous aspect in which the beam shaper is configured to reshape the laser beam.

[0251] Aspect 190 generally concerns the system of any previous aspect in which the field stop is located betw een the beam expander and the beam shaper.

[0252] Aspect 191 generally concerns the system of any previous aspect in which the beam shaper is configured to produce the laser beam with a circular shape.#3371089 OS <11 9-000025

[0253] 27

[0254] Aspect 192 generally concerns the system of any previous aspect in which the beam shaper is configured to produce the laser beam with a rectangular shape.

[0255] Aspect 193 generally concerns the system of any previous aspect in which the beam shaper is configured to produce the laser beam with a square shape.

[0256] Aspect 194 generally concerns the system of any previous aspect in which the beam shaper is configured to distort the laser beam from the pillow-shaped field aperture to the square shape.

[0257] Aspect 195 generally concerns the system of any previous aspect in which the beam shaper includes a refractive flat-top beam shaper.

[0258] Aspect 196 generally concerns the system of any previous aspect in which the beam shaper is positioned to shine the laser beam onto the slide.

[0259] Aspect 197 generally concerns the system of any previous aspect in which the beam shaper is positioned to shine the laser beam onto the coverslip.

[0260] Aspect 198 generally concerns the system of any previous aspect in which the temperature sensor includes a thermal camera.

[0261] Aspect 199 generally concerns the system of any previous aspect in which the thermal camera is configured to capture a thermal image of the slide.

[0262] Aspect 200 generally concerns the system of any previous aspect in which the thermal camera is configured to monitor a spatial temperature distribution across the slide.

[0263] Aspect 201 generally concerns the system of any previous aspect in which the slide holder is configured to orient the slide at a transverse angle to promote drainage.

[0264] Aspect 202 generally concerns the system of any previous aspect in which the slide holder is configured to hold the slide at about a 45-degree angle.#3371089 OS <11 9-000025

[0265] 28

[0266] Aspect 203 generally concerns the system of any previous aspect in which the slide holder includes a slide cap configured to cover the slide.

[0267] Aspect 204 generally concerns the system of any previous aspect in which the slide holder is configured to hold two or more slides.

[0268] Aspect 205 generally concerns the system of any previous aspect in which the slide holder includes a fan.

[0269] Aspect 206 generally concerns the system of any previous aspect in which the fan is configured to blow a gas across the slide.

[0270] Aspect 207 generally concerns the system of any previous aspect in which the slide holder includes a slide sensor configured to sense the slide.

[0271] Aspect 208 generally concerns the system of any previous aspect including a visible laser configured to produce a visible laser beam.

[0272] Aspect 209 generally concerns the system of any previous aspect including a beam combiner configured to combine the visible laser beam with the laser beam from the laser to produce a combined laser beam.

[0273] Aspect 210 generally concerns the system of any previous aspect in which the absorptive material is configured to absorb wavelengths outside those produced by carbon dioxide lasers.

[0274] Aspect 21 1 generally concerns the method of any previous aspect in which the beam shaper includes a diffractive optical element (DOE).

[0275] Aspect 212 generally concerns the system of any previous aspect in which the diffractive optical element includes a hologram.#3371089 OS <11 9-000025

[0276] 29

[0277] Aspect 213 generally concerns the system of any previous aspect in which the diffractive optical element includes a computer-generated hologram (CGH).

[0278] Aspect 214 generally concerns the system of any previous aspect in which the beam shaper includes a computer-generated hologram.

[0279] Aspect 215 generally concerns the system of any previous aspect in which the beam expander and the beam shaper are integrated together in a single computer-generated hologram.

[0280] Aspect 216 generally concerns a method.

[0281] Aspect 217 generally concerns the method of any previous aspect including placing a tissue sample on a slide.

[0282] Aspect 218 generally concerns the method of any previous aspect including staining the tissue sample on the slide.

[0283] Aspect 219 generally concerns the method of any previous aspect including processing the tissue sample on the slide for analysis by heating with a laser beam.

[0284] Aspect 220 generally concerns the method of any previous aspect in which the processing includes baking the tissue sample on the slide.

[0285] Aspect 221 generally concerns the method of any previous aspect in which the baking includes heating the slide by shining the laser beam onto the slide.

[0286] Aspect 222 generally concerns the method of any previous aspect in which the baking includes heating the slide by shining the laser beam onto the base surface of the slide.

[0287] Aspect 223 generally concerns the method of any previous aspect in which the baking includes avoiding shining the laser beam onto the tissue sample.#3371089 OS <11 9-000025

[0288] 30

[0289] Aspect 224 generally concerns the method of any previous aspect in which the baking includes flash baking the slide with the laser beam.

[0290] Aspect 225 generally concerns the method of any previous aspect including generating the laser beam at 40% power.

[0291] Aspect 226 generally concerns the method of any previous aspect including scanning the laser beam with a square beam path.

[0292] Aspect 227 generally concerns the method of any previous aspect in which the square beam path has 0.25 mm spacing.

[0293] Aspect 228 generally concerns the method of any pre vious aspect including scanning the laser beam at a scanning speed of about 800 mm / s.

[0294] Aspect 229 generally concerns the method of any previous aspect including heating with the laser beam until a peak temperature is reached.

[0295] Aspect 230 generally concerns the method of any previous aspect in which the peak temperature is 120 degrees Celsius.

[0296] Aspect 231 generally concerns the method of any pre vious aspect including scanning the laser beam for at least six passes.

[0297] Aspect 232 generally concerns the method of any previous aspect including measuring temperature of the slide with a temperature sensor.

[0298] Aspect 233 generally concerns the method of any previous aspect in which the baking includes gentle baking the slide with the laser beam.

[0299] Aspect 234 generally concerns the method of any pre vious aspect including generating the laser beam with at most 5% power.#3371089 OS <11 9-000025

[0300] 31

[0301] Aspect 235 generally concerns the method of any previous aspect including scanning the laser beam at a scanning speed of about 3,500 mm / s.

[0302] Aspect 236 generally concerns the method of any previous aspect including heating with the laser beam until a steady state temperature is reached.

[0303] Aspect 237 generally concerns the method of any previous aspect in which the steady state temperature is about 60 degrees Celsius.

[0304] Aspect 238 generally concerns the method of any previous aspect including heating the slide with the laser beam for at most 20 seconds.

[0305] Aspect 239 generally concerns the method of any previous aspect including heating the slide with the laser beam for at least 12 seconds.

[0306] Aspect 240 generally concerns the method of any previous aspect including expanding spot size of the laser beam by defocusing the laser beam,

[0307] Aspect 241 generally concerns the method of any previous aspect including expanding spot size of the laser beam with a beam expander.

[0308] Aspect 242 generally concerns the method of any previous aspect in which the processing includes dewaxing the tissue sample on the slide,

[0309] Aspect 243 generally concerns the method of any previous aspect including performing the baking and the dewaxing simultaneously.

[0310] Aspect 244 generally concerns the method of any previous aspect in which the dewaxing includes heating the slide with, the laser beam.

[0311] Aspect 245 generally concerns the method of any previous aspect in which the dewaxing includes heating the base surface of the slide with the laser beam.#3371089 OS <11 9-000025

[0312] 32

[0313] Aspect 246 generally concerns the method of any previous aspect in which the dewaxing includes heating the slide with the laser beam using a flash heating process.

[0314] Aspect 247 generally concerns the method of any previous aspect in which the dewaxing includes heating the slide with the laser beam using a gentle heating process.

[0315] Aspect 248 generally concerns the method of any previous aspect in which the dewaxing includes melting wax in the tissue sample with heat from the laser beam.

[0316] Aspect 249 generally concerns the method of any previous aspect in which the dewaxing includes applying a solvent to the tissue sample.

[0317] Aspect 250 generally concerns the method of any previous aspect including forming a puddle of the solvent on the slide,

[0318] Aspect 251 generally concerns the method of any previous aspect including forming a puddle of the solvent over the tissue sample.

[0319] Aspect 252 generally concerns the method of any previous aspect including applying the solvent to the slide with a nozzle.

[0320] Aspect 253 generally concerns the method of any previous aspect including draining a mixture of the solvent and the wax from the slide.

[0321] Aspect 254 generally concerns the method of any previous aspect including draining a mixture of the solvent and the wax from the slide into a drain channel.

[0322] Aspect 255 generally concerns the method of any previous aspect including sweeping the mixture of the wax and the solvent with an air knife.

[0323] Aspect 256 generally concerns the method of any previous aspect including applying a transfer fluid with a vaporization temperature above 100 degrees Celsius to the slide to form a puddle.#3371089 OS <11 9-000025

[0324] 33

[0325] Aspect 257 generally concerns the method of any previous aspect including irradiating the slide with the laser beam to heat the slide to a steady state temperature that removes tissue crosslinking from fixation to promote antigen retrieval.

[0326] Aspect 258 generally concerns the method of any previous aspect in which the steady state temperature is between about 110 to 118 degrees Celsius.

[0327] Aspect 259 generally concerns the method of any previous aspect in which the processing includes curing a coverslip on the slide.

[0328] Aspect 260 generally concerns the method of any previous aspect in which the curing includes heating the coverslip with the laser beam.

[0329] Aspect 261 generally concerns the method of any previous aspect including applying absorptive material to the coverslip.

[0330] Aspect 262 generally concerns the method of any previous aspect including removing the absorptive material from the coverslip after the curing.

[0331] Aspect 263 generally concerns the method of any previous aspect in which the curing includes heating the absorptive material with the laser beam.

[0332] Aspect 264 generally concerns the method of any previous aspect including cooling the slide via air convection cooling.

[0333] Aspect 265 generally concerns the method of any previous aspect including cooling the slide for at most 20 seconds.

[0334] Aspect 266 generally concerns the method of any previous aspect including scanning the laser beam along a laser scan path on the slide.#3371089 OS <11 9-000025

[0335] 34

[0336] Aspect 267 generally concerns the method of any previous aspect including generating the laser beam with a laser.

[0337] Aspect 268 generally concerns the method of any previous aspect including regulating power of the laser with a controller in response to the measuring the temperature of the slide to maintain a temperature set point.

[0338] Aspect 269 generally concerns the method of any previous aspect including detecting cessation of evaporative cooling on the slide with the controller based on a drop in the power of the laser required to maintain the temperature set point.

[0339] Aspect 270 generally concerns the method of any previous aspect including dispensing a cell conditioning reagent onto the tissue sample on the slide.

[0340] Aspect 271 generally concerns the method of any previous aspect including covering the cell conditioning reagent with a liquid coverslip.

[0341] Aspect 272 generally concerns the method of any previous aspect including heating the slide with the laser beam,

[0342] Aspect 273 generally concerns the method of any previous aspect in which the heating includes heating the slide to about 120°C.

[0343] Aspect 274 generally concerns the method of any previous aspect in which the heating occurs for about 8 to about 12 minutes.

[0344] Aspect 275 generally concerns the method of any previous aspect in which the heating occurs for about 10 minutes,

[0345] Aspect 276 generally concerns the method of any previous aspect in which the heating occurs for about 12 minutes.#3371089 OS <11 9-000025

[0346] 35

[0347] Aspect 277 generally concerns the method of any previous aspect in which the tissue sample includes a vimentin type ti ssue sample.

[0348] Aspect 278 generally concerns the method of any previous aspect in which the tissue sample includes a B-cell lymphoma 2 (BCL2) type tonsil tissue sample.

[0349] Aspect 279 generally concerns the method of any previous aspect in which the tissue sample includes an Anaplastic Lymphoma Kinase (ALK) type appendix tissue sample.

[0350] Aspect 280 generally concerns the method of any previous aspect in which the tissue sample includes a cluster of differentiation 10 (CD 10) type tonsil tissue sample.

[0351] Aspect 281 generally concerns the method of any previous aspect including expanding and flattening the laser beam to promote uniform heating while the laser beam is stationary,

[0352] Aspect 282 generally concerns the method of any previous aspect including expanding the laser beam with a beam expander.

[0353] Aspect 283 generally concerns the method of any previous aspect including shaping the laser beam with a beam shaper.

[0354] Aspect 284 generally concerns the method of any previous aspect including forming the laser beam to have a rectangular shape with the beam shaper.

[0355] Aspect 285 generally concerns the method of any previous aspect including drying the slide by blowing a gas across the slide.

[0356] Aspect 286 generally concerns the method of any previous aspect including monitoring a spatial temperature distribution across the slide with a thermal camera.

[0357] Aspect 287 generally concerns the method of any previous aspect including adjusting power of the laser beam at different locations on the slide based on the spatial temperature distribution.#3371089 OS <11 9-000025

[0358] 36

[0359] Aspect 288 generally concerns the method of any previous aspect including processing multiple slides with the laser beam in an asynchronous manner.

[0360] Aspect 289 generally concerns the method of any previous aspect including inducing thermocapillary convection on the slide with the laser beam.

[0361] Aspect 290 generally concerns the method of any previous aspect in which the thermocapillary convection sweeps moisture off the slide.

[0362] Aspect 291 generally concerns the method of any previous aspect including scanning the laser beam to form concentric loops with different radii.

[0363] Aspect 292 generally concerns the method of any previous aspect including analyzing real-time thermal curves from the slide to adapt laser heating protocols based on individual slide wetness.

[0364] Aspect 293 generally concerns the method of any previous aspect including scanning the laser beam in a radially outward direction on the slide.

[0365] Aspect 294 generally concerns the method of any previous aspect including combining the laser beam with a visible laser beam at a beam combiner to form a combined laser beam.

[0366] Aspect 295 generally concerns the method of any previous aspect including shining the combined laser beam onto the slide.

[0367] Further forms, objects, features, aspects, benefits, advantages, and embodiments of the present invention will become apparent from a detailed description and drawings provided herewith.#3371089 OS <11 9-000025

[0368] 37

[0369] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of a system with one or more modules for processing tissue samples for analysis.

[0370] FIG. 2 is a block diagram of select modules within the system that utilize a direct heating technique according to one example.

[0371] FIG. 3 is a chart showing examples of a standard processing workflow and a laser processing workflow.

[0372] FIG. 4 is a block diagram of a laser module according to one example.

[0373] FIG. 5 is a graph showing absorption of light by various slide materials at different frequencies.

[0374] FIG. 6 is a perspective view of a model of a slide irradiated by a laser beam.

[0375] FIG. 7 is a block diagram of a heating station with a laser according to one example.

[0376] FIG. 8 is a graph of temperature readings at different locations of the slide during a quick flash laser baking process,

[0377] FIG. 9 is a graph of temperature readings at different locations of the slide during a gentle laser baking process.

[0378] FIG. 10 is an image comparing various laser baking processes to a control process.

[0379] FIG. 11 is a block diagram of a laser system with a laser configured to dewax a tissue sample on the slide.

[0380] FIG. 12 is a perspective view of the FIG. 11 laser system.

[0381] FIG. 13 is an enlarged perspective view of a beam expander with a slide holder configured to hold the slide.

[0382] FIG. 14 is an enlarged perspective view of the FIG. 14 slide holder.

[0383] FIG. 15 is an enlarged perspective view of a dispenser head used in the FIG. 11 laser system. FIG. 16 is a graph of temperature readings at different locations of the slide during a dewaxing process.

[0384] FIG. 17 is a perspective view of the FIG. 13 beam expander and the slide holder with a temperature sensor positioned to measure the temperature at the bottom of the slide.

[0385] FIG. 18 is a perspective view of the FIG. 13 beam expander and the slide holder with a temperature sensor positioned to measure the temperature at the top of the slide.

[0386] FIG. 19 is an image of slides using laser dewaxing processes.

[0387] FIG. 20 is an image of further slides using laser dewaxing processes.#3371089 OS <11 9-000025

[0388] 38

[0389] FIG. 21 is an enlarged cross-sectional view of a coverslip and a slide during curing via a laser beam.

[0390] FIG. 22 is an enlarged cross-sectional view of a coverslip covered with an absorptive material during curing via a laser beam,

[0391] FIG. 23 is a side view of a laser system for curing the coverslip with a laser beam.

[0392] FIG, 24 is an enlarged perspective view of the FIG. 23 laser system.

[0393] FIG, 25 is a diagram of a control process and a test process according to one example.

[0394] FIG. 26 is a diagram of a slide assembly with absorpti ve bars and processing parameters according to one example.

[0395] FIG. 27 is a top view of a slide assembly with an absorptive material that includes a machine- readable identifier.

[0396] FIG. 28 is a top view of a slide assembly with absorptive material in the form of dots.

[0397] FIG. 29 is an image of slides stained with two different stains and cured using the laser curing process.

[0398] FIG, 30 is an image of slides damaged during cover slipping,

[0399] FIG, 31 is a chart illustrating results of a feasibility study.

[0400] FIG. 32 is an image of slides processed with laser-based processes.

[0401] FIG. 33 is an image of slides processed with laser-based processes.

[0402] FIG. 34 is an image of slides processed with a. laser-based dewaxing and curing process. FIG. 35 is another image of slides processed with a laser-based dewaxing and curing process. FIG, 36 is a perspective view of a laser system with a holder assembly according to another example.

[0403] FIG. 37 is a perspective view of a laser system with a holder assembly according to a further example,

[0404] FIG, 38 is a graph il lustrating light absorption of various material types of slides and coverslips at different wavelengths.

[0405] FIG. 39 is a block diagram of an adaptive laser heating system according to still yet another example.

[0406] FIG, 40 is a top view' of a laser scan path generated by the FIG. 39 adaptive laser heating system.

[0407] FIG. 41 is a diagram depicting the operation of a proportional-integral-derivative controller (" PID controller") in the FIG. 39 adaptive laser heating system for controlling temperature.#3371089 OS <11 9-000025

[0408] 39

[0409] FIG. 42 is a graph showing a first example of a heating ramp and power usage when heating the slide with the FIG. 39 adaptive laser heating system.

[0410] FIG. 43 is a graph showing a second example of a heating ramp and power usage when heating the slide with the FIG. 39 adaptive laser heating system.

[0411] FIG. 44 is a block diagram of an antigen retrieval system according to a further example. FIG. 45 is a perspective view of an optics system with passive components for laser heating without scanning the laser beam.

[0412] FIG. 46 is a first side view of the beam path in the FIG. 45 optics system.

[0413] FIG. 47 is a diagram illustrating lateral wavefront distortion in a beam shaper of the FIG. 45 optics system.

[0414] FIG. 48 is a diagram illustrating a Merit function for the simulated raytracing for the FIG. 45 optics system.

[0415] FIG. 49 is a diagram showing the simulated raytracing for the FIG. 45 optics system.

[0416] FIG. 50 is a diagram of an optics system according to another example that is able to output a square or rectangular beam.

[0417] FIG. 51 is a diagram providing information concerning the field masking of the FIG. 50 optics system for producing the square or rectangular beam.

[0418] FIG. 52 is a block diagram of a laser heating system according to a further example.

[0419] FIG. 53 is a cross-sectional view of the FIG. 52 system,

[0420] FIG. 54 is a side view of the FIG. 52 system.

[0421] FIG. 55 is an enlarged perspective view of the FIG. 52 system.

[0422] FIG. 56 is a top view of beam paths generated by the FIG. 52 system.

[0423] FIG. 57 is a top view of a slide with a combined laser beam from the FIG. 52 system.

[0424] FIG. 58 is a perspective view of the FIG. 57 slide during laser heating with the FIG. 52 system.

[0425] FIG. 59 is a flowchart of a method for heating two or more slides with the FIG. 52 system. FIG. 60 is a flowchart of a method for performing spatial temperature distribution control with the FIG. 52 system.#3371089 OS <11 9-000025

[0426] 40

[0427] DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

[0428] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to descri be the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments and any further applications of the principles of the in vention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. One embodiment of the invention is shown In great detail, although it will, be apparent to those skilled in the relevant art dial some features that arc not relevant to the present invention may not be shown for the sake of clarity.

[0429] The reference numerals in the following description have been organized to aid the reader in quickly identifying the drawings where various components are first shown. In particular, the drawing in which an element first appears is typically indicated by the left-most digit(s) in the corresponding reference number. For example, an element identified by a " 100” series reference numeral will likely first appear in FIG. 1, an element identified by a "200” series reference numeral will likely first appear in FIG, 2, and so on,

[0430] FIG. 1 show's a block diagram of a tissue analysis system 100 according to one example. As shown, the system 100 includes one or more modules 102 for processing and analyzing tissue samples. The modules 102 in the depicted example include an input module 105, a staining module 110, a coverslip module 115, and an imaging module 120, but the system 100 can include other types of modules 102 in other examples. In one form, the input module 105. the staining module 110, the coverslip module 115, and the imaging module 120 are integrated together into a single unit. In other examples, one or more of these modules 102 are separate from one another, and in stil l yet other examples, some of these modules 102 can be eliminated or optional. For example, the staining module 110 and the coverslip module 115 can be incorporated together in a tissue preparation system like the VENTANA HE® 600 brand system or instrument (Roche Diagnostics GmbH, Mannheim, Germany). The input module 105 is designed to prepare a tissue sample on a slide for subsequent staining. For instance, the tissue sample can be baked on the slide and / or dewaxed or deparaffinized in the input module 105. These processes traditional ly require heating of the tissue sample on the slide. Commonly, the slides are gathered in batches and placed into an oven that inefficiently#3371089 OS <11 9-000025

[0431] 41

[0432] heats the slides. This process of collecting the slides, heating the slides, and cooling the slides for subsequent handling and processing can require considerable amounts of time, energy, and effort. As will be explained further below, at least some of the modules 102 in the system 100 are configured to directly heat the slide, coverslip, and / or tissue sample to reduce processing times and energy usage. For example, instead of using an oven, a laser is used to bake and / or deparaffinize the tissue sample in the input module 105.

[0433] The staining module 110 is configured to stain the tissue sample on the slide. In one form, the staining module 110 includes an automated staining instrument, such as a BENCHMARK® brand staining instrument (Roche Diagnostics GmbH, Mannheim, Germany) or a VENTANA HE® 600 brand system, but it should be recognized that the staining module 110 can include other types of staining instruments. In some cases, the staining module 110 dispenses a single stain on the slide, and in other cases, the staining module 110 dispenses multiple stains onto the slide. The staining process in the staining module 110 sometimes includes heating the tissue sample to promote visibility of various tissue structures. For instance, antigen retrieval is used in some staining techniques, such as immunohistochemistry,, to help make certain proteins or molecules in the tissue sample more visible under a microscope. Over time or due to how the tissue sample is prepared, these proteins can become hidden or hard to detect. Antigen retrieval uses heat (and sometimes chemicals) to unmask these proteins so that special stains or antibodies can attach to them and make them visible. Instead of using traditional indirect heating techniques, the staining module 1 10 of system 100 in some cases uses a direct heating technique, such as via a laser, to heat the tissue sample during the antigen retrieval process or other staining processes in the staining module 1 10.

[0434] Once the stain is applied, the tissue sample may be dehydrated within the staining module 110 or in a downstream module. The tissue sample can be dehydrated in a number of manners. For example, the tissue sample can be dehydrated through a physical process, such as via evaporation caused by heating the tissue sample on the slide, and / or through a chemical process, such as by applying a dehydration solvent or fluid, to drive the water out of the tissue sample. In one example, a laser heats the tissue sample to promote dehydration. Alternatively or additionally, a series of alcohol solutions with increasingly higher concentrations of alcohol are dispensed onto the tissue sample in order to dehydrate the tissue sample by driving out the water from the tissue sample. In some cases, the dehydration#3371089 OS <11 9-000025

[0435] 42

[0436] solvent or drying agent, like alcohol, may remain in the tissue sample, and in those cases a clearing solvent or agent is applied to the tissue sample such as via dispensing or submersion techniques. Some common examples of clearing solvents include xylene, benzene, chloroform, toluene, and cedarwood oil. In most cases, the clearing solvent has a refractive index close to the proteins in the tissue sample. This reduces light scattering in the tissue sample, which in turn makes the tissue appear to be more transparent or clear. In a few cases, the clearing solvent is used to remove the drying agent. In most other cases, a clearing solvent is not used.

[0437] The coverslip module 115 is configured to prepare the tissue sample on the slide for analysis. At the coverslip module 1 15, the now dehydrated tissue sample on the slide is covered with a coverslip. In one version, the coverslip is a pre-glued type coverslip, and in one particular example, the pre-glued coverslip is a VENTANA HE® 600 brand glass coverslip (version / model / catalog number 06711138001) that is supplied by Ventana Medical Systems, Inc., but it should be recognized that other types of pre-glued coverslips or covertiles can be used. The pre -glued cover slip tends to have a more consistent thickness when applied to the tissue sample. Moreover, the pre-glued cover slip tends to overlay the slide and tissue sample in a more even manner. The pre-glued coverslip lays in a flat or parallel manner relative to the slide. In other words, the coverslip is not tilted or otherwise distorted relative to the rest of the slide. The coverslip module 115 in one example is an automated coverslipper. The automated coverslipper is able to place the coverslip over the tissue on the slide in a more consistent and even manner. Having the coverslip lying flat on the slide reduces distortions in the image captured at the imaging module 120. To promote curing of the glue or adhesive for the coverslip, the coverslip and / or slide in the coverslip module 115 is directly heated with a laser in some examples.

[0438] After coverslipping at the coverslip module 1 15, the imaging module 120 captures one or more images of the tissue sample on the slide. Wi thin the process, the imaging module 120 is located downstream of the coverslip module 115. In one version, the imaging module 120 includes a microscope that provides a magnified image of the tissue sample on the slide. In one version, the microscope is a fluorescent microscope with a camera that captures the image. The imaging module 120 in one form includes an image sensor in the form of a Charge-Coupled Device (CCD) that is optically coupled to a microscope. Based on the#3371089 OS <11 9-000025

[0439] 43

[0440] image, a pathologist, physician, or other personnel can analyze the various structural and / or other biological properties of the tissue sample. For example, the imaging module 120 can generate an image of the tissue sample using a birefringence technique. Based on the image, the presence or absence of amyloids in a tissue sample, like a breast tissue sample, can be analyzed.

[0441] FIG. 2 shows one or more processing stages or stations 205 in the modules 102 of the system 100 that may use this direct laser heating process. It should be noted that only some of the modules 102 and stations 205 that incorporate this direct heating technique are shown, but it should be appreciated that other tissue processing or analysis stations 205 that require heating may utilize this heating technique. Moreover, it should be recognized that the individual modules 102 typically include other processing stations 205 than the selected heating stations 205 that are shown. In the illustrated example, each of the stations 205 includes a laser 210 that generates a laser beam 215 for heating the slide, coverslip, and / or tissue sample as well as other associated materials.

[0442] As shown, the stations 205 in the modules 102 that require heating each have a laser 210 that generate a laser beam 215. While each station 205 is shown to have a single laser 210 that generates a single laser beam 215, it should be appreciated that individual stations 205 can have more than one laser 210 and can utilize more than one laser beam 215 for heating and / or other purposes. In the illustrated example, the input module 105 includes a baking station 220 and a dewaxing station 225 where the lasers 210 are used to provide direct heating of the slide and / or the tissue. Likewise, the staining module 110 has an antigen retrieval station 230, and the laser 210 at the antigen retrieval station 230 is used to heat the tissue sample to promote antigen retrieval. The coverslip module 115 has a curing station 235, and the laser 210 at the coverslip module 115 is configured to promote curing of the coverslip on the slide. As should be appreciated, the input module 105, the staining module 1 10, and the coverslip module 115 typically include other processing stations 205 than are shown in FIG, 2. Besides the ones shown in FIG. 2, other types of stations 205 in the modules 102 can utilize the lasers 210 for heating and other purposes. Some of the stations 205 can be combined together. In one example, the baking and dewaxing processes performed at the baking station 220 and the dewaxing station 225 are combined together into a single station 205. Using the laser 210 with this example, the combined baking and dewaxing processes are performed in about 15minutes, which is considerably shorter than traditional approaches that can take over an hour to perform. The stations 205 can also be located in other modules 102. For instance, the antigen retrieval station 230 can be located in the input module 105, In one particular example, the heating processes for the baking station 220, the dewaxing station 225, and the antigen retrieval station 230 are integrated into a single stations 205 within the input module 105 or the staining module 110. While the lasers 210 in FIG. 2 are illustrated as being located at the stations 205, the lasers 210 in some versions are located upstream from, downstream from, or between the processing stations 205.

[0443] FIG. 3 shows a chart 300 comparing a standard processing workflow 305 to a laser processing workflow 310 that uses the laser 210 at various stations 205 to process tissue samples. In the laser processing workflow 310, the baking station 220, the dewaxing station 225, and the curing station 235 each use the laser 10 for various heating purposes. As can be seen, the laser processing workflow 310 eliminates five manual touch points found in the standard processing workflow 305, and the laser processing workflow 310 saves approximately 2 hours of processing time as compared to the standard processing workflow 305.

[0444] FIG. 4 illustrates one example of a laser module 400 with the laser 210 that can be used in the system 100. The laser module 400 includes a laser head 405 and a controller 410 that controls the operation of the laser head 405. In one form, the system 100 has a single controller 410 that controls two or more of the lasers 210, and in another form, each of the lasers 210 has a dedicated controller 410. The control ler 410 controls the generation of the laser beam 215 that is directed onto a target 415.

[0445] The laser head 405 of the laser 210 in the depicted example includes a laser or beam generator 420 that generates the laser beam 215 and a beam positioner 425 that positions and moves the laser beam 215 along the target 415. The beam positioner 425 in the depicted example includes two galvanometer scanners that direct movement of the laser beam 215 along two axes. In one form, the beam positioner 425 includes motorized mirrors that are tilted to change the path of the laser beam 215. The beam positioner 425 can include other types of beam positioning or deflector systems such as acousto-optic or electro-optic type deflectors. The laser head 405 further includes a power control module 430 that controls the#3371089 OS <11 9-000025

[0446] 45

[0447] power of the laser beam 215 emitted by the beam generator 420. The power control module 430 is operatively connected to the beam generator 420 to control the power of the laser beam 215. In some cases, the laser beam 215 can be a continuous beam or a pulsed beam depending on the requirements. In one version, the power control module 430 is in the form of an electronic power control circuit, but it should be recognized that the power can be controlled in other manners.

[0448] To control the position and / or movement of the laser beam 215 via the beam positioner 425, the laser head 405 has a position control module 435 that is operatively connected to the beam positioner 425. The position control module 435 in the depicted example is in the form of an electronic position control circuit, but the position control module 435 can be configured differently in other variations. As shown, the laser head 405 further includes a communication module 440 that is configured to communicate with the controller 410. Via the communication module 440, the controller 410 is able to control the power of the laser beam 215 emitted by the beam generator 420 through the power control module 430, and the controller 410 is able to control the position of the laser beam 215 via the beam positioner 425 and the position control module 435. In one form, the communication module 440 is in the form of an electronic communication circuit that is operatively coupled to the controller 410, the position control module 435, and the power control module 430, but the communication module 440 can be configured differently in other variations.

[0449] In some versions, the laser module 400 utilizes a feedback control system to control the laser beam 215. In the depicted example, the laser module 400 includes a temperature sensor 445 that is operatively connected to the controller 410. The temperature sensor 445 is positioned proximal to the target 415 to measure the temperature of the target 415 where the laser beam 215 heats the target 415 and / or elsewhere on the target 415, In one version, the temperature sensor 445 is an infrared (IR) type temperature sensor, but the temperature sensor 445 can include other types of temperature sensors in other variations. The target 415 in the depicted example includes a coverslip 450. However, as will be explained further below, the target 415 can come in other forms such as a slide. Based on the temperature measured of the target 41 measured with the temperature sensor 445, the controller 410 is able to control the power of the laser beam 215 applied to the target 415 via the power control module 430 and the movement of the laser beam 215 along the target 415 via the position control module 435.#3371089 OS <11 9-000025

[0450] 46

[0451] In one version, the beam positioner 425 includes one or more mirrors controlled by galvanometer motors to direct the laser beam 215 to scan a predefined path to evenly heat up the target 415, such as a glass slide, during the baking process at the bakin station 220 and the dewaxing process at the dewaxing station 225, For the beam positioner 425, projection optics with galvanometers are chosen such that the rotational range of the mirrors can rapidly cover the full length and width of the target 415, In one form, a purposefully defocused laser optical system is used to irradiate a larger area without risk of reaching the damage threshold of the glass, and the defocused laser beam 215 provides a more homogeneous heat distribution. The temperature sensor 445 provides a feedback mechanism by measuring the surface temperature of the target 415 during laser treatment. The controller 410 via the temperature measurements from the temperature sensor 445 can be used to avoid excess heating. In turn, the controller 410 via the power control module 430 can control the energy deposited onto the target 415 and scan times as well as the scan paths of the laser beam 215 on the target 415 via the beam positioner 425 and the position control module 435,

[0452] FIG. 5 shows a graph 500 that depicts the transmission or absorption for different glasses at different frequencies. In the graph 500 of FIG. 5, the glasses are borosilicate glass, quartz glass, and soda-lime glass. Borosilicate and soda-lime glasses are commonly used to form microscope slides due to their optical properties. In one form, the laser 210 is a carbon dioxide (CO2) laser, because the carbon dioxide laser has a peak intensity at about 10.6 pm. This ensures that most of the power of the laser beam 215 is absorbed by the glass target 415 and instantly converted into heat. Moreover, carbon dioxide lasers are affordable and widely accessible. The use of the laser 210 aids in a more direct heating process being applied to the glass target 415, In other words, borosilicate and soda-lime glass slides more fully absorb the laser beam 215 from the carbon dioxide laser generated light due to the long wavelength (i. e., of about 10 um) of the laser beam 215, in such a way that the light of the laser beam 215 is converted into a localized heat source that increases the temperature of the glass slide located in the vicinity of the focus area,

[0453] FIG, 6 shows a model of a slide 605 that is one example of the target 415 that is heated by the laser 210, For quickly moving laser beams 215, a transient model can be derived by simplifying the model to a finite slab. As shown, the laser beam 215 forms a spot 610 on an#3371089 OS <11 9-000025

[0454] 47

[0455] exterior surface of or inside the slide 605 depending how the laser beam 215 is focused and aimed at the slide 605. The spot 610 of the laser beam 215 has a spot size which in the depicted example is defined by a spot radius 615. In one variation, the slide 605 is heated from the top exterior surface where the tissue sample rests, and in another variation, the slide 605 is heated from the bottom side or surface. In some cases, the laser beam 215 is focused so that heat is created inside the slide 605 or other target 415. When the slide 605 is irradiated from the bottom, the tissue sample on the top surface is safeguarded from being directly irradiated with the laser beam 215. Only the heat from the laser beam 215 at the bottom is conducted through the slide 605 to the tissue sample on the top surface.

[0456] In one example, the laser beam 215 is purposefully defocused. With the laser beam 215 defocused, the energy from the laser beam 215 is deposited at a wider area without reaching the damage threshold of the glass in the slide 605, and the defocused laser beam 215 tends to heat the glass of the slide 605 more evenly. In one variation, an optical array in the laser 210 is used to control the focal length. The focal length of the laser beam 215 can be managed to optimize space constraints, spot size, and target positioning tolerance. For example, the focal distance and beam correction in one version is managed by an F-Theta lens that also provides a fiat field focusing, a linear relationship between the scan angle and the projected path height, and minimizes distortion and aberrations that are typically found in galvanometer scanning systems.

[0457] Referring to FIGS. 4 and 6, the focal length impacts the space needed between the galvanometer and the sample, which in turn impacts the volume required for the laser module 400, and these space requirements may be a constraint in certain configurations. For example, these focal length constraints may inhibit integration of the laser module 400 into existing instruments. The focal length also impacts the positioning tolerance of the spot 610 of the laser beam 215 on the slide 605. The shorter the focal length in relation to the target 415 tends to make the spot 610 of the laser beam 215 move quicker as compared to longer focal lengths. In other words, the shorter focal lengths make the tolerance for spot position larger. The controller 410 is configured to manage these two factors (i.e., space constraints and tolerance to position), to enhance heating of the slide 605 or other target 415.#3371089 OS <11 9-000025

[0458] 48

[0459] The controller 410 for the laser 210 is further designed to control the size of the spot 610 for the spot 610. Spot size of the laser beam 215 impacts the distribution of energy on the slide 605. If the spot size is too small, the spot 610 of the laser beam 215 may concentrate too much energy in a small point. The thermal conduction properties of the glass of the slide 605 and the resulting overheating of the small spot 610 increases the risk of damage to the glass of the slide 605.

[0460] In traditional heating methods, a furnace or kiln for example needs to be preheated which consumes both time and energy. In contrast, the laser 210 can be swi tched on and off very quickly via the power control module 430 such that the time and energy for preheating is no longer required. The controller 410 through the power control module 430 is able to switch or change the power of the laser 210 at the speed in terms of kilohertz frequencies. This rapid changing of power delivered by the laser 210, such as via pulse width modulation (PWM), facilitates fine tuning of the power that is deli vered to the laser head 405. Combined with the ability to redirect the laser beam 215 around the area to be heated via the beam positioner 425 and the position control module 435, the controller 410 for the laser 210 is able to control the temperature gradient in the slide 605 by controlling the power, speed, and path of the laser beam 215. This control also allows the use of high power, which speeds up the heating to achieve the desired temperature in seconds. This in turn reduces the energy needed to reach a steady state at the desired temperature.

[0461] The controller 410 can control temperature using an open loop type control technique or a closed loop type control technique. When temperature control of the target 415 is performed using an open loop technique, the controller 410 in one version calculates the power, path, and speed of the laser beam 215 that is needed to reach steady state for the desired temperature. When the closed loop control technique is used, the temperature sensor 445. like an infrared sensor, feeds temperature readings for the laser head 405 to the controller 410. In one example, the controller 410 includes a proportional-integral-derivative (PID) controller, but the controller 410 can include other types of controllers. Based on the temperature readings, the controller 410 can adjust the power of the laser beam 215 via the power control module 430 and the path or speed of the laser beam 215 via the position control module 435 to adjust the pow'er delivered by the laser beam 215 to the coverslip 450, the slide 605, and / or other target 415 in order to reach a desired temperature. In other variations, the laser module#3371089 OS <11 9-000025

[0462] 49

[0463] 400 includes an array of temperature sensors 445, like in the form of a thermal camera, to con trol the power of the laser 210 as a function of the temperature gradient of the slide 605, ensuring more even heating,

[0464] The thermal mass or inertia of the slide 605 is substantially lower than a heating plate or pad that is sometimes used during heating. The relatively large exposed faces of the slide 605 facilitate faster air cooling, which allows for faster processing and handling. This faster air cooling for example allows higher temperatures to be used during baking or other heating processes to evaporate residual water, but the slide 605 is able to be cooled quickly from natural convection and prevents tissue damage immediately after the irradiation has stopped (or Is reduced).

[0465] Baking is a preparatory process that greatly enhances the reliability and clarity of histological analysis so as to support accurate diagnosis. To accelerate staining procedures in a pathology lab, the laser 210 is used for various thermal processes, including baking. Generally, baking in a histopathology lab involves heating paraffin -embedded tissue sections affixed to glass slides 605. Baking generally occurs after sectioning and before any staining procedures. Baking ensures that the tissue sections firmly adhere to the slides 605, which prevents detachment during staining and handling, The baking process also assists in removing any residual water from the tissue sections, which helps to prevent artifacts during staining. The heat provided by the laser beam 215 helps in melting excess paraffin, which promotes better adherence of the tissue and makes the sections more robust for subsequent processes.

[0466] FIG. 7 shows one example of a heating station 700 with the laser 210 that can be used at various stations 205 for heating and other processing purposes. The heating station 700 will be described as being used at the baking station 220 and the dewaxing station 225, but it should be recognized that the heating station 700 can be used at other stations 205, such as at the antigen retrieval station 230 and the curing station 235. As depicted, the heating station 700 includes the laser module 400 with the components of the laser module 400 discussed before, like the laser head 405, the beam generator 420, and the beam positioner 425.

[0467] The heating station 700 includes a position adjuster 705 configured to adjust the relative position of the laser 210. In the depicted example, the position adjuster 705 is configured to#3371089 OS <11 9-000025

[0468] 50

[0469] adjust the position of the laser head 405 along a single dimension as indicated by double arrow 710. In one version, the position adjuster 705 is a knob with a threaded connection to the laser head 405 that moves the laser head 405 in a vertical direction that is indicated with the double arrow 710. It should be appreciated that the position adjuster 705 in other variations can be configured to move the laser 210 in more than one dimension. For instance, the position adjuster 705 is designed to position the laser 210 in a vertical direction, a horizontal direction, a longitudinal direction, and'or a rotational direction.

[0470] As described before, the laser module 400 in the heating station 700 includes the beam generator 420 to generate the laser beam 215 and the beam positioner 425 to direct the scanning motion / position of the laser beam 215 along the target 415, which in this case is the slide 605. The laser module 400 in the heating station 700 of FIG. 7 further includes the other components of the laser module 400, such as the controller 410, the beam generator 420, the power control module 430. the position control module 435. and the communication module 440. The laser head 405 of the laser 210 in the heating station 700 includes a lens 715 that focuses or defocuses the laser beam 215. Tire lens 715 is able to adjust the focal length of the laser beam 215. In one form, the lens 715 is an F -Theta lens of the type described before that provides flat field focusing, a linear relationship between the scan angle and the projected path height and minimizes distortion and aberrations that are typically found in galvanometer scanning systems. The lens 715 in other variations can include other types of lenses.

[0471] From the lens 7.15, the laser beam 215 travels along a beam path 720 to a mirror 725. The mirror 725 reflects the laser beam 215 onto the slide 605. The mirror 725 in one form is a silver mirror, but the heating station 700 in other examples can include other types of mirrors and other optical components to reflect and / or refract the laser beam 215 along the beam path 720. As shown in FIG. 7, the slide 605 supports a tissue sample 730. The slide 605 has a sample surface 735 where the tissue sample 730 is placed and a base surface 740 that is positioned opposite to the sample surface 735, The heating station 700 further has a slide holder 745 that holds the slide 605 in place. As can be seen, the mirror 725 directs the laser beam 215 to shine on the base surface 740 of the slide 605. Shining the laser beam 215 onto the base surface 740 of the slide 605 rather than the sample surface 735 facilitates warming of the tissue sample 730 on the slide 605 via conduction without having the damage#3371089 OS <11 9-000025

[0472] 51

[0473] associated with directly shining the laser beam 215 onto the tissue sample 730 at the sample surface 735.

[0474] In one example, the laser 210 in the healing station 700 is the carbon dioxide type laser of the type described before, and the lens 715 for the laser 210 is the F-Theta lens that was previously described. During use, the laser beam 215 is directed to the glass slide 605 in such a way that laser beam 215 interferes with the underside or the base surface 740 of the slide 605 where the laser beam 215 Is fully absorbed typically within less than 10 um. This shallow penetration depth is due to the strong absorption of the laser beam 215 wi th the far-infrared wavelength (commonly 10.6 gm) by the silicon-oxygen (Si-O) bonds within the glass structure of the slide 605. 'Hie laser energy i s deposited very close to the base surface 740 of the borosilicate glass slide 605. Consequently, the laser energy is primarily converted to heat at the immediate surface. The focal properties of the F-Theta lens used by the carbondioxide heating station 700 allows control of the focal plane to be projected as a flat plane throughout the whole scan area (rather than as a curved plane when using a standard lens).

[0475] The distance between the glass slide 605 and the optimal focal plane of the F-Theta lens 715 is purposely chosen to achieve a defocused beam with a larger spot size at the glass interface. This is to avoid the laser beam 215 being too sharply focused and reaching the damaging temperature threshold of the borosilicate glass. This defocus reduces the power densi ty such that glass damage or engraving can be avoided. Since the wider beam waist of the defocused laser beam is still absorbed close to the immediate surface, but effectively irradiating a slightly larger surface area, this allows for more homogeneously distributed energy.

[0476] Once more, the use of the laser 210 provides greater direct heating of the glass substrate forming the slide 605, Borosilicate and soda-lime glass slides 605 tend to fully absorb carbon dioxide laser light due to the long infrared wavelength of about 10 um produced by carbon dioxide lasers. The carbon dioxide laser light is converted into a localized heat source that increases the temperature of the glass of the slide 605 located in the vicinity of the focus area of the laser beam 215.

[0477] The healing station 700 in FIG. 7 can be for example used at the baking station 220 to perform a baking process for the tissue sample 730 on the slide 605. A couple of different#3371089 OS <11 9-000025

[0478] 52

[0479] baking techniques can be used. In one version, a flash baking technique is used. During flash baking, the slide 605 with the tissue sample 730 is placed in the slide holder 745. The slide 605 is placed in the slide holder 745 in such a way that only a small area in the corners is touching the slide 605. The slide holder 745 allows for a precise positioning of the slide 605 (e.g., between + / - 0.5 mm). The slide holder 745 is constructed to provide a clear view of the laser beam 215 from the laser 210 and the mirror 725 to the working area at the base surface 740 of the slide 605. The irradiated area is generally not obscured by the label or other obstruction. The base surface 740 of the slide 605 is irradiated with consecutive passes of the laser beam 215 un til the irradiated area of the slide 605 reaches a predefined peak temperature (e.g., 60, 90 or 120 degrees Celsius, depending on the protocol). In one particular example of the flash heating process, the laser 210 irradiates the slide 605 from the bottom or base surface 740 (i.e.. pointing toward the bottom face of the slide 605) in a square path. In one form, this square path of the laser beam 215 is in the form of concentric squares separated by 0.25 mm from each other. The laser beam 215 is scanned with a speed of 800 mm / s and at around 40% power of the laser 210 (i.e., about 15 watts of light power). The irradiated area covers exclusively the working area or base surface 740 of the slide 605. Upon reaching the target peak temperature, the slide 605 is cooled by natural convection. In this process, the heating stage takes about twenty (20) seconds, and the cooling stage takes about one (1) minute.

[0480] FIG. 8 shows a graph 800 of an example of temperature readings for the flash baking technique where the peak temperature reached up to 120 degrees Celsius. In this example, an open loop temperature control technique was used for flash baking. For FIG. 8, the slide 605 was heated at a predefined power (i.e., 40% power), the beam scanning path was a square beam path with 0,25 mm spacing, and the scanning speed was about 800 mm / s. By the sixth pass of the laser beam 215, the slide 605 reached a peak or top temperature of 120 degrees Celsius at the center of the slide 605. The lines in the graph 800 represent different temperature sensor readings on or around the slide 605, In the graph 800, line 805 and line 810 are temperature readings from temperature sensors placed in the center and the edge of the slide 605, respectively. Further, line 815 is temperature readings from a temperature sensor placed at a tag for the slide 605 and line 820 is infrared sensor readings measuring the bottom or base surface 740 of the slide 605. While not certain, the peaks or spikes in line 820#3371089 OS <11 9-000025

[0481] 53

[0482] are presumed to be specular reflection of the laser beam 215 from the base surface 740 of the slide 605 to the infrared sensor.

[0483] In another version of the baking technique, a gentle baking technique is used. With this gentle baking technique, the slide 605 is irradiated in such a way that the slide 605 reaches a steady state at a predefined target temperature (i.e., 60 degrees Celsius), and the temperature is held for a calculated amount of time, which lets excess water to escape and wax to gently melt. This temperature can be reached by using open or close loop control techniques using the temperature sensor 445, such as an infrared sensor, to provide feedback.

[0484] FIG. 9 show a graph 900 of an example of temperature readings for the gentle baking, technique. In this example, an open loop temperature control technique was used for the gentle baking process. For FIG. 9, the slide 605 was heated at a predefined power (i.e., 5% power), the beam scanning path was a square beam path with 0.25 mm spacing, and the scanning speed was about 3,500 mm / s. The slide 605 reached a steady state temperature of 60 degrees Celsius. The lines in the graph 900 represent different temperature sensor readings on or around the slide 605. In the graph 900. line 905 and line 910 are temperature readings from temperature sensors placed in the center and the edge of the slide 605, respecti vely. Further, line 915 is temperature readings from a temperature sensor placed at a tag for the slide 605 and line 920 is infrared sensor readings measuring the bottom or base surface 740 of the slide 605. The peaks or spikes in the line 920 are again presumed to be specular reflection of the laser beam 215 from the base surface 740 of the slide 605 to the infrared sensor.

[0485] FIG. 10 show's an image 1000 of processed slides 605 using different baking techniques. The image 1000 include a first row 1005 of slides 605 that compare laser baked slides 605 to freshly cut tissue samples that were stained. In particular, breast ti ssue was freshly cut and stained for the control slides 605 in the first row 1005, The laser baked slides 605 with breast tissue samples in the first row 1005 were laser baked for 1 to 20 seconds.

[0486] The image 1000 of the slides 605 further Includes a second row 1010 of slides 605 with skin, liver, colon, kidney, and tonsil tissue samples. The control slides 605 in the second row 1010 include freshly cut samples that were baked for one (1 ) hour and five (5) hours at 60 degrees#3371089 OS <11 9-000025

[0487] 54

[0488] Celsius. In the second row 1010. the missing tissue is from the harsh effects of a dual rinse process. The image 1000 includes a third row 1015 with unsuccessful attempts.

[0489] FIGS. 11 and 12 illustrates a laser system 1 100 that is configured to perform baking and / or deparaffinization. In one version, the laser system 1 100 is a single unit that incorporates both the baking station 220 and the dewaxing station 225 into a single station 205. In another version, the baking station 220 and the dewaxing station 225 each have separate laser systems 1 100. The laser system 1100 has the laser 210 of the type described before.

[0490] Removing paraffin from the tissue sample 730 allows staining agents and antibodies to penetrate die tissue, highlighting cellular and structural details needed for accurate diagnosis and research. Deparaffinization followed by rehydration ensures that tissues are in a suitable state for staining as most staining reagents are water-based. Proper deparaffinization facilitates the successful detection of proteins, nucleic acids, and other biomolecules, enabling researchers and pathologists to observe and analyze the biological and pathological state of the tissue. The laser system 1100 and the direct laser heating deparaffinization process ensure that the tissue samples 730 are adequately prepared for subsequent analysis so as to enhance the reliabili ty and accuracy of histological and immunohistochemical studies.

[0491] As can be seen, the laser sy stem 1100 includes a number of features in common with the lasers 210 described in the previous examples. For example, the laser system 1 100 includes the controller 410, the beam generator 420, the beam positioner 425, the temperature sensor 445, and the lens 715 of the type described before. For the sake of brevity and clarity, these common components will not be described in detail, but please refer to the previous description. As shown, the controller 410 is operatively coupled to the beam generator 420, the beam positioner 425, and the temperature sensor 445. In one form, the beam generator 420 of the laser 210 inc ludes a carbon dioxide type laser source that produces the laser beam 215 having the infrared wavelength (e.g., 10.6 pm). The beam positioner 425 in the illustrated variation is a two-dimensional type galvanometer that is able to direct the movement and position of the laser beam 215 in two dimensions (e.g., X / Y galvanometer), and the lens 715 is an F-Theta type lens. It should be recognized that the laser 210 can be configured differently in other variations.#3371089 OS <11 9-000025

[0492] 55

[0493] Between the lens 715 and the base surface 740 of the slide 605, the laser system 1100 has a beam expander 1105 that is configured to defocus the laser beam 215, Defocusing the laser beam 215 with the beam expander 1105 promotes more even heating of the slide 605. The beam expander 1105 helps to reduce the risk of hotspots being formed on the slide 605 that could damage the slide 605 and / or the tissue sample 730, In one form, the beam expander 1105 includes a tube assembly, but the beam expander 1105 can include other types of optical devices that control the focus of and / or the spread of the laser beam 215 to create larger and / or more diffuse spot sizes on the slide 605.

[0494] In the laser system 1100, the laser beam 215 irradiates the base surface 740 of the slide 605 that is opposite to the sample surface 735 where the tissue sample 730 rests,. Again, heating the base surface 740 of the slide 605 rather than the sample surface 735 with the laser beam 215 reduces the risk of damaging the tissue sample 730 as well as facilitates more uniform heating. In other words, when the slide 605 is irradiated from below, the top tissue sample 730 remains protected from direct laser exposure, allowing only heat transmission (e.g„ via conduction).

[0495] To provide closed loop temperature control, the temperature sensor 445 monitors the tempera ture of the base surface 740 of the slide 605. In the depicted example, the temperature sensor 445 has a field of view (T’O V) 11 10 that generally covers the base surface 740 under the tissue sample 730, The field of view 1110 can have a different size andor be viewing differen t locati ons of the slide 605. The tem perature sensor 445 in on e form i s an i nfrared type temperature sensor, but the temperature sensor 445 can include other types of temperature sensors. While FIG. 1 1 shows a single temperature sensor 445, the laser system 1100 in other versions can have two or more temperature sensors 445 positioned to sense temperature at different locations in the laser system 1100. Via the temperature readings from the temperature sensor 445, the controller 410 is able to control the scanning speed, the scanning direction, the scanned area, the intensity, and / or other properties of the laser beam 215 to control heating of the slide 605.

[0496] The laser system 1100 further includes a dispenser head 1 115 positioned proximal to the sample surface 735 of the slide 605. Along with the laser 210, which heats the tissue sample 730, the dispenser head 1115 among other things facilitates removal of paraffin or wax from#3371089 OS <11 9-000025

[0497] 56

[0498] the tissue sample 730 on the slide 605. The dispenser head 1115 is operatively coupled to the con troller 410 so that the con troller 410 is able to control the operation of the dispenser head 1115, As shown in FIG. 11, the dispenser head 1115 has a nozzle 1120 that is configured to dispense a solvent 1 125 onto the tissue sample 730 on the slide 605. 'Hie nozzle 1 120 in other versions can dispense other liquids such as reagents and / or stains. The solvent 1125 faci litates the removal of wax from the tissue sample 730. When the tissue sample 730 on the slide 605 is heated by the laser beam 215 from the laser 210, the wax in the tissue sample 730 tends to melt. The solvent 1125 helps to dissolve and Hush the molten wax from the tissue sample 730. The dispenser head 1115 further includes an air knife 1130 that is configured to blow a gas, such as air. onto the sample surface 735 of the slide 605 to sweep the paraffin- solvent mixture from the slide 605. The dispenser head 1115 is configured to move along a longitudinal axis 1135 of the slide 605. As the nozzle 1120 di spenses the solvent 1125. the dispenser head 1115 moves the nozzle 1120 along the longitudinal axis 1135 of the slide 605. Before and / or at the same time, the laser 210 heats the tissue sample 730 by irradiating the base surface 740 of the slide 605 so as to melt the paraffin in the tissue sample 730. When applied to the sample surface 735 of the slide 605, the solvent 1125 helps to dissolve the molten paraffin, The air knife 1130 blows the air or other gas against the sample surface 735 of the slide 605. As the dispenser head 1115 moves longitudinally along the slide 605, the air knife 1130 sweeps the slide 605 in a lengthwise fashion to remove the dissolved paraffin-solvent mixture.

[0499] Referring to FIG. 13, the laser system 11 0 further includes a slide holder 1305 that is configured to hold the slide 605. In the depicted example, the slide holder 1305 is integrated into the beam expander 1105. Having the slide holder 1305 integrated into the beam expander 1 105 establishes a proper distance to achieve the focal length and spot size needed to evenly heat the slide 605, The slide holder 1305 defines a beam aperture 1310 through which the laser beam 215 reaches the base surface 740 of the slide 605. The illustrated example of the beam aperture 1310 is sized and sh aped to generally correspond to the sha pe of the slide 605. In the illustrated example, the beam aperture 1310 has a rectangular shape that corresponds generally to the size and shape of the slide 605, but the beam aperture 1310 can be shaped and sized differently in other examples.#3371089 OS <11 9-000025

[0500] 57

[0501] To support the slide 605, the slide holder 1305 has one or more posts 1315. In one form, the slide holder 1305 has at least three posts 1315 positioned to support the corners of the slide 605. The posts 131 can support the slide 605 at other locations in other variations. In the depicted example, the slide holder 1305 has four (4) posts 1315 positioned around the perimeter and al the corners of the beam aperture 1310 so as to support the slide 605 at the corners of the slide 605. The posts 1315 are configured to form an air gap 1320 between the slide holder 1305 and the slide 605 to thermally isolate the slide 605. The gap 1320 gaps between the slide holder 1305 and the slide 605 are established via the posts 1315 at the comers of the slide 605. In one variation, the posts 1315 are dimensioned to minimize the risk of wi eking of the solvent 1125 and / or melted paraffin to the base surface 740 of the slide 605 due to capillary action.

[0502] Around the perimeter of the beam aperture 1310, the slide holder 1305 defines one or more drain channels 1325 that promote wax and solvent 1125 drainage. In the depicted example, the slide holder 1305 has a single drain channel 1325 defined around the beam aperture 1310, but in other examples, the slide holder 1305 can have more than one drain channel 1325. In one version, the slide holder 1305 is constructed io only touch the comers of the slide 605 and has the drain channel 1325 around the perimeter of the beam aperture 1310 for the molten paraffin -sol vent mixture to properly drain into a waste chute. FIG. 14 has arrows 1405 that show the flow of the molten paraffin-solvent mixture along the waste path. In some examples, the laser system 1100 further includes one or more sensors to detect the presence of the slide 605 in order to avoid starting the laser 210 without the slide 605 being present.

[0503] FIG. 15 shows a perspective view of the dispenser head 1115. The dispenser head 1115 is once more to designed to apply the solvent 1125. The dispenser head 1115 is able to extend and retract along the longitudinal axis 1135 of the slide 605 so that the air knife 1130 is able to sweep the molten paraffin -sol vent mixture from the slide 605. The molten paraffin-solvent mixture is then drained via the drain channels 1325,

[0504] For the deparaffinization process, the slide 605 is placed on the slide holder 1305 in such a way that only a small area in the corners is touching the slide 605. The posts 1315, which in one form are machined pins, allow the precise positioning of the slide 605 (e.g., in between / / - 0.5 mm). The beam aperture 1310 in the slide holder 1305 provides a clear view for the#3371089 OS <11 9-000025

[0505] 58

[0506] laser beam 215 from the laser 210 to the base surface 740 a t the bottom of the working area of the slide 605. This area of the slide 605 is not obscured such as by a label.

[0507] Several deparaffinization or dewaxing techniques using the laser 210 have been developed. In a first version, the laser 210 irradiates the base surface 740 of the slide 605 in a square path (e.g., concentric squares separated by 0.25 mm from each other). The beam positioner 425 scans the laser beam 215 along the base surface 740 of the slide 605 at a speed of about 800 mm / s, and the beam generator 420 emits the laser beam 215 at around 40% power (e.g., about 15 watts of light power). The area irradiated covers exclusively the working area at the base surface 740 of the slide 605. The slide 605 is irradiated for a couple of cycles in such a way that the paraffin is completely molten or until reaching the desired temperature ( typically 60 - 64 degrees Celsius). Next, the nozzle 1120 dispenses the solvent 1125 (e.g., 1-2 mL of DOW ANOL.® solvent) onto the sample surface 735 of the slide 605 to dissolve the liquid wax in the tissue sample 730. The dispenser head 1 1 15 is moved along the longitudinal axis 1135, and the air knife 1130 then sweeps the mixture of solvent 1125 and melted wax away from the slide 605. This mixture drains from the slide 605 into the drain channels 1325 in the slide holder 1305.

[0508] In a second version, the solvent 1125 is deposited first to create a puddle on top of the slide 605. The puddle of the solvent 1125 on the sample surface 735 of the slide 605 is then heated at the same time as the working area of the slide 605 at the base surface 740 is heated by the laser beam 215. The puddle of the solvent 1125 is heated until the puddle reaches the target temperature and at time to ensure full paraffin dilution in the solvent 1 125. The air knife 1130 then sweeps the mixture after a predetermined time.

[0509] In a third version, the slide 605 is constantly irradiated by the laser beam 215 to reach a controlled temperature using an open or close loop control process. Once the temperature is reached (or after a calculated time), the slide 605 is rinsed with a transfer fluid (e.g,, DOW ANOL® solvent), a solvenVsurfactant (e.g., Ezprep), and'or any other liquid that dissolves the molten wax. Afterwards, the air knife 1130 removes any excess transfer fluid from the slide 605.#3371089 OS <11 9-000025

[0510] 59

[0511] In a fourth version, the slide 605 is irradiated with the laser beam 215 to perform the baking process (i.e., to evaporate or sweep away the trapped humidity / Iiquid water in the slide 605). This process typically melts the wax, and dcparaffinization with one of the above-discussed methods can be performed in a rapid sequence.

[0512] In a fifth version, the transfer fluid has a vaporization temperature above 100 degrees Celsius (e.g., DOWANOL® solvent at 120 degrees Celsius). The transfer fluid is poured onto the slide 605, such as via the nozzle 1120, forming a puddle. The slide 605 is irradiated by the laser beam 215 until reaching a steady state temperature at or above about 110 to 118 degrees Celsius for a predefined time that is typically longer than previously described methods. The air knife 1 130 removes the hot mixture from the slide 605 in the same fashion as in previous methods. The higher heat helps to remove tissue crosslinking from fixation and reduces the time required for antigen retrieval. In this variation, the processing for the antigen retrieval station 230 is in essence performed in the input module 105 via the depara ffinization process.

[0513] In one version, the laser system 1100 performs the dewaxing of the tissue sample 730 process using the flash heating technique in a fashion similar to the heating technique described with reference to the graph 800 in FIG. 8. In another version of the dew-axing technique, a gentle dewaxing technique is used which is similar to the heating technique described with reference to FIG. 9. With this gentle heating technique for dewaxing, the slide 605 is irradiated in such a way that the slide 605 reaches steady state at a predefined target temperature (e.g., 60 degrees Celsius), and the temperature is held for a calculated amount of time, which lets the wax or paraffin to gently melt. This temperature can be reached by using open or close loop control techniques using the temperature sensor 445, such as an infrared sensor, to provide feedback.

[0514] FIG. 16 show a graph 1600 of an example temperature readings for the gentle dewaxing technique. In this example, an open loop temperature control technique was used for the gentle heating process. For FIG. 16, the slide 605 was heated at a predefined power (i.e., 5% power), the beam scanning path was a square beam path with 0,25 mm spacing, and the scanning speed was about 3,500 mm / s. The slide 605 reached a steady state temperature of 60 degrees Celsius. The lines in the graph 1600 represent different temperature sensor readings on or around the slide 605. In the graph 1600, line 1605 and line 1610 are#3371089 OS <11 9-000025

[0515] 60

[0516] temperature readings from temperature sensors placed in the center and the edge of the slide 605, respectively. Further, line 1615 is temperature readings from a temperature sensor placed at a tag for the slide 605 and line 1620 is infrared sensor readings measuring the bottom or base surface 740 of the slide 605. FIGS. 17 and 18 show example setups where the temperature sensor 445 measures the temperature of the slide 605 from the bottom and the top, respectively. Once more, the peaks or spikes in the line 920 are presumed to be specular reflection of the laser beam 215 from the base surface 740 of the slide 605 to the infrared sensor.

[0517] FIG. 19 shows an image 1900 of slides 605 with tissue samples 730 that were stained after being dew-axed using the laser system 1100 and techniques described above. FIG. 20 shows an image 2000 of slides 605 from a series of tests to enhance the laser dew- axing process. It was discovered that the dewaxing process in some cases was enhanced when the power of the laser 210 was set to forty-percent (40%).

[0518] This laser heating approach can be used in other processes. For example, the laser 210 can create heat for curing at the curing station 235 in the eoverslip module 115. In one form, the coverslip 450 is a pre-glued coverslip that includes an adhesive for adhering the coverslip 450 to the slide 605. The laser beam 215 heats the coverslip 450 to cure the adhesive.

[0519] FIG. 21 shows an enlarged view- of one example of the laser beam 215 heating the laser head 405 during curing. In one example, the laser 210 used to perform the curing is a carbon dioxide type laser. As shown, the coverslip 450 is placed on the slide 605. The coverslip 450 in one form is a pre-glued type coverslip that has an adhesive that adheres the coverslip 450 to the slide 605. Between the coverslip 450 and the slide 605, a mounting media 2105, such as a mounting solvent, is used to enhance coverslipping. To cure the adhesive on the coverslip 450, the laser beam 215 irradiates the coverslip 450 to produce a heated area 2110 that is hotter than the surroundings to cure the mounting media 2105. As can be seen, portions of the laser beam 215 are absorbed, reflected, refracted, and pass through the coverslip 450 and the slide 605.

[0520] In other examples, different types of lasers can be used. For example, the laser 210 in other variations can include a visible light type laser (e.g. a blue laser). The coverslip 450 and the#3371089 OS <11 9-000025

[0521] 61

[0522] slide 605 are generally designed to be transparent relative to visible light, As such, the laser beam 215 from a visible light laser would generally shine through the coverslip 450 and the slide 605 such that very little heat would be produced for curing, Turning to FIG. 22, an absorptive material 2205, such as in the form of a visible light absorpti ve ink (e.g., black ink), is applied to the coverslip 450, With most of the energy from the laser beam 215 being absorbed by the absorpti ve material 2205, the laser beam 215 produces a heated area 2210 in the absorptive material 2205 and the coverslip 450 that promotes curing of the adhesive. In other words, the absorptive material 2205 is temporarily deposited to obscure the glass so as to absorb the visible light from the laser beam 215 that otherwise will pass through the coverslip 450, the mounting media 2105, and the slide 605. The absorptive material 2205 is temporarily applied during curing and removed afterwards so as to not interfere with analysis of the tissue sample 730. In other variations, the absorptive material 2205 is permanently applied to the coverslip 450 and / or the slide 605 at locations that do not obscure viewing of the tissue sample 730 during analysis.

[0523] As another example, the laser 210 is configured to generate deep ultraviolet (UV) light. When in the form of deep ultraviolet light, the laser beam 215 is absorbed almost completely by the glass forming the coverslip 450 and / or the slide 605. In one form, the ultraviolet laser beam 215 is produced by diode lasers that are typically efficient and provide a reduced form factor. The laser 210 in one variation includes an array of ultraviolet diodes.

[0524] As noted before, the laser 210 in other examples includes infrared type lasers. Infrared lasers are readily available and are able to provide sufficient pow'er. For example, high power infrared laser engravers are configured to already have many of the features identified above for the laser heating system (e.g., a light source, galvanometer scanner, F-Theta lens, controller, and software). Infrared lasers are considered to have a lower risk of interacting with stains or dyes as compared to ultraviolet lasers. In other variations, the laser 210 includes fiber lasers that have a peak radiation in the near infrared regions where the glass of the coverslip 450 or the slide 605 is still transparent. In some cases, the glass or diluent may be doped with a material that is opaque at those wavelengths. These types of fiber lasers provide flexible routing of fiber optics or other optical guides around obstacles, such as those caused by other stations 205 in the system 100, to redirect the laser beam 215.#3371089 OS <11 9-000025

[0525] 62

[0526] The beam positioners 425 in other examples can include other types of scanners besides ones using galvanometer positioning control, for example, the beam positioner 425 can include rotating mirrors / hexagons. like the ones used in barcode scanners, that can be modified to achieve the desired scanning path and scanning speed control for the laser beam 215, It also should be recognized that other types of optic con figurations can be used to produce the scanning line of the laser beam 215, to reduce the scanning motion axis to just one, and / or to cover the whole square scanning area.

[0527] FIGS. 23 and 24 show a laser system 2300 configured to act as the curing station 235 for the curing process. As shown, the laser system 2300 includes several components in common with the previously described systems, such as the laser 210 and the laser head 405. For the sake of clarity as well as brevity, these common components will not be described in detail, but please refer to the previous discussion.

[0528] The illustrated laser system 2300 includes a base 2305, such as in the form of a beadboard, and a slide holder 2310 mounted to the base 2305. The slide holder 2310 is configured to hold the slide 605, In the depicted example, tape 2315, like aluminum tape, is positioned between the slide 605 and the slide holder 2310. The laser head 405, which shines the laser beam 215, is powered by a power cable 2320. The laser head 405 and the slide holder 2310 as well as other components of the laser system 2300 are housed within an enclosure 2325 for safety purposes. To facilitate viewing, the enclosure 2325 includes a window 2330. such as in the form of safety glass, and the enclosure 2325 in one form is an acrylic enclosure. It should be appreciated that the enclosure 2325 and the window 2330 can be made from different materials.

[0529] As shown in FIG. 23, the laser head 405 in one example shines the laser beam 215 onto the absorptive material 2205 on the coverslip 450 in the manner depicted in FIG. 22. In another example, the laser head 405 shines the laser beam 215 onto the coverslip 450 without the absorptive material 2205, such as in the manner as depicted in FIG. 21. Once more, the laser beam 215 provides the heat to cure the adhesive for the pre-glued coverslip 450.

[0530] FIG. 25 shows a diagram 2500 that compares the chemistry and methodologies between a control process 2505 and a test process 2510 for curing. The control process 2505 uses#3371089 OS <11 9-000025

[0531] 63

[0532] traditional coverslipping and curing processes (i.e.. via an oven), The test process 2510 uses the laser curing technique described above. In the test process 2510, the coverslip 450 is a pre-glued covertile (c.g., HE600 cover tile). For each test slide, 40 nL of DOWANOL® (DPGPE) sol vent was applied to the slide and the covertile was added by hand or automatically cover slipped (e.g,, using an HE600 coverslipper). The slides in the test process 2510 were then cured using the laser 210.

[0533] FIG, 26 shows a slide assembly 2605 and processing parameters 2610 for laser curing the slide assembly 2605. As shown, the slide assembly 2605 includes the slide 605 and the coverslip 450. Tlie coverslip 450 has one or more absorptive bars 2615 that are configured to absorb the laser beam 215 to promote heating during curing. In the illustrated example, two absorptive bars 2615 extend along the lateral sides of the slide assembly 2605, Tlie absorptive bars 2615 are in the form of black ink, but the slide assembly 2605 can include other types of materials for absorbing the energy’ from the laser beam 215. Wi th the absorptive bars 2615 located along the sides, the absorptive bar's 2615 are positioned to heat the coverslip 450, and at the same time, the absorptive bars 2615 do not obstruct subsequent viewing and analysis of the tissue sample 730. As a result, the absorptive bars 2615 do not need to be removed after curing, but in some cases, the absorptive bars 2615 can be removed after curing.

[0534] FIG. 27 show’s another example of a slide assembly 2705 with an absorptive material 2710 on the coverslip 450 of the slide 605. In the depicted example, the absorpti ve material 2710 includes a machine-readable identifier 2715 in the form of a QR code. The machine-readable identifier 2715 can include other types of machine-readable codes like barcodes. The machine-readable identifier 2715 can be used to track the slide 605 through various processes. The machine-readable identifier 2715 in the depicted example is positioned over the tissue sample 730 such that the view of the tissue sample 730 is obstructed, so after curing, the machine-readable identifier 2715 is removed. In other examples, the machine-readable identifier 2715 can be positioned elsewhere on the coverslip 450 and or the slide 605 such that the machine-readable identi fier 2715 does not need to be removed prior to analysis at the imaging module 120.#3371089 OS <11 9-000025

[0535] 64

[0536] FIG. 28 shows another example of a slide assembly 2805 with an absorptive material 2810 on the coverslip 450 of the slide 605. In the depicted example, the absorptive material 2810 includes one or more dots 2815 positioned to not obstruct the view of the tissue sample 730 so that the absorptive material 2810 does not need to be removed after curing (but can be removed if so desired). In the depicted example, the dots 2815 are positioned at the comers of the coverslip 450, but the dots 2815 can be positioned elsewhere on the coverslip 450 and / or the slide 605.

[0537] FIG. 29 shows an image 2900 of slides 605 stained with differen t stains (i.e., Ki67 and ECAD stains) and cured using the laser curing process discussed before. The coverslips were pre-glued covertiles (e.g., HE600 cover tile). For each test slide, 40 uL of DOW ANOL® (DPGPE) solvent was applied to the slide and the covertile was added via an automated coverslipper (e.g., using an IIE600 coverslipper).

[0538] FIG. 30 is an image 3000 of slides 605 that show' some potential issues if the process parameters are not set correctly. For example, if too much power is applied by the laser beam 215 andor if the scanning speed of the laser beam 215 is too slow, the coverslip 450 can be damaged such as in the manner as is shown. The coverslip 450 can melt to form ridges or grooves in the coverslip 450. These optical imperfections in the coverslip 450 can make analysis of the tissue sample 730 difficult or even impossible.

[0539] FIG. 31 shows a chart 3100 illustrating results from a feasibility study. As can be seen, when the power of the laser beam 215 was too low' (i.e., 10-20% power), incomplete curing occurred, and when the power of the laser beam 215 was too high (i.e., 35-50% power), the coverslips 450 were damaged. With the laser 210 at medium power (i.e., 20-35% power) and the mounting media 2105 having middle to high viscosity, complete curing occurred. Using the laser 210 for heating during the baking, dewaxing, and cover slipping process stages considerably reduced processing time as compared to conventional methods, such as heating via oven. For example, baking of the tissue sample 730 on the slide 605 can be performed in about 15to 20 seconds, and dewaxing of the tissue sample 730 can occur in about 12 seconds. The curing for the cover slipping process can occur in about 12 seconds.#3371089 OS <11 9-000025

[0540] 65

[0541] FIG. 32 shows an image 3200 of the tissue samples 730 on the slides 605 with coverslip 450 processed using the laser processing techniques described above. FIG. 33 show's an image 3300 of another set of slides 605 processed using various laser processing and staining techniques along with control slides 605, FIG, 34 is an image 3400 of slides 605 processed using various laser rastering techniques, FIG. 35 is an image 3500 of slides 605 processed using different variations of the previously described laser- based dewaxing and curing techniques.

[0542] FIG, 36 illustrates a laser system 3600 according to another example. As can be seen, the laser system 3600 shares a number of components in common with and operates in a fashion similar to the previously discussed examples. For example, the laser system 3600 includes the beam generator 420 that produces the laser beam 215 and the temperature sensor 445 of the type described before. For the sake of brevity as well as clarity, these common features will not be described again, but please refer to the previous discussion.

[0543] The laser system 3600 includes a holder assembly 3605 that is configured to hold the slide 605, The holder assembly 3605 of the laser system 3600 can be used to hold the slide 605 as the beam generator 420 irradiates and heats the slide 605 with the laser beam 215 at the various stations 205, such as for example at the baking station 220, the dewaxing station 225, the antigen retrieval station 230, and the curing station 235, In the illustrated example, the holder assembly 3605 has an overall cylindrical shape, but the holder assembly 3605 can be shaped differently in other examples. The holder assembly 3605 includes a distance adjuster 3610, a holder body 3615. and a slide holder 3620. The distance adjuster 3610 is positioned on or proximal to the beam generator 420. The holder body 3615 is positioned between the distance adjuster 3610 and the slide holder 3620, Inside, the holder body 3615 is hollow or generally transparent relative to the laser beam 215 so that the laser beam 215 is able to shine onto the slide 605,

[0544] The distance adjuster 3610 is configured to adjust the relative position of the beam generator 420 and the slide holder 3620 that supports the slide 605. In the illustrated example, the distance adjuster 3610 is configured to adjust the vertical position (e.g., Z-di recti on) of the slide 605 relative to the beam generator 420. With the distance adjuster 3610. the spot size anri'or focus of the laser beam 215 on the slide 605 can be adjusted. In one version, the#3371089 OS <11 9-000025

[0545] 66

[0546] distance adjuster 3610 and the holder body 3615 have a threaded connection such that the relative distance between the beam generator 420 and the slide holder 3620 can be changed by rotating the distance adjuster 3610 relative to the holder body 3615 (or vice-versa). It should be recognized that the distance adjuster 3610 and the holder body 3615 can be connected in other manners to facilitate distance adjustment between the beam generator 420 and the slide 605. The holder assembly 3605 further has a sensor holder 3625 that secures the temperature sensor 445 to the holder body 3615. The sensor holder 3625 holds the temperature sensor 445 at a position where the temperature sensor 445 is able to measure the temperature of one or more parts of the slide 605 as well as elsewhere.

[0547] The laser system 3600 is again configured to heat the slide 605 with the laser beam 215 in the manner as described above. Like in the earlier examples, the slide holder 3620 has the posts 1315 that support the slide 605, and the slide holder 3620 defines a beam aperture 3630 through which the laser beam 215 shines to heat the slide 605. The posts 1315 in some versions space the slide 605 from the slide holder 3620 to form a gap to thermally isolate the slide 605 from the slide holder 3620. The slide holder 3620 is generally Hat and extends in a horizontal direction such that the slide 605 is generally supported in a horizontal plane. To promote drainage of wax, solvent, and / or other fluids from the slide 605, the slide holder 3620 defines a drain channel 3635 around the perimeter of the beam aperture 3630. The drain channel 3635 has a drain funnel 3640 for drainage into a waste chute, for example, a paraffin-solvent mixture from the slide 605 is able to drain into the waste chute via the drain channel 3635 and the drain funnel 3640.

[0548] FIG. 37 illustrates a laser system 3700 according to a further example. As can be seen, the laser system 3700 shares a number of components in common with and operates in a fashion similar to the previously discussed examples. For example, the laser system 3700 includes the beam generator 420 that produces the laser beam 215 and the temperature sensor 445 of the type described before. For the sake of brevity as well as clari ty, these common features wi ll not be described again, but please refer to the previous discussion.

[0549] The laser system 3700 includes a holder assembly 3705 that is configured to hold the slide 605. The holder assembly 3705 of the laser system 3700 can be used to hold the slide 605 as the beam generator 420 irradiates and heats the slide 605 with the laser beam 215 at the#3371089 OS <11 9-000025

[0550] 67

[0551] various stations 205, such as for example at the baking station 220, the dewaxing station 225, the antigen retrieval station 230, and the curing station 235. In the illustrated example, the holder assembly 3705 has an overall cylindrical shape, but the holder assembly 3705 can be shaped differently in other examples. The holder assembly 3705 includes a distance adjuster 3710, a holder body 3715. and a slide holder 3720. The distance adjuster 3710 is positioned on or proximal to the beam generator 420. The holder body 3715 is positioned between the distance adjuster 3710 and the slide holder 3720. Inside, the holder body 3715 is hollow or generally transparent relative to the laser beam 215 so that the laser beam 215 is able to shine onto the slide 605.

[0552] The distance adjuster 3710 is configured to adjust the relative position of the beam generator 420 and the slide holder 3720 that supports the slide 605. In the illustrated example, the distance adjuster 3710 is configured to adjust the vertical position (e.g., Z-direction) of the slide 605 relative to the beam generator 420. With the distance adjuster 3710. the spot size and / or focus of the laser beam 215 on the slide 605 can be adjusted. In one version, the distance adjuster 3710 and the holder body 3715 have a threaded connection such that the relative distance between the beam generator 420 and the slide holder 3720 can be changed by rotating the distance adjuster 3710 relative to the holder body 3715 (or vice-versa). It should be recognized that the distance adjuster 3710 and the holder body 3715 can be connected in other manners to facilitate distance adjustment between the beam generator 420 and the slide 605. The holder assembly 3705 further has a sensor holder 3725 that secures the temperature sensor 445 to the holder body 3715. The sensor holder 3725 holds the temperature sensor 445 at a posi tion where the temperature sensor 445 is able to measure the temperature of one or more parts of the slide 605 as well as elsewhere.

[0553] The laser system 3700 is again configured to heat the slide 605 with the laser beam 215 in the manner as described above. Like in the earlier examples, the slide holder 3720 defines a beam aperture 3730 through which the laser beam 215 shines to heat the slide 605, To promote drainage of wax, solvent, and / or other fluids from the slide 605, the slide holder 3720 defines a drain channel 3735 around the perimeter of the beam aperture 3730. The drain channel 3735 has a drain funnel 3740 for drainage into a waste chute. For example, a paraffin-solvent mixture from the slide 605 is able to drain into the waste chute via the drain channel 3735 and the drain funnel 3740.#3371089 OS <11 9-000025

[0554] 68

[0555] The slide holder 3720 is configured to further promote drainage from the slide 605 into the drain funnel 3740, As shown in FIG, 37. the slide holder 3720 has a base 3745 secured to the holder body 3715 and a stand 3750 extending from the base 3745. In the illustrated example, the drain channel 3735 extends around the perimeter of the stand 3750 so as to form a moat around the stand 3750. The base 3745 extends along a horizontal plane, and the stand 3750 extends from the base 3745. The stand 3750 has a rest surface 3755 that supports the slide 605. As shown, the rest surface 3755 is tilted such that the rest surface 3755 extends transverse to the base 3745. In one form, the rest surface 3755 extends at about forty-five degrees (45°) relative to the base 3745, but the rest surface 3755 can be tilted at other angles relative to the base 3745. With the rest surface 3755 tilted in such a manner, the slide 605 is likewise tilted to promote drainage. In the depicted example, the slide 605 on the rest surface 3755 is tilted down towards the drain funnel 3740 so as to promote drainage into the waste chute via the drain funnel 3740.

[0556] FIG. 38 includes a graph 3800 illustrating light absorption of various material types of slides 605 and coverslips 450 at different wavelengths.

[0557] As should be recognized, the above-discussed laser baking techniques, such as those occurring at the baking station 220, address several issues found in traditional slide baking methods. It was found that traditional baking methods, such as those performed with traditional ovens, had issues with tissue sample adhesion quality, process efficiency, temperature control, and energy use. One of the main challenges is ensuring a strong, lasting bond between the Formalin-Fixed, Paraffin-Embedded (FFPE) tissue section and the glass slide.

[0558] It was found that conventional techniques often fail to completely remove residual water trapped under the tissue alter microtomy, which is the root cause of poor adhesion and artifacts like wrinkles. These older baking methods typically relied on angled or vertical slide orientations to aid water drainage, complicating furnace setups. It was further found that prior baking approaches had issues related to process inefficiencies. Since there is no way to directly measure the humidity of the slide, baking protocols were often over-engineered for the worst-case scenario, which resulted in extensive overbaking that wasted time and energy.#3371089 OS <11 9-000025

[0559] 69

[0560] Conversely, underbaking using conventional methods created a high risk of the tissue detaching during subsequent processing. Traditional methods, whether utilizing conduction (e.g,, hot plates) or convection (e.g., ovens), were found to be inherently slow or inefficient, Conduction is a diffusion -limi ed mechanism, making it difficult to control the temperature gradient across the slide and often leading to poor thermal contact due to insulating air gaps. Once more, convection ovens are typically faster but suffer from poor energy efficiency, as the entire oven must be heated and maintained, with only a small fraction of the power actually heating the slide.

[0561] It was also discovered that inaccurate temperature control was another issue with conventional baking methods. Most existing baking methods estimated the slide temperature indirectly by measuring the temperature of the hot pla te or recirculating air. This lack of direct slide temperature measurement caused severe inefficiencies and delays in the reaction time of the traditional baking systems. This inability to monitor the slide directly forced operators to limit the energy input to avoid the risk of overheating the delicate tissue, which in turn lead to slow heating ramps and prolonged processing times. Furthermore, the high thermal inertia of these heating paths required management of excess energy, which negatively impacted the temperature stability and performance of adjacent processing chambers.

[0562] One example of an adaptive laser heating system 3900 for use in the baking station 220 that addresses these as well as other issues is illustrated in FIG. 39. As can be seen, the adaptive laser heating system 3900 shares a number of features in common with the previously described systems, such as the laser 210 for producing the laser beam 215, the laser head 405, the controller 410, the beam generator 420, the beam positioner 425, the temperature sensor 445, and the lens 715. Like before, the laser beam 215 irradiates the base surface 740 of the slide 605 to heat the tissue sample 730 on the sample surface 735 of the slide 605. For the sake of brevity as well as clarity, these common features and their function will not be again described in detail, but please refer to the previous discussion of these features. As can be seen, the adaptive laser heating system 3900 further includes a slide holder 3905 that positions and holds the slide 605. In one variation, the slide holder 3905 is in the form of a clip or clamp that holds the end of the slide 605 that has a label or tag 3910, but it should be recognized from the previous discussion that the slide holder 3905 can be configured#3371089 OS <11 9-000025

[0563] 70

[0564] differently in other variations. Like before, the tissue sample 730 is located on the sample surface 735 of the slide 605. In the illustrated example, the sample surface 735 of the slide 605 further has a puddle 3915 of liquid that covers the tissue sample 730.

[0565] As compared to the traditional slow, inefficient, and inaccurate slide baking systems and techniques, the adapti ve laser heating sy stem 3900 provides a fast, precise, and verifiable drying endpoint. This is achieved by utilizing a controlled power laser source, like the power control module 430 in FIG. 4, and the beam positioner 425 or other beam steering mechanisms, such as galvanometers or fast steering mirrors, to dynamically scan and heat the slide 605. This targeted heating allows the adaptive laser heating system 3900 to not only efficiently evaporate excess humidity but also leverage the thermocapillary convection effect through a specially designed scanning path, which moves residual water in the puddle 3915 and wax from the tissue center to the edges, significantly boosting process efficiency and adhesion quality.

[0566] To achieve this, the adaptive laser heating system 3900 utilizes a unique adapti ve process control method or technique. The controller 410 of the adaptive laser heating system 3900 actively monitors critical thermal cycle variables, including heating curves, the power required to maintain the slide 605 at a steady set temperature, and the total process time. By continuously tracking the power required, the controller 410 of the adaptive laser heating system 3900 can detect the moment when the power demand drops, signaling the cessation of evaporative cooling and confirming the slide 605 is fully dry. This feedback mechanism allows the baking protocol to be adapted in real-time, drastically reducing the time needed to process slides 605 compared to fixed-time protocols and substantially increasing the probability of successful tissue attachment of the tissue sample 730 to the slide 605.

[0567] The adaptive laser heating system 3900 uses the steerable laser beam 215 directed at the base surface 740 of the slide 605. with the pa th of the laser beam 215 precisely controlled to scan the working area and heat the slide 605 uniformly. The power input of the laser beam 215 on the slide 605 is managed by controlling the intensity of the laser beam 215, the speed of the laser beam 215 across the slide 605, and the frequency of scans so as to ensure the entire working area of the slide 605 is scanned multiple times per second to guarantee an even temperature distributi on.#3371089 OS <11 9-000025

[0568] 71

[0569] FIG. 40 shows one example of a laser scan path 4000 for the laser beam 215 on the base surface 740 of the slide 605. In the illustrated example, the laser scan path 4000 is in the form of an outbound square or rectangular hatch pattern. As indicated by arrow 4005, the laser beam 215 traces a series of concentric rectangles or squares that are scanned in an outward radial direction. In one particular example, the rectangles of the laser scan path 4000 are spaced by 0.25 mm. and the laser beam 215 scans the slide 605 at 3.000 mm / s for 1 0 seconds to create a temperature of about 85 degrees Celsius on the slide 605. It should be appreciated that other scanning parameters for scanning the laser beam 215 can be used in other examples. This laser scanning path is designed to leverage thermocapillary convection, an effect that moves excess humidity and wax from the center of the section toward the edges of the slide 605. further enhancing adhesion and quality of the tissue sample 730 on the slide 605. In other words, the adaptive laser heating system 3900 leverages the physical phenomenon of thermocapi llary convection to further enhance the process. The power, speed, and scanning path of the laser beam 215 are carefully controlled to create thermal gradients that intentionally sweep excess humidity and paraffin wax from the center of the slide 605 toward the edges. This directed removal of residual water and wax significantly enhances tissue-slide adhesion and overall section quality.

[0570] Referring again to FIG. 39, the touchless temperature sensor 445, such as an infrared pyranometer, is strategically positioned to report the current temperature of the slide 605. The temperature sensor 445 has the field of view 1110 for measuring the temperature of the slide 605, which is at least the part of the base surface 740 that is underneath the tissue sample 730 and / or the puddle 3915 on the slide 605. This direct measurement by the temperature sensor 445 overcomes the inaccuracies of prior methods that rely on indirect air or hot plate temperatures. If the laser 210 and the temperature sensor 445 operate near similar wavelengths, the temperature reading is coordinated with the lasing process to mitigate errors from laser reflections. Alternatively or additionally, the same temperature sensor 445 or a second temperature sensor 445 may be placed to read the temperature from the top or sample surface 735 of the slide 605, opposite to the lasing or base surface 740.

[0571] The sophisticated controller 410 receives the temperature feedback and utilizes a proportional-integral -derivative (PID) control algorithm to regulate the laser's power, often#3371089 OS <11 9-000025

[0572] 72

[0573] via a pulse-width modulation (PWM) signal. The control routine can adjust the laser power, scanning speed, or the interval between scanning cycles. The controller maintains radiation until the conditions for completion are met. These conditions include reaching a set temperature and detecting a sudden drop or threshold reduction in the required power to maintain that temperature. This drop signals the cessation of evaporative cooling, confirming that all excess humidity has been removed. Other completion criteria include a predetermined time limit or a temperature-over-time curve that shows a sudden temperature rise, indicating that water is no longer present to sink power through evaporation.

[0574] FIG. 41 shows a diagram 4100 of how the controller 410 in the adaptive laser heating system 3900 controls the temperature of the slide 605 according to one example. In the illustrated example, the controller 410 in the adaptive laser heating system 3900 includes a proportional-integral-derivative controller (PID controller) 4105. The PID controller 4105 is a sophisticated controller that receives the temperature feedback from the temperature sensor 445, and the PID controller 4105 of the controller 410 uti lizes a PID control algorithm to regulate the power of the laser 210 to achieve a temperature set point 4110. In most cases, the power of the laser beam 215 from the laser 210 is regulated via a pulse-width modulation (PWM) signal, but the power of the laser beam 215 can be regulated in other ways. The control routine can adjust the laser power, scanning speed, and / or the interval between scanning cycles to achieve the set point 4110. The controller 410 maintains radiation until the conditions for completion are met. In the FIG. 41, the diagram 4100 includes a graph 4115 that represents the closed loop temperature control that is used to detect the various temperatures of the slide 605 via the temperature sensor 445.

[0575] A unique technique has been developed to control temperature and generally detect when the tissue sample 730 on the slide 605 is dry. FIG. 42 shows a graph 4200 that generally illustrates this technique. The FIG. 42 graph 4200 has a time axis 4205, a temperature axis 4210, and a power axis 4215. Plotted on the graph 4200 is a temperature data line 4220 that corresponds to the temperature of the slide 605 measured by the temperature sensor 445 and a power data line 4225 that represents the power level signal from the PID controller 4105 to the laser 210 for controlling power of the laser beam 215. The graph 4200 has a line that depicts the temperature set point 41 10, and the graph 4200 in FIG. 42 has a power drop line 4230 that represents a detection of a sudden drop or threshold reduction in the required power#3371089 OS <11 9-000025

[0576] 73

[0577] from the controller 410 (e.g., PID controller 4105) needed to maintain the set point 4110 of the slide 605. As can be seen, the power data line 4225 precipitously drops around the power drop line 4230. This drop in the power data line 4225 at the power drop line 4230 signals the cessation of evaporative cooling on the slide 605, which in turn confirms that all excess humidity has been removed from the tissue sample 730 on the slide 605. In one variation, the power drop threshold is at least a 5% drop in laser power to detect cessation of evaporative cooling (i.e., the slide 605 is sufficiently dry), and in other variations, the power drop thresholds are at least a 10%, 20%, or 30% drop in power for the laser 210 to generally maintain the temperature set point 4110. Once the threshold is reached, the laser 210 can stop the heating process, and the slide 605 can be transferred to the next station 205 for further processing.

[0578] FIG. 43 shows another example of a graph 4300 that illustrates this power signal drop for the laser 210 from the controller 410 when the excess humidity has been removed. Like in the FIG. 42 graph 4200, the graph 4300 in FIG. 43 has a temperature data line 4320 and a power data line 4325. At a power drop line 4230 in the FIG. 43 graph 4300, the power data line 4325 from the PID controller 4105 or other type of controller for controlling power of the laser beam 215 from the laser 210 (e.g., via the power control module 430 in FIG. 4) dramatically drops. Again, this dramatic drop in power indicates that evaporative cooling of the slide 605 has stopped and any excess humidity from the slide 605 has been removed. At this point, the baking process is complete, and the controller 410 can stop the baking process, such as by turning off the laser 210. It should be recognized that other baking completion criteria can be used. In addition to or as an alternative to this laser power drop criteria, the controller 410 in the adaptive laser heating system 3900 can use for the baking completion criteria a predetermined time limit or a temperature-over-time curve that shows a sudden temperature rise, indicating that water is no longer present to sink power through evaporation.

[0579] As should be appreciated, other alternative approaches can be used to control and validate the slide baking process. In another approach, a thermal camera instead of a simpler, single-pixel pyranometer is used for the temperature sensor 445. A thermal camera offers the advantage of measuring and mapping the entire temperature gradient across the slide 605 simultaneously. This level of detail allows the controller 410 to be programmed to actively#3371089 OS <11 9-000025

[0580] 74

[0581] compensate for localized thermal differences, such as those caused by non-uniform convection cooling around the slide edges or by concentrated areas of water / melted wax mixture that tends to agglutinate into drops. By modulating the laser power based on the specific location the laser beam 215 is hitting, the adaptive laser heating system 3900 can achieve better heating uniformity.

[0582] In still yet another approach, a dedicated humidity sensor is positioned above the slide 605 to directly detect the evaporated water. The humidity sensor is able to monitor the humidity level and signal completion when the detected humidity drops below a certain threshold after the evaporation phase. It should be recognized that different combinations of these approaches can be used. For example, the humidity sensor can be used in conjunction with the laser power monitoring technique described above to act as a backup and confirm that the baking process is complete.

[0583] As will be explained further below, a passive optical array can be used to expand the beam and flatten the gaussian distribution in the slide 605, eliminating the need of active components like the galvanometer steering mirrors for scanning the laser beam 215. In other examples, a rotating mirror, similar to laser barcode readers, is used instead of the galvanometer to redirect the laser beam 215 following a predetermined path,

[0584] A unique laser-based heating system and technique for promoting antigen retrieval (AR) will now be described with reference to FIG. 44, As can be seen in FIG. 44, a laser-based antigen retrieval system 4400 uses the laser beam 215 to heat the slide 605 in a fashion similar to that described before. The antigen retrieval system 4400 in some examples can be incorporated into the antigen retrieval station 230 of FIG. 2, but in other examples, the antigen retrieval system 4400 is incorporated into the baking station 220 and / or the dewaxing station 225 of the input module 105. For instance, the baking, deparaffinization, and antigen retrieval processes can be performed as a single process in a single station 205. In other examples, the antigen retrieval process is performed separately from the baking and'or deparaffinization processes.

[0585] It should be recognized that the antigen retrieval system 4400 in FIG. 44 shares a number of features in common w ith and operates In a fashion similar to the previously described laser#3371089 OS <11 9-000025

[0586] 75

[0587] heating systems, such as the adaptive laser heating system 3900 in FIG. 39, For example, the antigen retrieval system 4400 includes the laser head 405 with the beam positioner 425 and the lens 7.15 that scans the laser beam 215 on the base surface 740 of the slide 605 to heat the slide 605. At end of the slide 605 with the tag 3910, the slide holder 3905 holds the slide 605 with the tissue sample 730 in a similar fashion as described before. In one version, the laser head 405 scans the laser beam 215 on the base surface 740 of the slide 605 in the same manner as the laser scan path 4000 depicted in FIG. 40. The antigen retrieval system 4400 has the temperature sensor 445 with the field of view 1110 so as to monitor the temperature of the base surface 740 of the slide 605. In this version, the controller 410 uses the PID controller 4105 as a closed-loop controller to control the temperature of the slide 605 in the same fashion as described above with reference to FIGS. 39, 40, 41, 42, and 43, and as should be appreciated, the laser beam 215 can heat the slide 605 using different patterns and manners. For the sake o f brevity as well as clarity, these common components, processes, and functions will not be again described in great detail, but please refer to the previous discussions of these features. As will be explained further below, a passive optical array can be used to expand the beam and flatten the gaussian distribution in the slide 605, eliminating the need of active components like the galvanometer steering mirrors for scanning the laser beam 215. In other examples, a rotating mirror, similar to laser barcode readers, are used instead of the galvanometer to redirect the laser beam 215 following a predetermined path.

[0588] Referring still to FIG, 44, the tissue sample 730 located on the sample surface 735 of the slide 605 is covered with a liquid puddle that includes a cell conditioning reagent 4405 and a liquid coverslip (LCS) 4410, During processing, the cell conditioning reagent 4405 is dispensed onto the sample surface 735 of the slide 605 so as to cover the tissue sample 730 on the slide 605. The cell conditioning reagent 4405 in some variations includes a high- temperature cell conditioning liquid, like a mixture of Tris-EDTA (pH 9.0) with ethylene glycol (EG) or ethylene glycol cell conditioning reagent (EEGCC1), to elevate the boiling point and allow high -temperature exposure without evaporation. After the cell conditioning reagent 4405 is applied, the liquid coverslip 4410 is applied to the sample surface 735 of the slide 605 so as to cover the puddle of the cell conditioning reagent 4405 on the slide 605. In one version, the liquid coverslip 4410 is mineral oil, but other types of coverslip materials can be used in other examples. Applying the liquid coverslip 4410 over the cell conditioning reagent 4405 forms a seal that maintains an isolated microenvironment, which in turn#3371089 OS <11 9-000025

[0589] 76

[0590] prevents or minimizes evaporation during heating with the laser beam 215. During heating of the slide 605 with the laser beam 215, the heat from the laser beam 215 is transferred to the liquid puddle containing the cell conditioning reagent 4405. In one example, the antigen retrieval system 4400 heats the puddle with the cell conditioning reagent 4405 up to 120 degrees Celsius.

[0591] This rapid heating up to 120 degrees Celsius enables IHC antigen retrieval in a matter of minutes (e.g., 8 to 12 minutes) instead of the typical 64 to 104 minutes on current instruments, reducing overall turnaround time ( TAT) for IHC workflows. The feedback- controlled laser heating ensures precise and consistent temperatures. The direct and uniform heat transfer prevents overheating, scorching, and other damage to tissue samples. The antigen retrieval system 4400 can be optimized for various reagents and tissue types by fine- tuning laser parameters and liquid formulations so as to promote compatibility. It was unexpectedly discovered that this antigen retrieval system 4400 and processing technique surpasses conventional barriers in liquid puddle AR. speed and efficiency by using targeted microscale laser heating, potentially revolutionizing high-throughput histopathology.

[0592] In one example, the cell conditioning reagent 4405 includes EGCC1 (Ethylene Glycol Cell Conditioning Reagent), The EGCC1 is a pre-mixed solution of Tris-EDTA (pH 9.0) and ethylene glycol that is applied to the tissue sample 730 on the slide 605. The formulation of the cell conditioning reagent 4405 in this example is composed of at least 60% ethylene glycol (EG) to raise the boiling temperature to approximately 110°C. The high -temperature CC1 was formulated to mimic CC1 without boric acid. In one formulation, the 60% ethylene glycol CC1 (CC1E) recipe included: about 300mL of water, 30mM Tris Base (i.e., about 3.6g), l. OmL Tween 20, and ImM EDTA (i.e., about 0,37g). In this example, the liquid coverslip 4410 included a layer of mineral oil applied over the reagent paddle to maintain a sealed microenvironment. The laser 210 forming the laser beam 215 included a carbon dioxide type laser, and the focused laser beam 215 was applied to the base surface 740 of the slide 605 to directly heat the glass substrate forming the slide 605. A closed-loop feedback control system is used, and the temperature sensor 445 includes one or more Infrared (IR) temperature sensors to monitor and adjust the laser power for precise thermal regulation.In another example, the laser antigen retrieval system 4400 employs carbon dioxide laser heating applied directly to the glass slides 605 containing formalin-fixed, paraffin-embedded (FFPE) tissue samples 730. The cell conditioning reagent 4405 or liquid reagent puddle (CCiE) is composed of at least 60% ethylene glycol (EG) and is used to achieve high- temperature stability without evaporation. The liquid coverslip 4410 (mineral oil) is added atop the cell conditioning reagent 4405 to maintain an isolated microenvironment and prevent evaporation. The slides 605 are then exposed to the laser beam 215, which heats the glass slide 605 directly while the closed-loop system maintains precise thermal control (e.g., at 120 degrees Celsius for 8 to 12 minutes).

[0593] During some initial tests, the cell conditioning reagent 4405 included CC1G (60% Glycerol) and CC1E (60% Ethylene Glycol), and no liquid coverslip 4410 was used. The slides 605 were heated for 8 and 12 minutes at 120°C. Without the liquid coverslip 4410, the results were unacceptable as antigen retrieval appeared to be incomplete when compared to a 64-minute traditional heating reference process. While not certain, it has been hypothesized that evaporation was lowering the puddle temperature on the slides 605. The experiment was generally repeated, but in this experiment, the slide 605 included the liquid coverslip 4410. In this repeat experiment with the liquid coverslip 4410, the cell conditioning reagent 4405 included C E (Ethylene Glycol). The slide 605 was heated to 120c‘C for 8 minutes, and this process achieved an equivalent staining intensity and no morphological damage compared to the 64-minute reference. This laser-heating antigen retrieval process represented an equivalent staining in one-eighth ( 1 / 8) the antigen retrieval time over the traditional reference method. It was further found that the Glycerol (CC IG) conditions were inferior and did not show acceptable staining, and heating to 130°C for 8 minutes resulted in a slight decrease in staining intensity, which suggested that the tissue samples 730 were overcooked.

[0594] Further experiments have been performed for different types of tissue samples. For vimentin (placenta) type tissue samples, successful antigen retrieval was achieved in 8 minutes at 120°C using this laser-based technique. It was found that the staining quality matched or surpassed BENCHMARK® ULTRA control samples, and the tissue morphology was intact. It was further discovered that the optimal retrieval processing or laser heating time was between 8 to 12 minutes at 120°C.#3371089 OS <11 9-000025

[0595] 78

[0596] For B-cell lymphoma 2 (BCL2) type tonsil tissue samples, the processing or reference time on a BENCHMARK® ULTRA brand system is typically around 64 minutes for antigen retrieval. Using the above-discussed laser-based heating antigen retrieval technique, the slides 605 were heated to 120°C and tested after four (4) minutes of heating. These laser- heated slides 605 showed equivalent staining to the control slides from the BENCHMARK® ULTRA brand system. For these types of tissue samples, it was found that the optimal heating or processing was from 8 to 12 minutes at 120°C

[0597] For Anaplastic Lymphoma Kinase (ALK) type appendix tissue samples, the processing or reference time on a BENCHMARK® ULTRA brand system is typically around 104 minutes. Using the above-discussed laser-based heating antigen retrieval technique, the slides 605 were heated to 120°C. The slides 605 were heated for 12 minutes and 16 minutes. The 12- minute condition showed the most equivalent staining to the reference of control BENCHM ARK® ULTRA brand systems, though one repeat failed. The 16-minute laser heating processing time resulted in much higher or prominent background, which suggests the antigen retrieval laser- heating process was at temperature for too long.

[0598] In cluster of differentiation 10 (CD 10) type tonsil tissue samples, the processing or reference time on a BENCHMARK® ULTRA brand system is typically around 92 minutes. Using the above-discussed laser-based heating antigen retrieval technique, the slides 605 were heated from 10 to 14 minutes at 120°C. The 10-minute heating time condition was found to be the closest to the staining of the reference BENCHMARK® ULTRA brand system. The CD10 antigen is very sensitive to overcooking; the longer heating times tend to cause issues, such as decreased staining due to epitope loss.

[0599] For the above, it should be appreciated that the laser-based antigen retrieval technique dramatically reduces turnaround or processing times. Once more, the traditi onal BENCHMARK® ULTRA brand systems typically required antigen retrieval processing times of around 64 minutes for BCL2 tissue samples, 64 minutes for vimentin tissue samples, 92 minutes for CD 10 tissue samples, and 104 minutes for ALK tissue samples. In contrast the laser heating technique required dramatical ly shorter runtimes of around 8 to 12 minutes. Even with these shorter runtimes, the laser heating method produced comparable antigen#3371089 OS <11 9-000025

[0600] 79

[0601] retrieval outcomes across all markers. The shorter antigen retrieval processing times can enable a significant reduction in laboratory turnaround time while preserving tissue integrity.

[0602] It should also be recognized from the above explanation that the composition of the cell conditioning reagent 4405 and the use of the liquid coverslip 4410 enhanced the laser-based antigen retrieval process. For instance, having the cell conditioning reagent 4405 composed of ethylene glycol-infused Tris-EDTA solution ( EGCC1) enabled high -temperature retrieval by increasing the boiling threshold. Moreover, the liquid coverslip 4410 of mineral oil was found to consistently decrease evaporation during high-temperature cycles used in the laser-based antigen retrieval process.

[0603] The previously described laser heating systems and techniques for baking, dewaxing, antigen retrieval, and coverslip curing scan the laser beam 215 to promote even heating. In most cases (but not all), a commercially available carbon dioxide laser beam engraving system is used to perform the scanning and heating. As noted before, these types of systems facilitate fast thermal control directly on the slide 605. With the slide 605 only being heated with the laser beam 215, such systems can provide 10 times in terms of energy savings. With less moving parts as compared to conventional ovens or other heating systems, the reliability'’ and uptime is enhanced. Since the lasers 210 are extensively used in different industries, the lasers 210 tend to be very reliable. Moreover, the laser-heating technique promotes faster heating ramps, convective heating, mixing, and single-piece processing, and these systems reduce or even eliminate most, if not all, reagent replenishment activities. However, the commonly used laserbeam engraving system still uses some moving parts, such as two galvo scanners in the beam positioner 425 for deflecting the laser beam 215, which can be costly and increase complexity of the system. Typically, the laser scanner needs a controller and power electronics, such as the power control module 430 and the position control module 435 in FIG. 4, for driving the beam positioner 425 (e.g., galvo scanners), which again are costly and increase complexity, As previously depicted in FIGS. 7. 11, 39, and 44, the lens 715 is required for assuring a linear relation between scan angle and beam position on the slide 605. Using the F-Theta lens 715 increases the cost and complexity of the system.

[0604] Referring now to FIG. 45, an optics system 4500 has been developed to eliminate the need for the beam positioner 425 and the F-Theta lens 715. This optics system 4500 typically is#3371089 OS <11 9-000025

[0605] 80

[0606] located between the beam generator 420 and the slide 605, and the optics system 4500 eliminates the need for scanning the laser beam 215 from the beam generator 420 by expanding and shaping the laser beam 215 that is shone onto the slide 605, the tissue sample 730, and' or other items. Instead of scanning the slide 605 with a focused laser beam 215 having a diameter that is much smaller than the whole slide 605, one or more slides 605 are illuminated by and heated with a static and massively expanded laser beam 215. To facilitate this expansion, the original laser beam 215 from the beam generator 420, which has an extremely nonuni form Gaussian profile, is converted into a uniform flat top laser beam 215 of appropriate size.

[0607] Looking at FIG. 45, the optics system 4500 includes a beam expander 4505 to increase the beam diameter of the laser beam 215, an aperture or field stop 4510 that helps in shaping the laser beam 215, and a beam shaper 4515 configured to achieve homogeneous illumination of the slide 605 (the coverslip 450 and or tissue sample 730) with the laser beam 215. In one particular example, the beam expander 4505 includes a Galilean beam expander that includes a short focal length spherical diverging lens followed by a long focal length spherical converging lens. The beam shaper 4515 in this example includes a refractive flat-top beam shaper, which is also sometimes referred to as a Gaussian-to-top-hat converter or aspheric beam homogenizer, Tire refractive flat-top beam shaper is commonly identified by the brand name "jiShaper" for a series of beam shapers that are sold by AdlOptica Optical Systems GmbH of Berlin, Germany. With this construction, no moving parts for the galvo scanners in the beam positioner 425 are needed, and no controller (e.g., the position control module 435 in FIG. 4) for the galvo scanners is required. Moreover, the F-Theta lens 715 is not required for assuring a linear relation between scan angle and beam position on the slide 605.

[0608] As can be seen, the profile and size of the laser beam 215 changes as the laser beam 215 passes through the optics system 4500 of FIG. 45. The laser beam 215 that is generated at the beam generator 420 is directed to the beam expander 4505. At the input or entrance end of the beam expander 4505, the laser beam 215 has an input beam waist 4520. In one form, the input beam waist 4520 is about 0.90mm. Between the output of the beam expander 4505 and the input of the field stop 4510, the laser beam 2.15 has an expander beam waist 4525.

[0609] Returning to the previous example, the expander beam waist 4525 is about 4.72mm, and the field stop 4510 has an aperture diameter of about 12mm. Upon exi ting the output end of the#3371089 OS <11 9-000025

[0610] 81

[0611] beam shaper 4515, the laser beam 215 has a Held of view 4530 that is now considerably larger and more uniform. Referring to the previous example, the field of view 4530 has a waist diameter of about 70mm. In other words, the beam waist is clipped at a diameter of 12mm, and the diameter of the field of view 4530 is 70mm. In this case, the beam shaper 4515 may be referred to as a "aShaper 12_70” where the ”1 " refers to the beam waist clipped diameter of 12mm and the "70" refers to the 70mm field of view diameter. The dimensions of the field stop 4510, the input beam waist 4520. the expander beam waist 4525. and the field of view 4530 can be different in other variations.

[0612] FIG. 46 shows a side view of the optics system 4500, and FIG. 46 further illustrates how the laser beam 215 is expanded in the optics system 4500. The beam expander 4505 magnifies the beam waist of the coherent laser beam 215. The beam expander 4505 includes a diverging lens 4605 and a converging lens 4610. In this example, the beam expander 4505 is a Galilean beam expander. In the illustrated example, the diverging lens 4605 is a short focal length spherical diverging lens, and the converging lens 4610 is a long focal length spherical converging lens. It should be recognized that the diverging lens 4605 and the converging lens 4610 can include different types of lenses, and the beam expander 4505 can have different combinations of lenses oriented in other manners.

[0613] The beam shaper 4515 converts the magnified beam from a gaussian profi le to a flat top profile. The beam shaper 4515 includes an input aspheric lens 4615 and an output aspheric lens 4620. In one version, the Input aspheric lens 4615 is a negative or diverging aspheric lens, and the output aspheric lens 4620 is a positive or converging aspheric lens, with no internal focus. The input aspheric lens 4615 and the output aspheric lens 4620 are oriented in a telescope-like configuration to redistribute the intensity profile of the laser beam 215. The beam shaper 4515 maps the rays of the input laser beam 215 such that a Gaussian (i.e., bell- shaped) intensity distribution is converted into a flat-top (uniform) profile at the output, while keeping the beam collimated. This is to achieve homogeneous illumination for improved efficiency and quality of the laser light illuminated on the coverslip 450, the slide 605, the tissue sample 730, or elsewhere on the slide 605. In one version, the beam shaper 4515 (e.g., pi-shaper) includes two even aspheres, and the lens material is zinc selenide (ZnSe) because standard glass materials do not transmit in the mid IR. Instead of grinding and polishing, the standard manufacturing processes for conventional glass lenses, the lenses are manufactured#3371089 OS <11 9-000025

[0614] 82

[0615] by diamond turning. In some cases, the two spherical lenses (i.e., the diverging lens 4605 and the converging lens 4610) of the beam expander 4505 can be omitted by fully integrating the expansion functionality into the beam shaper 4515 a t the expense of more challenging tolerances.

[0616] As can be seen In FIG. 46, the beam expander 4505 and the beam shaper 4515 generate planar wavefronts (i.e., at the expander beam waist 4525 and the field of view 4530). The beam expander 4505 maintains the gaussian profile. Using the beam expander 4505 makes manufacturability of the beam shaper 4515 easier, because the beam shaper 4515 does not require as tight of tolerances if the beam expander 4505 was not used. The beam shaper 4515 redistributes the intensity of the wavefront through excessive distortion. Looking at a diagram 4700 in FIG. 47, a diagram 4800 in FIG. 48, and another diagram 4900 in FIG. 49, the beam shaper 4515 is configured to redistribute the irradiance of the wavefront by excessive lateral distortion, and a planar inbound wave front is converted into a planar outbound wavefront.

[0617] It should be recognized that the optics system 4500 can be configured differently in other variations. For example, the beam shaper 4515 as well as other optics in the optics system 4500 can include a diffractive optical element (DOE), Diffractive optical elements are helpful in laser applications, because inherent chromatic aberrations play no role, and diffractive optical elements benefit from the monochromaticity of the laser radiation. One specialized type of diffracti ve optical element is a computer-generated hologram (CGH). Computergenerated holograms can be useful in the optics system 4500, because the computergenerated holograms encompass many more internal degrees of freedom, which accordingly means many more possibilities for beam shaping of the laser beam 215. In one variation, the beam shaper 4515 includes a diffractive optical element in the form of a computer- gen erated hologram. In another variation, the entire beam shaping optics in the optics system 4500 is a single computer-generated hologram. For example, the beam expansion and shaping functions of the beam expander 4505 and the beam shaper 4515 are performed by the single computer-generated hologram in one version. In other words, the beam expander 4505 and the beam shaper 4515 of the optics system 4500 are integrated together into a single unit in the form of the computer-generated hologram.#3371089 OS <11 9-000025

[0618] 83

[0619] FIG. 49 shows a diagram 4900 that depicts optical modeling of one version of the optics system 4500. The optical modeling was performed with a raytracing software. As can be seen, the optical model demonstrates that the beam expander 4505 in the form of a Galilean beam expander converts a gaussian beam into a gaussian beam with enlarged beam diameter. This enlarged gaussian beam is then converted into a flat top beam by the beam shaper 4515.

[0620] As can be seen in FIGS. 45 and 49. the optics system 4500 of FIG. 45 converts a circular gaussian beam into a circular flat top beam. However, a non-circular beam may be desirable in certain cases, such as for a square or rectangular slide 605. FIG. 50 shows a diagram 5000 of an optics system 5005 that is configured to produce a square or rectangular flat top beam for irradiating the coverslip 450, the slide 605, the tissue sample 730, or other part of the slide 605 for heating purposes.

[0621] Like before, the optics system 5005 in FIG. 50 includes the beam expander 4505 that is configured to expand the laser beam 215. Downstream from the beam expander 4505, the optics system 5005 further includes an aperture or field stop 5010 and a beam shaper 5015 that shape the laser beam 215 io form the laser beam 215 to have a square or rectangular shape. As shown, the expanded laser beam 215 after the beam expander 4505 has a circular shape, and the field stop 5010, which is located at the entrance of the beam shaper 5015. distorts or masks the laser beam 215 to form a pincushion shape. In another variation, the field stop 5010 distorts or masks the expanded laser beam 215 to create a pi llow or barrel shape.

[0622] Like in the previous example, the beam shaper 5015 converts the magnified beam from a gaussian profile to a flat top profile. The beam shaper 5015 in this example includes a refractive flat-top beam shaper, which is again sometimes referred to as a Gauss ian-to-top-hat converter or aspheric beam homogenizer. Once more, the refractive flat-top beam shaper is commonly iden tified by the brand name ’biSha per" or "pi-shaper" for a series of beam shapers that are sold by AdlOptica Optical Systems GmbH of Berlin, Germany. In this case, the beam shaper 5015 produces a square or rectangularly shaped beam. The beam shaper 5015 includes an input lens 5020 and an output lens 5025. The input lens 5020 and the output lens 5025 are oriented in a telescope- like configuration to redistribute the intensity profile of the laser beam 215. The beam shaper 5015 (e.g., a pi -shaper) maps the rays of the input laser#3371089 OS <11 9-000025

[0623] 84

[0624] beam 215 such that a Gaussian (i.e., bell-shaped) intensity distribution is converted into a flat-top (uniform) profile at the output, while keeping the beam collimated. Commonly, the ray remapping of the beam shaper 5015 causes a barrel-shaped distortion that in essence spreads the centra! Gaussian energy to the periphery to promote uniformity. In the illustrated example, the field stop 5010 is in the form of a pillow-shaped field aperture, and the input lens 5020 and the output lens 5025 in the beam shaper 5015 create a barrel-shaped distortion that converts the laser beam 215 from the pillow-shaped field aperture of the field stop 5010 to a square or rectangular shape at a field of view 5030. In other variations, the field stop 5010 can mask or distort the laser beam 215 into other shapes, and the beam shaper 5015 can distort these other shapes of the laser beam 215 into the square or rectangular shape at the field of view 5030. The square flat top beam at the exit of the beam shaper 5015 then can be shone onto the coverslip 450 and / or slide 605 for various heating purposes, such as for baking, dewaxing, antigen retrieval, and curing. Depending on the particular needs, the shape of the laser beam 215 at the field of view 5030 can be different.

[0625] FIG. 51 shows a diagram 5100 of how the beam shaper 5015 (e.g., the πShaper 12_70) distorts the pincushion shape of the laser beam 215 from the field stop 5010 to the square or rectangular shape at the exit of the beam shaper 5015. The slides 605 typically have a rectangular shape, and the coverslips 450 normally have a square or rectangular shape. The tissue sample 730 on the slide 605 commonly occupies a square or rectangular area on the slide 605. Having the expanded laser beam 215 with a square or rectangular shape minimizes energy loss and accidental heating of other components during processing of the ti ssue sample 730.

[0626] Once more, the optics system 4500 in FIG. 45 and the optics system 5005 in FIG. 50 eliminate the need for scanning the laser beam 215 from the beam generator 420 by expanding and shaping the laser beam 215 that is shone onto the coverslip 450, the slide 605, the tissue sample 730, and / or other items. For example, the optics system 4500 of FIG. 45 and the optics system 5005 of FIG. 50 can be incorporated into the various stations 205 described above, like the baking station 220, the dewaxing station 225. the antigen retrieval station 230, and the antigen retrieval station 230 in FIG. 2, so as to heat the coverslip 450, the slide 605, the tissue sample 730, and other items via laser heating without scanning. In one example, the FIG. 45 optics system 4500 or the FIG. 50 optics system 5005 replaces the#3371089 OS <11 9-000025

[0627] 85

[0628] beam positioner 425, the lens 715, and the mirror 725 in the heating station 700 of FIG. 7. In another example, the FIG. 45 optics system 4500 or the FIG. 50 optics system 5005 replaces the beam positioner 425 and the lens 715 in the laser system 1100 in FIG. 11 for dewaxing. The optics system 4500 of FIG. 45 or the optics system 5005 of FIG. 50 in still yet another example is incorporated into the laser system 2300 of FIG. 23 for curing. It should be appreciated that the laser heating technique described with reference to the optics system 4500 in FIG. 45 and the optics system 5005 of FIG. 50 generally eliminates the risk of damaging the coverslips 450 in the manner as shown in FIG. 30. Moreover, this laser heating technique can be used with the various laser absorptive materials described with reference to FIGS. 26, 27, and 28. In other examples, the FIG. 45 optics system 4500 or the FIG. 50 optics system 5005 replaces the beam positioner 425 and the lens 715 in the adaptive laser heating system 3900 of FIG. 39 for baking and the antigen retrieval system 4400 of FIG. 44 for antigen retrieval. As will be appreciated, the FIG. 45 optics system 4500 and the FIG. 50 optics system 5005 can be incorporated into other types of tissue processing activities that require heating.

[0629] FIGS. 52, 53, 54, and 55 depict an integrated adaptive laser heating system 5200 that enhances the laser processing methods described above by utilizing a multi -slide asynchronous architecture and spatially resolved thermal management. The system 5200 is designed to ensure consistent and robust tissue adhesion, and the system 5200 further provides a better confirmation of the removal of the w'atcr from the slide 605. As can be seen, the system 5200 shares several features in common with the previously described systems, like the controller 410, the beam generator 420, the beam positioner 425, the power control module 430, the position control module 435, and the temperature sensor 445. For the sake of clarity as well as brevity, these common features and their function will not be again described in grea t detail, but please refer to the previous discussion of these features. While the system 5200 will be described as performing baking and / or dewaxing, it should be recognized that the system 5200 can be adapted to perform laser heating in other types of processing stations 205.

[0630] As illustrated, the system 5200 includes a heating laser 5205 that produces the heating laser beam 215 for heating the slide 605 and a visible laser 5210 that produces a visible laser beam 5215 that aids in laser beam visibility on the slide 605. In the illustrated example, the heating#3371089 OS <11 9-000025

[0631] 86

[0632] laser 5205 includes the beam generator 420, such as in the form of an infrared (IR) laser source, and the power control module 430 that controls the power of the laser beam 215 generated by the beam generator 420. The heating laser 5205 in one version includes a carbon dioxide type laser, but it should be recognized that other types of lasers can be used. In the illustrated example, the visible laser 5210 produces a red visible laser beam 5215, but the visible laser 5210 can produce visible laser beams 5215 having different colors. In some cases, the visible laserbeam 5215 may not be visible to the human eye when initially produced by the visible laser 5210, but the visible laser beam 5215 may be modified at a later state so as to be visible on the slide 605. For instance, the slide 605 and or fluorescent chemicals on the slide 605 may fluoresce to produce visible light when exposed to the visible laser beam 5215.

[0633] The system 5200 further includes a beam combiner 5220 that is configured to combine the heating laser beam 215 from the heating laser 5205 with the visible laser beam 5215 from the visible laser 5210 to produce a combined laser beam 5225 that is configured to both heat the slide 605 and provide visibility of the combined laser beam 5225 on the slide 605. The heating laser beam 215 in the combined laser beam 5225 promotes heating of the slide 605. The visible laser beam 5215 part of the combined laser beam 5225 enhances safety, because the operator is able to see if the combined laser beam 5225 is being generated. By being visible, the visible laser beam 5215 further can assist in adjusting alignment of the combined laser beam 5225 shone on the slide 605. From the beam combiner 5220. the combined laser beam 5225 is directed through a focus lens 5230 of the sy stem 5200 so as to focus the combined laser beam 5225.

[0634] Like in the previously described examples, the system 5200 includes the beam positioner 425 with the position control module 435 and a steerable mirror 5235 that is moved by the position control module 435 to scan the combined laser beam 5225 from the focus lens 5230 onto the slide 605. For baking and dewaxing, the steerable mirror 5235 typically (but not always) directs the combined laser beam 5225 towards the base surface 740 of the slide 605. However, in other variations, the steerable mirror 5235 can shine the combined laser beam 5225 onto the coverslip 450 and even the sample surface 735 of the slide 605 to promote heating, such as during curing. As shown, the combined laser beam 5225 is reflected off the steerable mirror 5235 driven by the position control module 435. In one version, the steerable#3371089 OS <11 9-000025

[0635] 87

[0636] mirror 5235 includes voice-coil type actuators on X and Y axes that are driven so as to allow the combined laser beam 5225 to execute arbitrary scanning paths, such as increasing- radius concentric circles, ovals, or rectangles, on the base surface 740 of the slide 605 to enhance heat transfer.

[0637] The system 5200 further includes a slide holder 5240 that is configured to position and hold one or more slides 605. As shown in FIG, 55, the slide holder 5240 in the depicted example is configured to hold two slides 605, but the slide holder 5240 in other examples can hold a single slide 605 or more than two slides 605. By holding more than one slide 605, multiple slides 605 can be heated in parallel or together with the combined laser beam 5225. In other cases, the slides 605 can be heated by the combined laser beam 5225 in an asynchronous manner. As can be seen, the slide holder 5240 holds the slides 605 at a transverse angle relative to a level or horizontal plane to promote drainage of water and / or wax. In one particular example, the slide holder 5240 holds the slides 605 at an angle of about forty-five (45) degrees relative to the horizontal plane to facilitate drying and / or dewaxing. The slide holder 5240 uses gravity to assist in the removal of excess water and / or melted wax.

[0638] The slide holder 5240 is protected by a holder cap 5245. When on the slide holder 5240, the slides 605 are covered by the holder cap 5245. The holder cap 5245 provides operator safety from laser radiation. In the illustrated example, the holder cap 5245 is in the form of a door that is pivotally coupled to the slide holder 5240 via a hinge or other pivotal connection, In other variations, the holder cap 5245 can be secured to the slide holder 5240 in other manners. As shown in FIGS. 52. 53, and 54, the holder cap 5245 has a fan 5250. The slide holder 5240 and the holder cap 5245 form a cavity where the slides 605 are disposed during heating by the laser beam 215. In the internal cavity between the slide holder 5240 and the holder cap 5245, the fan 5250 induces airflow to remove air saturated with moisture. As depicted in FIGS. 52 and 53, the holder cap 5245 has an intake port 5255 where relatively dry air or other dry gas is received and an exhaust port 5260 where the humidified air or other gas is discharged. When inside the holder cap 5245, the air blows across the tissue sample 730 on the slide 605, which in turn helps to dry the slide 605. The now humidified air is discharged from the holder cap 5245 at the exhaust port 5260. The system 5200 will be described as blowing ambient air across the slides 605 to promote drying, but it should be recognized that other types of gases may be used. For example, instead of using the fan 5250,#3371089 OS <11 9-000025

[0639] 88

[0640] a compressed gas, such as compressed air or nitrogen, is blown across the slides 605 to promote drying. In the illustrated example, the fan 5250 is located at the intake port 5255 so tha t the fan 5250 is exposed to dry air so as to reduce the risk of damage caused by high humidity. In this example, the intake port 5255 is located at the lower end of the slide holder 5240 so as to create a dry air flow that is counter to the drainage direction of the water due to gravity. This counter flow further promotes drying of the slide 605. In other variations, the fan 5250 can be located elsewhere, and the flow of dry gas can be different.

[0641] Instead of using single-point sensors, the temperature sensor 445 in the system 5200 includes a thermal camera 5265 that monitors spatial temperature distribution across the slides 605 without physical contact. The digitized data from the thermal camera 5265 is utilized by multiple PID control loops to modulate laser power with spatial resolution. This allows the creation of controlled thermal gradients that ensure even drying and prevent localized artifacts or water pockets. To manage high-throughput workflows, the system 5200 includes one or more proximity or slide sensors 5270 for precise slide detection. In one version, the slide sensor 5270 is disposed on the slide holder 5240 to detect the presence of the slide 605. The slide sensor 5270 can be used to further promote safety, For instance, the system 5200 can deactivate the heating laser 5205 when the slide sensor 5270 fails to detect a slide 605 on the slide holder 5240. As illustrated, the slide sensor 5270 is communicatively or operatively coupled to a data acquisition unit (DAQ unit) 5275. The DAQ unit 5275 is further operatively coupled to the power control module 430.

[0642] Like in the other previously described examples, the system 5200 includes the controller 410 that is configured to control the operation of the system 5200. The controller 410 is operatively coupled to the position control module 435, the thermal camera 5265, and the DAQ unit 5275. The controller 410, either directly or through the DAQ unit 5275, is able to receive data from the position control module 435, the thermal camera 5265, and the slide sensor 5270. Based on this data, the controller 410 is able to control the power of the heating laser beam 215 via the power control module 430 and the scanning path of the combined laser beam 5225 via the position control module 435.

[0643] It was discovered that using adaptive control to detect dryness via the cessation of evaporative cooling often relied on single-sensor averaging that failed to account for#3371089 OS <11 9-000025

[0644] 89

[0645] localized temperature gradien ts caused by geom etric arti facts or the agglutinati on of water and wax. The thermal camera 5265 provides a broader picture to the controller 410 of the thermal gradients on the slide 605. By using turbulent air to replace saturated air near the slide surface via the fan 5250 and by utilizing specialized laser scanning paths to manage temperature gradients, the system 5200 is able to leverage thermocapillary convection to sweep excess moisture off th e slide 605 and further accelerate the drying process.

[0646] In particular, the controller 410 via the position control module 435 is able to control the scanning path of the combined laser beam 5225. The combined laser beam 5225 is reflected off the steerable mirror 5235 so as to allow the combined laser beam 5225 to execute arbitrary scanning paths, such as increasing- radius concentric circles, ovals, or rectangles, on the base surface 740 of the slide 605 to enhance heat transfer, FIG. 56 shows an example of concentric beam paths 5600 of the combined laser beam 5225 that are used to heat the base surface 740 of the slide 605. As depicted by the variations of the beam paths 5600. the power of the combined laser beam 5225 varies as a function of the radius of the beam paths 5600 according to the temperature distribution as sensed by the thermal camera 5265. It was unexpectedly discovered that the depicted beam paths 5600 were able to physically drive excess water and wax toward the side edges of the slide 605 via laser-induced thermocapillary convection, as is depicted in FIGS. 57 and 58. In essence, the laser heating system 5200 effectively sweeps the moisture away from the tissue sample 730 to enhance adhesion. The controller 410 of the system 5200 manages the entire protocol. The controller 410 analyzes real-time thermal curves from the thermal camera 5265 to adapt the baking protocols based on individual slide wetness, and the controller 410 is able to confirm successful dryness by detecting the drop in power demand that occurs once evaporative cooling stops in the fashion as described above with reference to FIGS. 41, 42, and 43.

[0647] In one example, the beam paths 5600 on the slides 605 are controlled by the following alternating current (AC) signals induced on each of the voice coils actuators in the steerable mirror 5235 using the equations below:

[0648] X1..n = a1..n*sin(bt+c) + d (Equation 1)

[0649] Y1..n = e1..n*sin(ft+g) + h (Equation 2)#3371089 OS <11 9-000025

[0650] 90

[0651] Where t goes from 0 to 2Π, and phase between x and y is 90 degrees apart.

[0652] In one version, the controller 410 via the power control module 430 modulates the power of the laser beam 215 as a function of the mirror positioning signal amplitudes with a pulse width modulation (PWM) signal on the beam generator 420 in which pulse width is determined by PID loops (P1..n). Using both signals, the controller 410 of the system 5200 controls the irradiated temperature profile. When the slide holder 5240 holds two or more slides 605, such as in the manner depicted in FIG. 55, an offset in the X signal controls which of the slides 605 is being irradiated with the combined laser beam 5225.

[0653] As depicted in FIG. 58. the thermocapillary convection effect is utilized in the system 5200 through a specifically designed scanning path to actively enhance tissue adhesion. By irradiating the slide 605 with a series of concentric oval beam paths 5600 that initiate at the center of the tissue sample 730 and expand outward, the system 5200 generates a localized zone of high thermal energy that is significantly hotter than the surrounding non-irradiated areas. This intentional temperature differential creates surface tension gradients across the liquid-slide interface, inducing a fluid flow that physically sweeps the excess water and molten wax mixture from the center of the slide 605 toward the edges of the slide 605. This directed removal of residual moisture and paraffin prevents the entrapment of humidity beneath the tissue sample 730 on the slide 605. This in turn accelerates the baking process and ensures a more robust, artifact-free bond between the specimen and the slide substrate.

[0654] A method for operating the FIG. 52 system 5200 will now be described with reference to a flowchart 5900 in FIG. 59 as well as FIGS. 52-58. The method illustrated in the flowchart 5900 in FIG. 59 allows multiple slides 605 on the slide holder 5240 to be laser heated or otherwise processed, such as for baking, dewaxing, or curing, in an asynchronous manner. In one particular version that will be described below; the flowchart 5900 represents the general logic or algorithm, such as in the form of software and / or firmware, performed by the controller 410 in the system 5200. The various actions in the method will be described as being performed by the controller 410 as well as other components of the system 5200. but it should be recognized that these actions can be performed by different components. It should#3371089 OS <11 9-000025

[0655] 91

[0656] be appreciated that the various activities can be performed in a different order as well as certain actions maybe omitted or additional actions may occur. For explanation purposes, the technique illustrated by the flowchart 5900 in FIG. 59 will be described with reference to processing two slides 605, such as is illustrated in FIG. 55, but this technique can be modified for use with a single slide 605 or more than two slides 605.

[0657] After starting in stage 5905, the controller 410 in the system 5200 performs a status check in stage 5910. Via the slide sensor 5270, the controller 410 is able to determine whether a first slide 605 (i.e., slide A) is present on the slide holder 5240 in stage 5915. If the slide sensor 5270 indicates the first slide 605 is present, the controller 410 of the system 5200 uses the thermal camera 5265 to capture a thermal image of the first slide 605 in stage 5920. Based on the thermal image of the first slide 605 from the thermal camera 5265, the controller 410 using a PID algorithm similar to those described before calculates an irradiance profile that should be used on the first slide 605 in stage 5925, and the controller 410 of the system 5200 in stage 5930 scans the first slide 605 using the irradiance profile calculated in stage 5925. The irradiance profile for the combined laser beam 5225 on the first slide 605 can be tailored based on the temperatures at various areas on the first slide 605. For example, the power and / or dwell time of the heating laser beam 215 in the combined laser beam 5225 can be increased or the beam paths 5600 can be changed to further heat a cooler area on the first slide 605. Conversely, the controller 410 via the power control module 430 and the position control module 435 can reduce heating at certain locations on the first slide 605. For example, the thermal heating generated by the heating laser 5205 can vary such as in the manner depicted in FIGS. 56 and 58. For instance, the center of the first slide 605 can be heated to a higher temperature as compared to the outer edges of the first slide 605.

[0658] After scanning the combined laser beam 5225 on the first slide 605, the thermal camera 5265 in stage 5935 captures another thermal image of the first slide 605. Based on the thermal image from the thermal camera 5265, the controller 410 of the system 5200 in stage 5940 estimates slide wetness from the thermal curves, such as in the manner as described before with respect to FIGS. 41, 42, and 43. If the first slide 605 is not dry (i.e., wet) in stage 5945. the controller 410 of the system 5200 proceeds to stage 5920. Otherwise, if the first slide 605 is determined to be dry, the controller 410 proceeds to stage 5950 where a flag is set in memory and / or a signal is sent to pick up the first slide 605. A robotic arm, conveyor, or#3371089 OS <11 9-000025

[0659] 92

[0660] other transfer device can be used to pick up and remove the first slide 605 from the slide holder 5240. Based on the slide sensor 5270, the controller 410 of the system 5200 is able to determine whether or not the first slide 605 has been removed in stage 5955. If the first slide 605 has been removed from the slide holder 5240, the controller 410 proceeds to stage 5910 where the controller 410 of the system 5200 again performs the status check and proceeds in the previously described manner.

[0661] Otherwise, if the first slide 605 has not been removed from the slide holder 5240 in stage 5955, the controller 410 via the slide sensor 5270 is able to determine whether a second slide 605 (i.e., slide B) is present on the slide holder 5240 in stage 5960. If the slide sensor 5270 indicates the second slide 605 is present, the controller 410 of the system 5200 uses the thermal camera. 5265 to capture a thermal image of the second slide 605 in stage 5965. Based on the thermal image of the second slide 605 from the thermal camera 5265, the controller 410 using a PID algorithm similar to those described before calculates an irradiance profile that should be used on the second slide 605 in stage 5970, and the controller 410 of the system 5200 in stage 5975 scans the second slide 605 using the irradiance profile calculated in stage 5970, The irradiance profile for the combined laser beam 5225 on the second slide 605 can be tailored based on the temperatures at various areas on the second slide 605. For example, the power and / or dwell time of the heating laser beam 215 in the combined laser beam 5225 can be increased or the beam paths 5600 can be changed to further heat a cooler area on the second slide 605. Conversely, the controller 410 via the power control module 430 and the position control module 435 can reduce heating at certain locations of the second slide 605. For example, the thermal heating generated by the heating laser 5205 can vary such as in the manner depicted in FIGS. 56 and 58. For instance, the center of the second slide 605 can be heated to a higher temperature as compared to the outer edges of the second slide 605.

[0662] After scanning the combined laser beam 5225 on the second slide 605, the thermal camera 5265 in stage 5980 captures another thermal image of the second slide 605. Based on the thermal image from the thermal camera 5265, the controller 410 of the system 5200 in stage 5982 estimates slide wetness from the thermal curves, such as in the manner as described before with respect to FIGS. 41, 42, and 43. If the second slide 605 is not dry (i.e., wet) in stage 5984, the con troller 410 of the system 5200 proceeds to stage 5965. Otherwise, if the#3371089 OS <11 9-000025

[0663] 93

[0664] second slide 605 is determined to be dry, the controller 410 proceeds to stage 5986 where a flag is set in memory and / or a signal is sent to pick up the second slide 605. The robotic arm, conveyor, or other transfer device can be used to pick up and remove the second slide 605 from the slide holder 5240. Based on the slide sensor 5270, the controller 410 of the system 5200 is able to determine whether or not the second slide 605 has been removed in stage 5988. If the second slide 605 has not been removed from the slide holder 5240, the controller 410 proceeds to stage 5910 where the controller 410 of the system 5200 again performs the status check and proceeds in the previously described manner.

[0665] When the controller 410 in stage 5988 determines via the slide sensor 5270 that the second slide 605 has been removed from the slide holder 5240, the controller 410 of the system 5200 proceeds to stage 5990 in order to determine whether or not more slides 605 need to be processed. If more slides 605 need to be processed, the controller 410 proceeds to stage 5910 to start the process again. Otherwise, when there are no slides 605 in the queue for laser heating, the process in stage 5995 ends.

[0666] A specific example of a method or technique for performing the thermocapillary con vection sweeping or brooming of the type discussed above, such as with respect to FIGS. 40, 56, 57, and 58, will now be described with reference to a flowchart 6000 in FIG, 60. For explanation purposes only, this method will be described as being performed by the system 5200 in FIG.

[0667] 52, but it should be recognized that other types of laser heating systems can be used to perform this method. Referring now to FIGS. 52, 53, and 60, after starting the process in stage 6005, the control ler 410 of the system 5200 retrieves or receives the laser scanning path parameters and maximum temperature information. For instance, the controller 410 in stage 6010 can retrieve this information from memory or receive the information from the operator of the system 5200. As an example, a barcode reader for the system 5200 can read a barcode on the slide 605 that identifies the type of ti ssue sample being processed, and the controller 410 can retrieve the scanning parameters and maximum temperature information from memory based on this tissue type. In another example, the operator manually selects and / or types in this information.

[0668] After retrieving the information in stage 6010, the thermal camera 5265 of the system 5200 captures a thermal image of the slide 605 in the slide holder 5240 in stage 6015. Based on the#3371089 OS <11 9-000025

[0669] 94

[0670] captured thermal image, the controller 410 of the system 5200 calculates the temperature averages at the areas of interest (AOI) on the slide 605 in stage 6020. For example, the areas of interest can include different locations around the center and edges of the slide 605 as well as various locations on the tissue sample 730. The controller 410 of the system 5200 then registers the temperature curves in stage 6025 at the areas of interest, and in stage 6030, the controller 410 determines the dryness level on the slide 605 based on the thermal curves. For example, the dryness level of the slide 605 can be determined in the manners as described above with respect to FIGS. 41, 42, and 43. Based on the estimates in stage 6030, the controller 410 of the system 5200 in stage 6035 determines whether or not the slide 605 is dry.

[0671] When the controller 410 of the system 5200 in stage 6035 determines the slide 605 is still wet, the controller 410 proceeds to stage 6040. The controller 410 of the system 5200 in stage 6040 calculates the laser power required in each area of interest on the slide 605. In one version, the controller 410 uses PID control algorithms to determine the power requirements at the various locations. The controller 410 in stage 6045 retrieves from memory or elsewhere a list of appropriate laser scan geometries and powers based on the requirements. When scanning in a loop pattern, such as in the manner depicted in FIGS. 40 and 56, the controller 410 retrieves a list of scan radii and pow ers at the different radii. Once this scanning information is retrieved in stage 6045, the controller 410 in stage 6050 controls the steerable mirror 5235 via the position control module 435 to scan the combined laser beam 5225 along a first beam path 5600 at a designated radius. For this first beam path 5600 at the first radius, the control ler 410 modulates the power of the heating laser 5205 via the power control module 430 in stage 6055. The power is selected based on the laser pow er designated for the particular beam path 5600 from stage 6045. For example, the center or radially inward beam paths 5600 in certain cases may have a higher power as compared to the peripheral or radially outward beam paths 5600, such as is shown in FIGS. 56 and 58.

[0672] The controller 410 in stage 6060 waits for the scanning loop or beam path 5600 for the combined laser beam 5225 to be completed. Once completed, the controller 410 of the system 5200 in stage 6065 uses the position control module 435 to position the steerable mirror 5235 to move to the next scanning loop or beam path 5600. As noted before, the scanning loops of the combined laser beam 5225 in one version are formed in a concentric#3371089 OS <11 9-000025

[0673] 95

[0674] outer radial direction to promote thermal sweeping of the water and / or wax in a radially outward direction, but in some cases, the laser scanning geometries and / or paths can be different than is illustrated. At the next scanning loop, the controller 410 in stage 6070 modulates the power of the heating laser 5205 based on the power set in stage 6045 for that loop.

[0675] In stage 6075. the controller 410 of the system 5200 determines whether or not scanning is completed. In other words, the controller 410 determines if additional scanning loops of the combined laser beam 5225 are required. If the combined laser beam 5225 needs to further heat the slide 605 by scanning along another beam path 5600 at a different radius, the controller 410 proceeds to stage 6065 so that additional scanning loops can be performed in a similar manner as described before. Otherwise, the con troller 410 of the system 5200 proceeds to stage 6015 to capture another thermal image of the slide 605 and process the slide 605 in a similar manner as described before. Once the controller 410 determines the slide 605 to be dry in stage 6035, the process in stage 6080 ends.

[0676] Glossary of Terms

[0677] The language used in the claims and specification is to only have its plain and ordinary meaning, except as explicitly defined below'. The words in these definitions are to only have their plain and ordinary meaning. Such plain and ordinary' meaning is inclusive of all consistent dictionary'' definitions from the most recently published Webster’s dictionaries and Random House dictionaries. As used in the specification and claims, the following definitions apply to these terms and common variations thereof identified below.

[0678] " About" with reference to numerical values generally refers to plus or minus 10% of the stated value. For example, if the stated value is 4.375, then use of the term "‘about 4.375” generally means a range between 3.9375 and 4.8125.

[0679] " Activator” generally refers to any non-metallic substance applied to one or both surfaces of two separate parts that binds them together and resists their separation. For example, an activator may be an adhesive and / or glue that can bond both mating surfaces through specific adhesion (e.g., molecular attraction), through mechanical anchoring (e.g., by flowing into holes in porous surfaces), and or through fusion (e.g., partial solution of both surfaces in the#3371089 OS <11 9-000025

[0680] 96

[0681] adhesive or its solvent vehicle). In a particular example, an activator can be a solvent for dissolving a pre-applied glue, which will then be cured by heat. Some non-limiting examples of activator include liquid activator, film activator, resin activator, rubber activator. limonene-based activator, silicone-based activator, mastics, metal-to-metal adhesives, plastic adhesives, rubber activator, sprayable activator, and hot melt activator.

[0682] " Adhesive" generally refers to any non-metallic substance applied to one or both surfaces of two separate parts that binds them together and resists their separation. For example, an adhesive can bond both mating surfaces through specific adhesion (e.g., molecular attraction), through mechanical anchoring (e.g., by flowing into holes in porous surfaces), and / or through fusion (e.g., partial solution of both surfaces in the adhesive or its solvent vehicle). Some non-limiting examples of adhesives include liquid adhesives, film adhesives, resin adhesives, rubber adhesives, silicone-based adhesives, mastics, metal-to-metal adhesives, plastic adhesives, rubber adhesives, sprayable adhesives, and hot melt adhesives, to name just a few.

[0683] " Air Knife" generally refers to a device that uses air or other gas to remove excess fluid, dry, and / or clean surfaces. 'Hie air knife in some cases uses a high-pressure gas (relative to ambient pressure) that is blown across the surface, and in other cases, the air knife draws a vacuum or creates suction (i.e., lower pressure relative to ambient pressure) along the surface so as to remove excess fluid, dry, and / or clean the surface. In one example, the air knife works by forcing a thin, high-velocity stream of air or other gas onto the surface. Air knives are commonly used for drying wet surfaces, removing dirt and other debris from surfaces, and blowing / vacuuming up excess materials, such as dust or chips, from surfaces. For example, the stream of air from the air knife can blow water or other liquids off the surface. In other examples, the air knife can vacuum the water or other liquids off the surface. In some (but not all) cases, the air stream further causes the water or other liquid to evaporate quickly. Typically, but not always, the air knife includes a compressed gas supply, a distribution chamber, and one or more nozzles. The compressed gas supply can include a compressor and / or an existing compressed gas system for supplying compressed gas, such as in the form of compressed air, ni trogen, or other gas, to the air knife. The distribution chamber inside the air knife collects the compressed gas and distributes the compressed gas to the nozzles. In some cases, the distribution chamber may include internal baffles or#3371089 OS <11 9-000025

[0684] 97

[0685] restrictors to control the flow and / or velocity of the gas blown from the air knife. The nozzles are typically narrow openings coupled to the distribution chamber through which the compressed gas is expelled. Depending on the requirements of the gas stream, the nozzles are shaped, sized, and arranged to form various gas flow patterns. In some cases, the air knife may further include a silencer to reduce the noise level created by the air knife.

[0686] " Barcode" generally refers to a visible arrangement of shapes, colors, lines, dots, or symbols fixed in some medium and arranged on the medium in a pattern configured to encode data. Examples include optical machine-readable representations of data relating to an object to which the barcode is attached such as a Universal Produce Code (UPC), or any visible patterns related to any type of Automatic Identification and Data Capture (AIDC) system. Another example of a barcode is a Quick Response Code (QR Code) which arranges various light and dark shapes to encode data. In still yet another example, the barcode is an ArUco marker. Any suitable medium is envisioned. Examples include an adhesive label, a physical page, a display device configured to display the barcode, or any other object such as a box, a machine, or other physical structure to which the barcode is affixed or upon which it is printed. For example, a barcode may be etched into metal, machined into plastic, or formed by organizing visible three-dimensional shapes into a pattern. The barcode may not be visible to humans but may be fixed using a substance or device that allows the barcode to be visible to sensors in a machine configured to read wavelengths of light outside those detectable by the human eye. Examples of this type of barcode include barcodes printed with ink that is only visible under ultraviolet (i.e., "black") light, or barcodes displayed using infrared light.

[0687] " Beam Expander" generally refers to an optical device or system that enlarges the size or diameter of a collimated laser beam while proportional ly reducing its divergence angle. Hie beam expander enables better beam propagation over long distances. The beam expander is also commonly used to match to optical apertures and / or reduce power density to prevent damage in high-power applications. A common structure for a beam expander includes two lenses arranged in a telescope configuration, an input lens that diverges the incoming rays and an output lens that recollimates them at a magni fied scale. The input lens spreads the beam and the output lens ensures the expanded beam remains parallel w ithout introducing significant aberrations. One example of a type of beam expander includes a Gali lean beam expander, which uses a diverging (negative) lens followed by a converging (positive) lens to#3371089 OS <11 9-000025

[0688] 98

[0689] avoid an internal focus and minimize risks in high-power lasers. Another type of beam expander includes a Keplerian beam expander. The Keplerian beam expander employs two converging lenses with an intermediate real focus that allows spatial filtering but can create intensity hotspots. Still yet another type of beam expander includes a reflective beam expander. The reflective beam expander utilizes curved mirrors for high-power or broadband applications without chromatic issues. A further type or example of a beam expander includes variable or zoom type beam expanders that have adjustable lens spacing for tunable magnification,

[0690] " Beam Shaper" generally refers to an optical device or system that redistributes the intensity profile of a laser beam from i ts typical Gaussian (bell-shaped) di stri bution to a more uniform or customized shape, such as a flat-top shape, to enhance uniformity. For example, the beam shaper can be used in applications like material processing, illumination, or lithography. A common structure of beam shapers in refractive designs includes two aspheric lenses, a first lens (often di erging) for mapping input rays to new radial positions for intensity equalization and a second lens (converging) for collimating the output and maintaining a flat wavefront to achieve energy -conserving redistribution with high efficiency, Some examples of different types of beam shapers include refractive beam shapers, diffractive beam shapers, diffusive or integrating beam shapers, and freeform or adaptive beam shapers. Refractive beam shapers, such as the πShaper brand series of beam shapers sold by AdlOptica Optical Systems GmbH of Berlin, Germany, typically use aspheric lens pairs for near-lossless Gaussian-to-flat-top conversion in collimated beams. Diffractive beam shapers normally employ diffractive optical elements (DOEs) with micro -structured surfaces to create complex patterns like lines, rings, or arrays via phase modulation and interference. Some types of diffractive optical elements include computer-generated holograms (CGHs), Diffusive or integrating beam shapers, such as microlens arrays or rod homogenizers, normally scatter and overlap rays for uniformity but may introduce speckle or lower efficiency. Freeform or adaptive beam shapers typically use deformable mirrors or spatial light modulators for dynamic, customizable intensity profiles in advanced systems,

[0691] " Camera" generally refers to a device that records visual images. Typically, a camera may record two- and or three-dimensional images. In some examples, images are recorded in the form of fi lm, photographs, image signals, and / or video signals. A camera may inchide one or#3371089 OS <11 9-000025

[0692] 99

[0693] more lenses or other devices that focus light onto a light-sensitive surface, for example a digital light sensor or photographic film. The light-sensitive surface may react to and be capable of capturing visible light or other types of light, such as infrared (IR) and / or ultraviolet (UV) light.

[0694] '’Channel" generally refers to a long, narrow groove in a surface of an object.

[0695] " Cleansing agent” typically refers to a substance used to remove dirt, impurities, and contaminants from surfaces. Cleansing agents can be found in various forms, including liquids, gels, and powders, and often contain surfactants that help break down oils and debris. In one example, the cleansing agent is a commonly used liquid dishwashing soap, but other types of cleansing agents can be used. For instance, the cleansing agent can include other types o f soaps, detergents, surfactants, and the li ke. In certain histological processes, the cleansing agent is a citrate buffer in a glycol ether and water solution configured to clean hematoxy lin lines and staining anifolds. In some other histological processes, the cleansing agent is diluted hydrochloric acid in propylene glycol and water solution.

[0696] " Conductor" generally refers to a material that allows energy in the form of heat, to transfer within the material, without any movement of the material itself. Put differently, conductors allow efficient transfer of energy in the form of heat. To name just a few' examples, conductors can include metals, such as copper, iron, gold, silver, aluminum, titanium, mercury, and / or steel.

[0697] " Controller” generally refers to a device, using mechanical, hydraulic, pneumatic electronic techniques, and / or a microprocessor or computer, which monitors and physically alters the operating conditions of a given dynamical system. In one non-limiting example, the controller can include an Allen Bradley brand Programmable Logic Controller (PLC). A controller may include a processor for performing calculations to process input or output. A controller may include a memory for storing values to be processed by the processor, or for storing the results of previous processing. A controller may also be configured to accept input and output from a wide array of input and output devices for recei ving or sending values. Such devices include other computers, keyboards, mice, visual displays, printers, industrial equipment, and sy stems or machinery of all types and sizes. For example, a controller can#3371089 OS <11 9-000025

[0698] 100

[0699] control a network or network interface io perform various network communications upon request. The network interface may be part of the controller or characterized as separate and remote from the controller. A controller may be a single, physical, computing device such as a desktop computer, or a laptop computer, or may be composed of multiple devices of the same type such as a group of servers operating as one device in a networked cluster, or a heterogeneous combination of different computing devices operating as one controller and linked together by a communication network. The communication network connected to the controller may also be connected to a wider network such as the Internet. Thus, a controller may include one or more physical processors or other computing devices or circuitry and may also include any suitable type of memory. A controller may also be a virtual computing platform having an unknown or fluctuating number of physical processors and memories or memory devices. A controller may thus be physically located in one geographical location or physically spread across several widely scattered locations with multiple processors linked together by a communication network to operate as a single controller. Multiple control lers or computing devices may be configured to communicate with one another or with other devices over wired or wireless communication links to form a network. Network communications may pass through various controllers operating as network appliances such as switches, routers, firewalls or other network devices or interfaces before passing over other larger computer networks such as the Internet. Communications can also be passed over the network as wireless data transmissions carried over electromagnetic waves through transmission lines or free space. Such communications include using Wi-Fi or other Wireless Local Area Network (WLAN) or a cellular transmitter / receiver to transfer data.

[0700] " Controller" generally refers to a device, using mechanical, hydraulic, pneumatic electronic techniques, and / or a microprocessor or computer, which monitors and physically alters the operating conditions of a given dynamical system. For example, the controller may be configured to control the behavior of another mechanical and / or electronic device. A controller may include a “control circuit” configured to provide signals or other electrical impulses that maybe received and interpreted by the controlled device to indicate how the controlled device should behave. A controller may include a processor for performing calculations to process input or output. A controller may include a memory for storing values to be processed by the processor, or for storing the results of previous processing, A controller may also be configured to accept input and output from a wide array of input and#3371089 OS <11 9-000025

[0701] 101

[0702] output devices for receiving or sending values. A controller may also be a virtual computing platform having an unknown or fluctuating number of physical processors and memories or memory devices. A controller may thus be physically located in one geographical location or physically spread across several widely scattered locations with multiple processors linked together by a communication network to operate as a single controller. Multiple control lers or computing devices may be configured to communicate with one another or with other devices over wired or wireless communication links to form a network,

[0703] ’'Coverslip" or " Covertile" generally refers to a thin, flat piece of glass or pl astic placed over a slide to protect the specimen and / or create a uniform thickness for histological analysis. The covertile facilitates flattening the tissue, reduces light refraction, and prevents contamination while allowing for clearer visualization of the sample under histological analysis. In some instances, the covertile may contain a pre-applied glue in dried form that can be activated by a solvent.

[0704] " Fluid" generally refers to a substance that docs not have a fixed shape. For example, a fluid includes a liquid nd'or a gas. Typically, fluids arc able to flow easily, such as air flowing over a wing, blood flowing through a circulatory system, water flowing through plumbing, or oil flowing through a motor as examples. In some cases, a fluid refers to a mixture of solids, liquids, and / or gases. For example, a slurry of solids and w-'ater, liquid droplets mixed with air. aerated solid particles, a mixture of solids with liquids and gases, and or other mixtures of different materials may be fluids.

[0705] " Gap" generally refers to a space between objects, surfaces, or points.

[0706] " Glass" generally refers to an amorphous material formed from a melt by cooling to rigidity without crystallization. Glass is usually a hard, brittle substance. Typically, but not always, glass is transparent or translucent. Glass is normally made by fusing sand with soda, lime. and sometimes other ingredients and cooling rapidly. Glass can occur synthetically or naturally. For example, glass can be naturally made as obsidian that is produced by fast cooling of magma.#3371089 OS <11 9-000025

[0707] 102

[0708] " Insulator" generally refers to a material and / or structure that has a low thermal conductivity. Put differently, an insulator is a material and / or structure that does not conduct heat well. For example, insulators can be made from glass, porcelain, and / or plastic materials, to name just a few. Insulator structures for example can include air gaps and / or vacuums to minimize thermal conduction.

[0709] The term " Laser" generally refers to any device or system that generates and emits electromagnetic radiation through the process of stimulated emission, resulting in a coherent, collimated, and often monochromatic beam of energy. The word "laser" is an acronym for " Light Amplification by Stimulated Emission of Radiation." A laser can include devices that produce electromagnetic radiation across a broad range of frequencies, including but not limited to ultraviolet, visible, infrared, microwave, and radio frequencies. This encompasses traditional optical lasers, such as diode lasers, solid-state lasers, and gas lasers (for example, carbon dioxide lasers emitting in the infrared spectrum), as well as devices that generate coherent radiation at microwave frequencies, commonly known as masers (" Microwave Amplification by Stimulated Emission of Radiation"). Some nonlimiting examples of lasers include carbon dioxide lasers, diode lasers, fiber lasers, Nd:YAG lasers, excimer lasers, ultraviolet lasers, visible light lasers, infrared lasers, and masers, to name just a few'.

[0710] " Lateral" generally refers to being situated on, directed toward, or coming from the side.

[0711] "Light" generally refers to electromagnetic radiation having any wavelength. Among other examples, light includes visible light, infrared, ultraviolet, gamma rays. X-rays, microwaves, and radio waves,

[0712] " Liquid" generally refers to a fluid that has no independent shape but has a definite volume and does not expand indefinitely and that is only slightly compressible.

[0713] " Longitudinal" general ly refers to the length or lengthwise dimension of an object, rather than across.

[0714] " Machine-Readable Identifier" generally refers to a marking, structure, and / or device that is readable by an electronic device such as by a computer with an optical and / or#3371089 OS <11 9-000025

[0715] 103

[0716] electromagnetic scanner. Typically, but not always, the machine-readable identifier identifies the object and / or some property of the object to which the machine-readable identifier is associated. Unless great effort is used, the machine-readable identifier is not easily read and / or understood by a human being. Some examples of machine-readable identifiers include barcodes. Quick Response (QR) codes, and / or Radio-Frequency Identification (RFID) tags, to name just a few.

[0717] " Mounting Sol vent" generally refers to a type of solvent incorporated into a mounting media that is used during adhering a coverslip to a slide with a stained tissue sample.

[0718] " Nozzle" generally refers to a device configured to control the flow and direction of a fluid such as a liquid or gas. The nozzle normally has one or more narrow openings that reduce the cross-sectional area of a stream of the fluid, thereby increasing the veloci ty and pressure of the fluid discharged from the nozzle. Nozzles can discharge the fluid in various forms such as in a spraying pattern to create droplets or a fine mi st, a jet pattern that produces a high velocity stream of the fluid, or an atomization pattern where the nozzle breaks up the fluid into tiny droplets. Some common types of nozzles include, bu t are not limi ted to. cone, hollow-cone, full-cone, fan, and jet nozzles.

[0719] " Opaque" generally refers to a material and / or article that has the physical property of blocking light or other forms of electromagnetic radiation from passing through the material. The material can be in the form of a solid, liquid, or gas. An opaque material is nei ther transparent nor translucent. Whether a material is opaque typically depends on the wavelength of the light and the nature of the material. For instance, some kinds of glass, while transparent in the visible light range, are largely opaque to ultraviolet light.

[0720] " Parallel" generally refers to coplanar straight lines in two-dimensional space or planes in the same three-dimensional space that never intersect and / or otherwise never meet.

[0721] " Plastic" generally refers to a synthetic or semi-synthetic material made from a wide range of organic polymers, such as polyethylene, PVC, nylon, and the like. Typically, but not always, plastics are mostly thermoplastic or thermosetting polymers of high molecular weight and#3371089 OS <11 9-000025

[0722] 104

[0723] that can be made into objects, films, or filaments. In some cases, plastics can be molded into shape while soft and then set into a rigid or slightly elastic form.

[0724] " Power Cable" generally refers to a cable configured to transfer electrical power as part of an electrical circuit. A power cable may be used exclusively to transfer power, or it may be used to also transfer signals, such as in the case of a Power Line Communication (PLC) system.

[0725] " Pre-Glued Coverslip" generally refers to a type of coverslip that comes with an adhesive already applied to one side of the coverslip. This adhesive allows for easier and quicker mounting of the coverslip onto a slide, The pre-applied glue helps to secure the coverslip in place. Once adhered to the slide, the pre-glued coverslip protects the tissue sample and ensures that the tissue sample remains flat and intact for examination.

[0726] " Prepared Slide" generally refers to a type of slide at a state after a tissue sample is applied to the slide and the coverslip has been mounted over the tissue sample on the slide. The prepared slide has been processed so that the tissue sample is able to be properly viewed under a microscope or similar device.

[0727] " Proportional-Integral-Derivative (PID) Controller" generally refers to a control loop feedback type mechanism or algorithm that uses feedback in an attempt to minimize error by adjusting process control inputs. PID controllers generally use three control tuning parameters, the proportional (P), integral (I), and derivative ( D) tuning parameters. PID controllers are a common architecture for implementing closed loop speed control (e.g,, cruise control) or other types of controls. The PID control ler continuously calculates an error value (e.g., e(t)) as the difference between a desired setpoint (SP) and a measured process variable (PV) and applies a correction based on proportional (P), integral (I), and derivative (D) control terms. The PID controller is configured to attempt to minimize error over time by adjusting a control variable (e.g., u(t)) to a new value via a weighted sum of the control terms. Loop tuning is used to balance the effects of the control terms to create an optimal control function through tuning constants (K), Several different techniques can be used to tune a PID control loop. These tuning techniques can include, but are not limited to, manual tuning methods, Ziegler-Nichols tuning methods, Tyreus Luybert tuning methods, software#3371089 OS <11 9-000025

[0728] 105

[0729] tuning methods, Cohen-Coon timing methods, relay tuning methods, and simple control rule (SIMC) tuning methods, to name just a few.

[0730] " Slide" general ly refers to a thin piece of fully or mostly transparent material, like glass, quartz, or plastic, that supports one or more objects for visual examination such as under a microscope. An example of a standard microscope slide is a flat, rectangular piece of glass having the dimensions of 75 mm by 26 mm with a thickness of about 1 mm, but slides in other examples can be shaped and dimensioned differently as well as can be made from other materials. The slide is typically transparent or clear, but some parts of the slide may contain translucent or even opaque sections. For instance, the slide can be frosted or coated with enamel to facilitate labelling and / or writing on the slide. Graticule slides, for example, are typically marked with grid lines to facilitate counting and / or sizing objects on the slide such as for cell counting. The slide may further have a special coating such as to enhance chemical inertness and / or promote cell adhesion. While slides are normally flat, some slides may contain shallow depressions or wells, such as in the case of concavity slides or cavity slides, that hold a specimen or other object in place. Often, the object, such as a biological specimen, is held in place using a small transparent cover like a glass cover slip. The specimen can be mounted on the slide in several ways such as using dry mount, wet mount, prepared mount, and / or strewn mount techniques.

[0731] ''Solvent” generally refers to a substance, typically a liquid, that is used to dissolve other substances so as to form a solution. In the context of tissue analysis, the solvent is a substance used to dissol ve other materials during the preparation of tissue samples for microscopic examination. Solvents can play a significant role in the various tissue processing stages. During dehydration of the tissue samples, sol vents can be used to remove water from the tissue samples. In the clearing stage, solvents for instance can be used to remove solvents previously applied during the dehydration stage, and solvents can be further used to make parts (or even all) of the tissue sample transparent. During staining, solvents can be used to help in the application and remo val of stains to highlight different tissue components in the tissue sample. Solvents may also be used during coverslipping to facilitate proper mounting of a coverslip or covertile to the slide to cover the tissue sample.#3371089 OS <11 9-000025

[0732] 106

[0733] " Stain" generally refers to a dye or other chemical substance used to enhance the visibility of specific structures and / or components within tissue samples. For example, the stain selectively binds to particular cellular elements or components to enhance contrast and / or visibility so as to differentiate structures within the tissue sample. The stain allows researchers, pathologists, and others to distinguish and study different parts of the tissue sample under a microscope or other device. The stained tissue samples for example are used for identifying cellular morphology, diagnosing diseases, and conducting detailed biological research.

[0734] " Temperature Sensor" or " Thermometer" generally refers to a device or instrument that measures temperature or a temperature gradient. The thermometer can include empirical or absolute type thermometers as well as primary or secondary based thermometers. Some non¬ limiting examples of thermometers include thermometers using thermal expansion, pressure, density, optical, electrical resistance, electrical potential, and / or electrical resonance techni ques for measuring temperature.

[0735] " Tissue Sample" or " Specimen" generally refers to a small piece of biological material that has been collected from a living or deceased organism for the purpose of examination such as under a microscope. These samples are typically obtained through procedures such as biopsies, surgeries, or autopsies. The tissue sample is typically prepared and examined to study cel lular structure, abnormalities, and composition of the biological material. This examination may help with diagnosing diseases, understanding biological processes, and conducting research.

[0736] " Translucent" generally refers to a materia! and / or article that has the physical property'’ of allowing light or other forms of electromagnetic radiation to pass through the material but appreciably scatters the light so that objects beyond cannot be seen clearly. The material can be in the form of a solid, liquid, or gas. A transparent ma terial is generally made up of components with different indices of refraction. Whether a material is translucent typically depends on the wavelength of the light and the nature of the material. Some examples of translucent materi als include some forms of glass and plastics.#3371089 OS <11 9-000025

[0737] 107

[0738] " Transparent" generally refers to a material and / or article that has the physical property of allowing light or other forms of electromagnetic radiation to pass through the material without appreciable scattering of light. The material can be in the form of a solid, liquid, or gas. A transparent material is generally made up of components with a uniform index of refraction. Transparent materials appear clear, with the overall appearance of one color, or any combination leading up to a brilliant spectrum of every color. Whether a material is transparent typically depends on the wavelength of the light and the nature of the material. Some examples of transparent materials include some forms of glass and plastics as well as air and liquid water.

[0739] " Transverse" generally refers to things, axes, straight lines, planes, or geometric shapes extending in a non-parallel and / or crosswise manner relative to one another. For example, when in a transverse arrangement, lines can extend at right angles or perpendicular relative to one another, but the lines can extend at other non- straight angles as well such as at acute, obtuse, or reflex angles. For instance, transverse lines can also form angles greater than zero (0) degrees such that the lines are not parallel. When extending in a transverse manner, the lines or other things do not necessarily have to intersect one another, but they can.

[0740] " Window" generally refers to an opening or generally transparent panel in a wall of an object that facilitates interior viewing of the object.

[0741] It should be noted that the singular forms "a," "an," "the," and the like as used in the description and / or the claims include the plural forms unless expressly discussed otherwise. For example, if the specification and / or claims refer to "a device" or "the device", it includes one or more of such devices.

[0742] It should be noted that directional terms, such as "up," "down," "top," "bottom," "lateral," "longitudinal," "radial." "circumferential," "horizontal," "vertical," etc., are used herein solely for the convenience of the reader in order to aid in the reader's understanding of the illustrated embodiments, and it is not the intent that the use of these directional terms in any manner limit the described, illustrated, and / or claimed features to a specific direction and / or orientation.#3371089 OS <11 9-000025

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[0744] It is noted that terms like "preferably ’ "commonly," and "typically" are not utilized herein to limit the scope of the claimed embodiments or to imply that certain features are critical, essential, or even important to the structure or function of the claimed embodiments. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure.

[0745] For the purposes of describing and defining the present disclosure, it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0746] While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes, equivalents, and modifications that come within the spirit of the inventions defined by the following claims are desired to be protected. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and set forth in its entirety herein.

[0747] Reference Numbers

[0748] 100 system

[0749] 102 modules

[0750] 105 input module

[0751] 110 staining module

[0752] 115 coverslip module

[0753] 120 imaging module

[0754] 205 stations

[0755] 210 laser#3571089 054149-000025

[0756] 109 215 laser beam

[0757] 220 baking station

[0758] 225 dewaxing station

[0759] 230 antigen retrieval station

[0760] 235 curing station

[0761] 300 chart

[0762] 305 standard processing workflow310 laser processing workflow400 laser module

[0763] 405 laser head

[0764] 410 controller

[0765] 415 target

[0766] 420 beam generator

[0767] 425 beam positioner

[0768] 430 power control module

[0769] 435 position control module

[0770] 440 communication module

[0771] 445 temperature sensor

[0772] 450 coverslip

[0773] 500 graph

[0774] 605 slide

[0775] 610 spot#3571089 054149-000025

[0776] 110 615 spot radius

[0777] 700 heating station

[0778] 705 position adjuster

[0779] 710 double arrow

[0780] 715 lens

[0781] 720 beam path

[0782] 725 mirror

[0783] 730 tissue sample

[0784] 735 sample surface

[0785] 740 base surface

[0786] 745 slide holder

[0787] 800 graph

[0788] 805 line

[0789] 810 line

[0790] 815 line

[0791] 820 line

[0792] 900 graph

[0793] 905 line

[0794] 910 line

[0795] 915 line

[0796] 920 line

[0797] 1000 image#3571089 054149-000025

[0798] in 1005 first row

[0799] 1010 second row

[0800] 1015 third row¬ 1 100 laser system

[0801] 1105 beam expander

[0802] 1110 field of view

[0803] 1115 dispenser head

[0804] 1120 nozzle

[0805] 1125 solvent

[0806] 1130 air knife

[0807] 1135 longitudinal axis

[0808] 1305 slide holder

[0809] 1310 beam aperture

[0810] 1315 posts

[0811] 1320 gap

[0812] 1325 drain channels

[0813] 1405 arrows

[0814] 1600 graph

[0815] 1605 line

[0816] 1610 line

[0817] 1615 line

[0818] 1620 line#3371089 OS <11 9-000025

[0819] 112 1900 image

[0820] 2000 image

[0821] 2105 mounting media

[0822] 2110 heated area

[0823] 2205 absorptive material

[0824] 2210 heated area

[0825] 2300 laser system

[0826] 2305 base

[0827] 2310 slide holder

[0828] 2315 tape

[0829] 2320 power cable

[0830] 2325 enclosure

[0831] 2330 window

[0832] 2500 diagram

[0833] 2505 control process

[0834] 2510 test process

[0835] 2605 slide assembly

[0836] 2610 processing parameters

[0837] 2615 absorptive bars

[0838] 2705 slide assembly

[0839] 2710 absorptive material

[0840] 2715 machine -readable identifier#3571089 054149-000025113 2805 slide assembly

[0841] 2810 absorptive material

[0842] 2815 dots

[0843] 2900 image

[0844] 3000 image

[0845] 3100 chart

[0846] 3200 image

[0847] 3300 image

[0848] 3400 image

[0849] 3500 image

[0850] 3600 laser system

[0851] 3605 holder assembly

[0852] 3610 distance adjuster

[0853] 3615 holder body

[0854] 3620 slide holder

[0855] 3625 sensor holder

[0856] 3630 beam aperture

[0857] 3635 drain channel

[0858] 3640 drain funnel

[0859] 3700 laser system

[0860] 3705 holder assembly

[0861] 3710 distance adjuster#3571089 054149-000025

[0862] 114 3715 holder body

[0863] 3720 slide holder

[0864] 3725 sensor holder

[0865] 3730 beam aperture

[0866] 3735 drain channel

[0867] 3740 drain funnel

[0868] 3745 base

[0869] 3750 stand

[0870] 3755 rest surface

[0871] 3800 graph

[0872] 3900 adaptive laser heating system 3905 slide holder

[0873] 3910 tag

[0874] 3915 puddle

[0875] 4000 laser scan path

[0876] 4005 arrow

[0877] 45100 diagram

[0878] 4105 PID controller

[0879] 4110 set point

[0880] 4115 graph

[0881] 4200 graph

[0882] 4205 time axis#3571089 054149-000025115 4210 temperature axis

[0883] 4215 power axis

[0884] 4220 temperature data line

[0885] 4225 power data line

[0886] 4230 power drop line

[0887] 4300 graph

[0888] 4320 temperature data line

[0889] 4325 power data line

[0890] 4400 antigen retrieval system

[0891] 4405 cell conditioning reagent 4410 liquid coverslip

[0892] 4500 optics system

[0893] 4505 beam expander

[0894] 4510 field stop

[0895] 4515 beam shaper

[0896] 4520 input beam waist

[0897] 4525 expander beam waist

[0898] 4530 field of view

[0899] 4605 diverging lens

[0900] 4610 converging lens

[0901] 4615 input aspheric lens

[0902] 4620 output aspheric lens#3571089 054149-000025

[0903] 116 4700 diagram

[0904] 4800 diagram

[0905] 4900 diagram

[0906] 5000 diagram

[0907] 5005 optics system

[0908] 5010 field stop

[0909] 5015 beam shaper

[0910] 5020 input lens

[0911] 5025 output lens

[0912] 5030 field of view

[0913] 5100 diagram

[0914] 5200 system

[0915] 5205 heating laser

[0916] 5210 visible laser

[0917] 5215 visible laser beam

[0918] 5220 beam combiner

[0919] 5225 combined laser beam

[0920] 5230 focus lens

[0921] 5235 steerable mirror

[0922] 5240 slide holder

[0923] 5245 holder cap

[0924] 5250 fan#3571089 054149-000025

[0925] 117 5255 intake port

[0926] 5260 exhaust port

[0927] 5265 thermal camera

[0928] 5270 slide sensor

[0929] 5275 DAQ unit

[0930] 5600 beam paths

[0931] 5900 flowchart

[0932] 5905 stage

[0933] 5910 stage

[0934] 5915 stage

[0935] 5920 stage

[0936] 5925 stage

[0937] 5930 stage

[0938] 5935 stage

[0939] 5940 stage

[0940] 5945 stage

[0941] 5950 stage

[0942] 5955 stage

[0943] 5960 stage

[0944] 5965 stage

[0945] 5970 stage

[0946] 5975 stage054149-000025

[0947] #3571089

[0948] 118 5980 stage

[0949] 5982 stage

[0950] 5984 stage

[0951] 5986 stage

[0952] 5988 stage

[0953] 5990 stage

[0954] 5995 stage

[0955] 6000 flowchart

[0956] 6005 stage

[0957] 6010 stage

[0958] 6015 stage

[0959] 6020 stage

[0960] 6025 stage

[0961] 6030 stage

[0962] 6035 stage

[0963] 6040 stage

[0964] 6045 stage

[0965] 6050 stage

[0966] 6055 stage

[0967] 6060 stage

[0968] 6065 stage

[0969] 6070 stage#3571089 054149-000025119 6075 stage

[0970] 6080 stage

Claims

1. #3571089 054149-000025120CLAIMSWhat is claimed is:

1. A system, comprising:a laser configured to produce a laser beam;a controller configured to control the laser; andwherein the laser beam is configured to create heat to prepare for analysis of a tissue sample on a slide.

2. The system of claim 1, wherein the laser is configured to bake the tissue sample on the slide.

3. The system of claim 2, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite to the sample surface; andthe laser beam is configured to heat the base surface of the slide.

4. The system of claim 3, wherein:the slide is made of glass; andthe laser includes an infrared type laser.

5. The system of claim 4, wherein:the laser is a carbon dioxide type laser: andthe laser beam has a wavelength of about 10.6 micrometers.

6. The system of claim 3, further comprising:a temperature sensor configured to measure temperature of the base surface of the slide.

7. The system of claim 6, wherein the controller is configured to control the laser based on the temperature sensed by the temperature sensor.#3371089 OS <11 9-0000251218. 'Hie system of claim 2, wherein the laser is configured to perform a flash baking technique.

9. The system of claim 2, wherein the laser is configured to perform a gentle baking technique.

10. The system of claim 1, wherein the laser is configured to dewax the tissue sample on the slide.

11. The system of claim 10, further comprising:a beam expander configured to defocus the laser beam.

12. The system of claim 11, wherein the beam expander includes a tube assembly.

13. The system of claim 11, further comprising;a slide holder being integrated with the beam expander.

14. The system of claim 13, wherein the slide holder is configured to engage the comers of the slide.

15. The system of claim 14, wherein:the slide holder includes one or more posts; andthe posts are positioned to support the comers of the slide.

16. 'Hie system of claim 15, wherein the posts are configured to form an air gap between the holder and the slide.

17. The system of claim 13, wherein:the holder defines a beam aperture; andthe laser beam shines through the beam aperture.

18. The system of claim 17. wherein:the holder defines a drain channel;#3371089 OS <11 9-000025122the drain channel extends around the beam aperture; andthe drain channel is configured to drain wax from the slide.

19. The system of claim 13, further comprising:a nozzle configured to dispense a solvent,20. The system of claim 19, further comprising:an air knife configured to sweep a mixture of the solvent and wax from the slide.

21. The system of claim 1, wherein the laser is configured to facilitate antigen retrieval.

22. The system of claim 1, wherein:the slide is configured to receive a coverslip; andthe laser is configured to cure the coverslip,23. The system of claim 22, wherein the coverslip is a pre-glued coverslip.

24. The system of claim 22, wherein the laser beam is configured to heat the coverslip.

25. Idle system of claim 24, wherein:the coverslip is at least partially covered with an absorptive material; andthe laser beam is configured to heat the absorptive material.

26. The system of claim 25, wherein:the laser is a visible light laser; andthe absorptive material is configured to absorb visible light,27. The system of claim 25, wherein:the laser is an ultraviolet type laser; andthe absorpti ve material is configured to absorb ultraviolet light.28, The system of claim 25, wherein the absorptive material includes black ink.#3371089 OS <11 9-00002512329. The system of claim 25, wherein the absorptive material is in the form of a machine- readable identifier.

30. The system of claim 25, wherein:the absorptive material includes one or more absorptive bars; andthe absorptive bars cover one or more edges of the coverslip.

31. The system of claim 25, wherein:the absorptive material includes one or more dots; andthe dots are positioned at the corners of the coverslip.

32. The system of claim 25, wherein the absorptive material is posi tioned to not obstruct visibility during tissue analysis.

33. The system of claim 1, wherein:the laser includes a lens; andthe lens is configured to project the laser beam with a flat focal plane.

34. The system of claim 33, wherein the lens includes an F-Theta lens,35. The system of claim 1, further comprising:a module configured to bake and dewax at the same time.

36. The system of claim 1, further comprising:a module configured to bake, dewax, and facilitate antigen retrieval in a single integrated unit.

37. The system of claim 1, wherein:the laser is configured to scan the laser beam along a laser scan path; andthe laser scan path generates thermal gradients to sweep excess humidity from the center of the slide to the edges of the slide.#3371089 OS <11 9-00002512438. The system of claim 1, wherein the controller is confi gured to perform closed loop temperature control.

39. The system of claim 1, further comprising:a temperature sensor operatively coupled to the controller;wherein the controller includes a proportional -integral-derivative (PID) controller; wherein the PID controller is configured to receive temperature feedback from the temperature sensor; andwherein the PID controller is configured to regulate power of the laser to achieve a temperature set point,40. The system of claim 39, wherein the controller is configured to detect dryness of the slide based on a threshold reduction in the power of the laser required to maintain the temperature set point.

41. The system of claim 1, further comprising:the slide;the tissue sample disposed on the slide;a cell conditioning reagent covering the tissue sample on the slide;wherein the cell conditioning reagent includes a high-temperature cell conditioning liquid; a liquid coverslip covering the cell conditioning reagent; andwherein the liquid coverslip is configured to reduce evaporation of the cel! conditioning reagent during heating with the laser beam.

42. The system of claim 1, further compri sing:an optical system configured to expand and flatten the laser beam; andwherein the optical system is disposed between the laser and the slide.

43. The system of claim 1, further comprising:a beam shaper configured to redistribute the intensity profile of the laser beam.

44. The system of claim 43, further comprising;a beam expander configured to expand the laser beam;#3371089 OS <11 9-000025125a field stop configured to clip the laser beam expanded by the beam expander; and wherein the field stop is located between the beam expander and the beam shaper.

45. The system of claim 44, wherein:the beam shaper is configured to reshape the laser beam; andthe beam shaper is configured to produce the laser beam with a rectangular shape.

46. The sy stem of claim 1, further comprising:a thermal camera configured to monitor a spatial temperature distribution across the slide including.

47. The system of claim 1, further comprising:a slide holder including a slide cap configured to cover the slide.

48. The system of claim 47. wherein:the slide holder includes a fan; andthe fan is configured to blow a gas across the slide.

49. The system of claim 48, wherein the slide hol der is configured to orient the slide at a transverse angle to promote drainage.

50. The sy stem of claim 1, further comprising:a visible laser configured to produce a visible laser beam; anda beam combiner configured to combine the visible laser beam with the laser beam from the laser to produce a combined laser beam.

51. The system of claim 1, wherein:the slide is at least partially covered with an absorptive material; andthe absorptive material is configured to absorb wavelengths outside those produced by carbon dioxide lasers.

52. The system of claim 1, further comprising:a beam shaper including a diffractive optical element (DOE).#3371089 OS <11 9-00002512653, The system of claim 52, wherein the diffractive optical element includes a computer¬ generated hologram (CGH).54, The system of claim 1, further comprising:a beam expander configured to expand the laser beam;a beam shaper configured to redistribute the intensity profile of the laser beam; and wherein the beam expander and the beam shaper are integrated together in a single computergenerated hologram.55, A method, comprising:placing a tissue sample on a slide; andprocessing the tissue sample on the slide for analysis by heating with a laser beam,56. The method of claim 55, wherein the processing includes baking the tissue sample on the slide,57. fhe method of claim 56, wherein the baking includes heating the slide by shining the laser beam onto the slide.

58. The method of claim 57, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite to the sample surface; andthe baking includes heating the slide by shining the laser beam onto the base surface of the slide,59. The method of claim 56, wherein the baking includes flash baking the slide w ith the laser beam.60, The method of claim 59, further comprising:generating the laser beam at 40% power:#3371089 OS <11 9-000025127scanning the laser beam with a square beam path:wherein the square beam path has 0.25 mm spacing;scanning the laser beam at a scanning speed of about 800 mm / s; andheating with the laser beam until a peak temperature is reached.

61. The method of claim 60, wherein the peak temperature is 120 degrees Celsius.

62. The method of claim 56, further compri sing:measuring temperature of the slide with a temperature sensor.

63. Idle method of claim 56, wherein the baking includes gentle baking the slide with the laser beam.

64. The method of claim 56, further comprising:generating the laser beam with at most 5% pow er;scanning the laser beam with a square beam path;scanning the laser beam at a scanning speed of about 3.500 mm / s; andheating with the laser beam until a steady state temperature is reached.

65. Hie method of claim 64, wherein the steady state temperature is about 60 degrees Celsius.

66. The method of claim 56, further comprising:heating the slide with the laser beam for at most 20 seconds.

67. file method of claim 56, further comprising:dewaxing by heating the slide with the laser beam.

68. The method of claim 67, further comprising:performing the baking and the dewaxing simultaneously.

69. The method of claim 55, wherein the processing includes dewax ing the tissue sample on the slide.#3371089 OS <11 9-00002512870. The method of claim 69, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite to the sample surface; andthe dewaxing incl udes heating the base surface of the slide with the laser beam.

71. fhe method of claim 69, wherein the dewaxing includes heating the slide with the laser beam using a flash heating process.

72. The method of claim 69, wherein the dewaxing includes heating the slide with the laser beam using a gentle heating process.

73. The method of claim 69, further comprising:wherein the dewaxing includes melting wax in the tissue sample with heat from the laser beam;wherein the dewaxing includes applying a solvent to the tissue sample; anddraining a mixture of the solvent and the wax from the slide,74. The method of claim 73, further comprising:sweeping the mixture of the wax and the sol ent with an air knife.

75. The method of claim 55, further comprising:applying a transfer fluid with a vaporization temperature above 100 degrees Celsius to the slide to form a puddle; andirradiating the slide w'ith the laser beam to heat the slide to a steady state temperature that removes tissue crosslinking from fixation to promote antigen retrieval.

76. The method of claim 75, wherein the steady state temperature is between about 110 to 118 degrees Celsius.

77. The method of claim 55, wherein the processing includes curing a coverslip on the slide.#3371089 OS <11 9-00002512978, The method of claim 77, wherein the curing includes heating the coverslip with the laser beam.79, The method of claim 78, further comprising:applying absorptive material to the coverslip; andwherein the curing includes heating the absorptive material with the laser beam.80, The method of claim 79, further compri sing:removing the absorptive material from the coverslip after the curing.

81. The method of claim 55, further comprising:scanning the laser beam along a laser scan path on the slide; andwherein the laser scan path leverages thermocapillary convection.

82. The method of claim 55, further comprising:measuring temperature of the slide with a temperature sensor;regulating power of the laser with a controller in response to the measuring the temperature of the slide to maintain a temperature set point; anddetecting cessation of evaporative cooling on the slide with the controller based on a drop in the power of the laser required to maintain the temperature set point,83, The method of claim 55, further comprising:dispensing a cell condi ioning reagent onto the tissue sample on the slide;covering die cell conditioning reagent with a liquid coverslip;heating the slide with the laser beam; andwherein the liquid coverslip is configured to reduce evaporation of the cel! conditioning reagent during the heating with the laser beam,84. The method of claim 83, wherein:the heating includes heating the slide to about 120°C; andthe heating occurs for about 8 to about 12 minutes.#3371089 OS <11 9-00002513085. The method of claim 84, wherein the tissue sample includes a vimentin type tissue sample.

86. fhe method of claim 84, wherein the tissue sample includes a B~cel! lymphoma 2 (BCL2) type tonsil tissue sample,87. The method of claim 83, wherein:the heating includes heating the slide to about 120°C;the tissue sample includes an Anaplastic Lymphoma Kinase (ALK) type appendix tissue sample; andthe heating occurs for about 12 minutes.

88. The method of claim 83, wherein:the heating includes heating the slide to about 100°C;the tissue sample includes a cluster of differentiation 10 (CD 10) type tonsi l tissue sample; andthe heating occurs for about 10 minutes.

89. The method of claim 55, further comprising:expanding and flattening the laser beam to promote uniform heating while the laser beam is stationary.

90. The method of claim 55, further comprising:shaping the laser beam with a beam shaper.

91. Tlie method of claim 90, further comprising:expanding the laser beam w'ith a beam expander.

92. The method of claim 90, further comprising:forming the laser beam to have a rectangular shape with the beam shaper.

93. The method of claim 55, further comprising:drying the slide by blow ing a gas across the slide.#3371089 OS <11 9-00002513194, The method of claim 55, further comprising:monitoring a spatial temperature distribution across the slide with a thermal camera; and adjusting power of the laser beam at different locations on the slide based on the spatial temperature distribution.

95. The method of claim 55, further comprising:processing multiple slides with the laser beam in an asynchronous manner.

96. The method of claim 55, further comprising:inducing thermocapillary convection on the slide with the laser beam.

97. The method of claim 96, further compri sing:scanning the laser beam to form concentric loops with different radii.

98. The method of claim 55, further comprising:analyzing real-time thermal curves from the slide to adapt laser heating protocols based on individual slide wetness.99, 'Hie method of claim 55, further comprising:combining the laser beam with a visible laser beam at a beam combiner to form a combined laser beam; andshining the combined laser beam onto the slide.

100. The system or method of any of cl aims 1 to 99, wherein the laser is configured to bake the tissue sample on the slide.

101. The system or method of any of claims 1 to 100, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite to the sample surface; andthe laser beam i s configured to heat the base surface of the slide.#3371089 OS <11 9-000025132102. I he system or method of any of cl aims 1 to 101, wherein:the slide is made of glass; andthe laser includes an infrared type laser,1 3. The system or method of any of claims 1 to 102, wherein:the laser is a carbon dioxide type laser; andthe laser beam has a wavelength of about 10.6 micrometers.1 4. The system or method of any of claims 1 to 103, further comprising:a temperature sensor configured to measure temperature of the base surface of the slide,105. The system or method of any of claims 1 to 104, wherein the controller i s configured to control the laser based on the temperature sensed by the temperature sensor.

106. The system or method of any of claims 1 to 105, wherein the laser is configured to perform a flash baking technique.

107. The system or method of any of claims 1 to 106, wherein the laser is configured to perform a gentle baking technique.

108. The system or method of any of claims 1 to 107, wherein the laser is configured to dewax the tissue sample on the slide.

109. The system or method of any of cl aims 1 to 108, further comprising:a beam expander configured to defocus the laser beam.

110. The system or method of any of claims 1 to 109, wherein the beam expander includes a tube assembly.

111. The system or method of any of claims 1 to 110, further comprising:a slide holder being integrated with the beam expander.#3371089 OS <11 9-000025133112. The system or method of any of claims 1 to 111, wherein the slide holder is configured to engage the corners of the slide.

113. The system or method o f any of claims 1 to 112, wherein:the slide holder includes one or more posts; andthe posts are posi tioned to support the corners of the slide.

114. The system or method of any of claims 1 to 113, wherein the posts are configured to form an air gap between the holder and the slide.

115. The system or method of any of claims 1 to 114, wherein:the holder defines a beam aperture; andthe laser beam shines through the beam aperture.

116. The system or method of any of claims 1 to 115, wherein:the holder defines a drain channel;the drain channel extends around the beam aperture; andthe drain channel is configured to drain wax from the slide.

117. The system or method of any of cl aims 1 to 116, further comprising:a nozzle configured to dispense a solvent.1 18. The system or method of any of claims 1 to 1 17, further comprising:an air knife configured to sweep a mixture of the solvent and wax from the slide.

119. The system or method of any of claims 1 to 118, wherein the laser is configured to facilitate antigen retrieval.

120. The system or method of any of claims 1 to 119, wherein:the slide is configured to receive a coverslip; andthe laser is configured to cure the coversli.#3371089 OS <11 9-000025134121. The system or method of any of claims 1 to 120, wherein the coversli is a pre-glued coverslip.

122. The system or method o f any of claims 1 to 121, wherein the laser beam is configured to heat the covershp.1 3. The system or method of any of claims 1 to 122, wherein:the coverslip is at least partial ly covered with an absorptive material; andthe laser beam is configured to heat the absorpti ve material.

124. The system or method of any of cl aims 1 to 123, wherein:the laser is a visible light laser; andthe absorptive material is configured to absorb visible light.

125. The system or method of any of claims 1 to 1 4, wherein:the laser is an ultraviolet type laser; andthe absorptive material is configured to absorb ultraviolet light.

126. The system or method of any of claims 1 to 125, wherein the absorptive material includes black ink.1 7. The system or method of any of claims 1 to 1 6, wherein the absorptive material is in the form of a machine-readable i dentifier.

128. The system or method of any of cl aims 1 to 127, wherein:the absorptive material includes one or more absorptive bars; andthe absorptive bars cover one or more edges of the coverslip.

129. The system or method of any of claims 1 to 1 8, wherein:the absorpti ve material includes one or more dots; andthe dots are positioned at the corners of the coverslip.#3371089 OS <11 9-000025135130. The system or method of any of claims 1 to 129, wherein the absorptive materia! is positioned to not obstruct visibility during tissue analysis.

131. The system or method o f any of claims 1 to 130, wherein:the laser includes a lens; andthe lens is configured to project the laser beam with a flat focal plane.

132. The system or method of any of claims 1 to 131, wherein the lens includes an F-Theta lens.

133. The system or method of any of cl aims 1 to 132, further comprising:a module configured to bake and dewax at the same time.

134. The system or method of any of claims 1 to 133, further comprising:a module configured to bake, dewax, and facilitate antigen retrieval in a single integrated unit.

135. The system or method of any of claims 1 to 134, wherein:the laser is configured io scan the lase beam along a laser scan path; andthe laser scan path generates thermal gradients to sweep excess humidity'’ from the center of the slide to the edges of the slide.

136. The system or method of any of claims 1 to 135, wherein the controller is configured to perform closed loop temperature control.

137. The system or method of any of claims 1 to 136, further co rising:a temperature sensor operatively'’ coupled to the controller;wherein the controller includes a proportional-integral-derivative (PID) controller; wherein the PID controller is configured to receive temperature feedback from the temperature sensor; andwherein the PID controller is configured to regulate power of the laser to achieve a temperature set point.#3371089 OS <11 9-000025136138. The system or method of any of claims 1 to 137, wherein the controller is configured to detect dryness of the slide based on a threshold reduction in the power of the laser required to maintain the temperature set point.

139. The system or method of any of claims 1 to 138, further comprising:the slide;the tissue sample disposed on the slide;a cell conditioning reagent covering the tissue sample on the slide;wherein the cell conditioning reagent includes a high -temperature cell conditioning liquid; a liquid coverslip covering the cell conditioning reagent; andwherein the liquid coverslip is configured to reduce evaporation of the cell conditioning reagent during heating w ith the laser beam.

140. The system or method of any of claims 1 to 139, further comprising:an optical s stem configured to expand and flatten the laser beam; andwherein the optical system is disposed between the laser and the slide.

141. The system or method of any of claims 1 to 140, further comprising:a beam shaper configured to redistribute the intensi ty profile of the laser beam.

142. The system or method of any of claims 1 to 141, further comprising:a beam expander configured to expand the laser beam;a field stop configured to clip the laser beam expanded by the beam expander; and wherein the field stop is located between the beam expander and the beam shaper.1 3. The system or method o f any of claims 1 to 142, wherein:the beam shaper is configured to reshape the laser beam; andthe beam shaper is configured to produce the laser beam with a rectangular shape.

144. The system or method of any of claims 1 to 143, further comprising:a thermal camera configured to monitor a spatial temperature distribution across the slide including.#3371089 OS <11 9-000025137145. The system or method of any of claims 1 to 144, further comprising:a slide holder including a slide cap configured to cover the slide.

146. The system or method o f any of claims 1 to 145, wherein:the slide holder includes a fan; andthe fan is configured to blow a gas across the slide.

147. The system or method of any of claims 1 to 146, wherein the slide holder i s configured to orient the slide at a transverse angle to promote drainage.

148. The system or method of any of cl aims 1 to 147, further comprising:a visible laser configured to produce a visible laser beam; anda beam combiner configured to combine the visible laser beam with the laser beam from the laser to produce a combined laser beam.1 9. The system or method of any of claims 1 to 148, wherein:the slide is at least partially covered with an absorptive material; andthe absorptive material is configured to absorb wavelengths outside those produced by carbon dioxide lasers.

150. The system or method of any of claims 1 to 149, further comprising:a beam shaper including a diffractive optical element (DOT).

151. The system or method of any of claims 1 to 150, wherein the di fractive optical element includes a computer-generated hologram (CGH).

152. The system or method of any of claims 1 to 151, further comprising:a beam expander configured to expand the laser beam;a beam shaper configured to redistribute the intensi ty profile of the laser beam; and wherein the beam expander and the beam shaper are integrated together in a single computergenerated hologram.#3371089 OS <11 9-000025138153. The system or method of any of claims 1 to 1 2, wherein the processing includes baking the tissue sample on the slide.

154. The system or method o f any of claims 1 to 1 3, wherein the baking includes heating the slide by shining the laser beam onto the slide.

155. The system or method of any of claims 1 to 154, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite to the sample surface; andthe baking includes heating the slide by shining the laser beam onto the base surface of the slide.

156. The system or method of any of claims 1 to 1 5, wherein the baking includes flash baking the slide with the laser beam.

157. The system or method of any of claims 1 to 156, further comprising:generating the laser beam at 40% power;scanning the laser beam with a square beam path;wherein the square beam path has 0.25 mm spacing;scanning the laser beam at a scanning speed of about 800 mm / s; andheating with the laser beam until a peak temperature is reached.

158. The system or method of any of cl aims 1 to 157, wherein the peak temperature is 120 degrees Celsius.

159. The system or method of any of claims 1 to 1 8, further comprising:measuring temperature of the slide with a temperature sensor.

160. The system or method of any of claims 1 to 159, wherein the baking includes gentle baking the slide with the laser beam.#3371089 OS <11 9-000025139161. The system or method of any of claims 1 to 160, further comprising:generating the laser beam with at most 5% power;scanning the laser beam with a square beam path;scanning the laser beam at a scanning speed of about 3,500 mm / s; andheating with the laser beam until a steady state temperature is reached.

162. The system or method of any of claims 1 to 161, wherein the steady state temperature is about 60 degrees Celsius.

163. The system or method of any of claims 1 to 162, further comprising:heating the slide with the laser beam for at most 20 seconds.

164. The system or method of any of claims 1 to 163, further comprising:dewaxing by heating the slide with the laser beam.

165. The system or method of any of claims 1 to 164, further comprising:performing the baking and the dewaxing simultaneously.

166. The system or method of any of claims 1 to 165, wherein the processing includes dewaxing the tissue sample on the slide.

167. The system or method of any of claims 1 to 166, wherein:the slide has a sample surface;the tissue sample is disposed on the sample surface;the slide has a base surface;the base surface is disposed opposite io the sample surface; andthe dewaxing includes heating the base surface of the slide w'ith the laser beam.

168. The system or method o f any of claims 1 to 167, wherein the dewaxing includes heating the slide with the laser beam using a flash heating process.

169. The system or method of any of claims 1 to 168, wherein the dewaxing includes heating the slide with the laser beam using a gentle heating process.#3371089 OS <11 9-000025140170. The system or method of any of cl aims 1 to 169, further comprising:wherein the dewaxing includes melting wax in the tissue sample with heat from the laser beam;wherein the dewaxing includes applying a solvent to the tissue sample; anddraining a mixture of the solvent and the wax from the slide.

171. The system or method of any of claims 1 to 170, further comprising:sweeping the mixture of the wax and the solvent with an air knife.

172. The system or method of any of cl aims 1 to 171, further comprising:applying a transfer fluid with a vaporization temperature above 100 degrees Celsius to the slide to form a puddle; andirradiating the slide with the laser beam to heat the slide to a steady state temperature that removes tissue crosslinking from fixation to promote antigen retrieval.

173. The system or method of any of claims 1 to 172, wherein the steady state temperature is between about 110 to 118 degrees Celsius.

174. The system or method of any of cl aims 1 to 173, wherein the processing includes curing a coverslip on the slide.

175. The system or method of any of claims 1 to 174, wherein the curing includes healing the coverslip with the laser beam.

176. The system or method of any of claims 1 to 175, further comprising:applying absorptive material to the coverslip; andwherein the curing includes heating the absorptive material with the laser beam.

177. The system or method of any of claims 1 to 176, further comprising:removing the absorptive material from the coverslip after the curing.

178. The system or method o f any of claims 1 to 177. further comprising:#3371089 OS <11 9-000025141scanning the laser beam along a laser scan path on the slide; andwherein the laser scan path leverages thermocapillary convection.

179. The system or method o f any of claims 1 to 178, further comprising:measuring temperature of the slide with a temperature sensor;regulating power of the laser with a controller in response to the measuring the temperature of the slide to maintain a temperature set point; anddetecting cessation of evaporative cooling on the slide with the controller based on a drop in the power of the laser required to maintain the temperature set point.

180. The system or method of any of cl aims 1 to 179, further comprising:dispensing a cell conditioning reagent onto the tissue sample on the slide;covering the cell conditioning reagent with a liquid coverslip;heating the slide with the laser beam; andwherein the liquid coverslip is configured to reduce evaporation of the cell conditioning reagent during the heating with the laser beam.

181. The system or method o f any of claims 1 to 180, wherein:the heating includes heating the slide to about 120°C; andthe heating occurs for about 8 to about 12 minutes.

182. The system or method of any of claims 1 to 181, wherein the tissue sample includes a vimentin type tissue sample.

183. The system or method of any of cl aims 1 to 182, wherein the tissue sample includes a B- cell lymphoma 2 (BCL2) type tonsil ti sue sample.

184. The system or method of any of claims 1 to 183, wherein:the heating includes heating the slide to about 120°C;the tissue sample includes an Anaplastic Lymphoma Kinase (ALK) type appendix tissue sample; andthe heating occurs for about 12 minutes.#3371089 OS <11 9-000025142185. The system or method of any of claims 1 to 184, wherein:the heating includes heating the slide to about 120°C;the tissue sample includes a cluster of differentiation 10 (CD 10) type tonsil tissue sample; andthe heating occurs for about 10 minutes.

186. The system or method of any of claims 1 to 185, further comprising:expanding and flattening the laser beam to promote uniform heating while the laser beam is stationary.

187. The system or method of any of cl aims 1 to 186, further comprising:shaping the laser beam with a beam shaper.

188. The system or method of any of claims 1 to 187, further comprising:expanding the laser beam with a beam expander.

189. The system or method of any of claims 1 to 188, further comprising:forming the laser beam to have a rectangular shape with the beam shaper.

190. The system or method of any of cl aims 1 to 189, further comprising:drying the slide by blowing a gas across the slide.

191. The system or method of any of claims 1 to 190, further comprising: monitoring a spatial temperature distribution across the slide with a thermal camera; and adjusting power of the laser beam at different locations on the slide based on the spatial temperature distribution,192. The system or method of any of claims 1 to 191, further comprising:processing multiple slides with the laser beam in an asynchronous manner.

193. The system or method of any of claims 1 to 192, further comprising:inducing thermocapillary convection on the slide with the laser beam.#3371089 OS <11 9-000025143194. The system or method of any of claims 1 to 193, further comprising:scanning the laser beam to form concentric loops with different radii.

195. The system or method o f any of claims 1 to 194, further comprising:analyzing real-time thermal curves from the slide to adapt laser heating protocols based on individual slide wetness.

196. The system or method of any of claims 1 to 195, further comprising:combining the laser beam with a vi sible laser beam at a beam combiner to form a combined laser beam; andshining the combined laser beam onto the slide.

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