Optical module with three or more color fluorescent light sources and methods of use thereof

Abstract

An imaging apparatus is provided that includes a fluorescence microscope (115), an imaging sensor (120), and an optical module (110) and phase light (125) to facilitate epifluorescence imaging of three (or more) color channels and to perform phase contrast and / or brightfield imaging of a sample without manual adjustment of the imaging apparatus. This allows for automated imaging of multiple samples over an extended period of time by the imaging apparatus located inside an incubator (180) without disturbing the incubator environment to manually adjust the apparatus. Embodiments are also provided that facilitate user replacement of the detachable optical module (110) and / or transillumination module (125) to allow the imaging apparatus to accommodate different combinations of assays and / or fluorescent indicators in order to increase the variety of experiments and / or fluorescent dyes that can be imaged using the imaging apparatus.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. nonprovisional patent application No. 16 / 854,756, filed April 21, 2020, the contents of which are incorporated herein by reference in their entirety. Technical Field Background Technology

[0004] Microscopic imaging of live cellular biological samples can be performed in various ways to assess the sample's growth, metabolism, morphology, or other properties at one or more time points. This microscopic imaging can include fluorescence imaging, in which fluorophores in the sample are excited by light at the fluorophore's excitation wavelength, causing them to emit fluorescence at the fluorophore's emission wavelength. In epifluorescence imaging, the excitation light is provided via the same objective lens used to collect the emitted light.

[0005] Achieving multichannel fluorescence imaging often involves moving different sets of filters and, occasionally, excitation sources to the appropriate positions each time a fluorescence image is acquired for a specific emission wavelength. However, such an arrangement results in larger, slower, more expensive, and less reliable systems due to the need to physically move components. Summary of the Invention

[0006] In a first aspect, an example optical module for imaging fluorophores in live cellular biological samples is disclosed. The optical module includes: (a) a first light source configured to emit first light in a first excitation wavelength band; (b) a first filter disposed in a first optical path of the first light source, the first filter being configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths; (c) a second light source configured to emit second light in a second excitation wavelength band; (d) a second filter disposed in a second optical path of the second light source, the second filter being configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths; (e) a third light source configured to emit third light in a third excitation wavelength band; and (f) a third filter. A filter is arranged in the third optical path of the third light source, the third filter being configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths, wherein the first, second, and third optical paths converge along a main transmission optical path, the main transmission optical path being configured to guide toward the live cell biological sample; and (g) an emission filter, the emission filter being arranged in the main emission optical path of light emitted by a fluorophore in the live cell biological sample, wherein the main emission optical path is configured to terminate at an imaging sensor, wherein the emission filter is configured to allow light in the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band to pass through, and the emission filter is configured to reflect light in the first excitation wavelength band, the second excitation wavelength band, and the third excitation wavelength band.

[0007] In a second aspect, an example system for measuring live cellular biological samples is disclosed. The system includes: (a) an optical module according to a first aspect of this disclosure; (b) a fluorescence microscope detachably coupled to the optical module, wherein the fluorescence microscope has at least one objective lens; (c) an imaging sensor arranged in the emission path of light emitted by fluorophores in the live cellular biological sample from the objective lens; and (d) a phase lamp detachably coupled to the fluorescence microscope and arranged at the end of a main transmission optical path.

[0008] In a third aspect, an example method for imaging fluorophores in a live cellular biological sample is disclosed. The method includes: (i) aligning a first biological sample and a fluorescence microscope such that the first biological sample is within the field of view of the fluorescence microscope, wherein the first biological sample comprises: (a) a first fluorophore that emits light in a first emission wavelength band in response to illumination by light in a first excitation wavelength band; (b) a second fluorophore that emits light in a second emission wavelength band in response to illumination by light in a second excitation wavelength band; and (c) a third fluorophore that emits light in a third emission wavelength band in response to illumination by light in a third excitation wavelength band; (ii) obtaining an image set of the first biological sample using the fluorescence microscope, wherein the images in the image set differ in terms of focus settings; and (iii) based on the image set, imaging the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band, respectively. The wavelength bands determine a first focusing setting, a second focusing setting, and a third focusing setting; (iv) during a first time period, the first biological sample is illuminated with light in the first excitation wavelength band using a first light source, and the fluorescence microscope is operated according to the first focusing setting to obtain a first image of light in the first emission wavelength band via the image sensor of the fluorescence microscope; (v) during a second time period, the first biological sample is illuminated with light in the second excitation wavelength band using a second light source, and the fluorescence microscope is operated according to the second focusing setting to obtain a second image of light in the second emission wavelength band via the image sensor; and (vi) during a third time period, the first biological sample is illuminated with light in the third excitation wavelength band using a third light source, and the fluorescence microscope is operated according to the third focusing setting to obtain a third image of light in the third emission wavelength band via the image sensor.

[0009] In a fourth aspect, an example non-transitory computer-readable medium is disclosed. This computer-readable medium stores program instructions that, when executed by a processor, cause the method of the third aspect to perform.

[0010] The features, functions, and advantages discussed can be implemented independently in various examples or combined with other examples. More details can be found in the following description and figures. Attached Figure Description

[0011] Figure 1 This is a functional block diagram of a system based on an example implementation;

[0012] Figure 2 A block diagram depicts a computing device and a computer network according to an example implementation.

[0013] Figure 3 A functional block diagram of a system (including an optical module) for measuring live cell biological samples according to an example embodiment is shown;

[0014] Figure 4 A functional block diagram of a system (including an optical module) for measuring live cell biological samples according to an example embodiment is shown;

[0015] Figure 5 A functional block diagram of a system (including an optical module) for measuring live cell biological samples according to an example embodiment is shown;

[0016] Figure 6 A front view of an optical module according to an example embodiment is shown;

[0017] Figure 7 It shows that according to Figure 6 Rear view of the optical module in an example implementation;

[0018] Figure 8 It shows that according to Figure 6 A side view of the optical module in an example implementation;

[0019] Figure 9 It shows that according to Figure 6 A front cross-sectional view of the optical module in an example implementation;

[0020] Figure 10 It shows that according to Figure 6 A side cross-sectional view of the optical module in an example implementation;

[0021] Figure 11 It shows that according to Figure 6 A side cross-sectional view of the optical module in an example implementation;

[0022] Figure 12 It shows that according to Figure 11 Detailed view of the axis of the optical module in the example implementation;

[0023] Figure 13 A functional block diagram of a system (including an optical module) for measuring live cell biological samples according to an example embodiment is shown;

[0024] Figure 14 A perspective view of a phase lamp according to an example embodiment is shown;

[0025] Figure 15 A cross-sectional view of a phase lamp coupled to a fluorescence microscope according to an exemplary embodiment is shown; and

[0026] Figure 16 A flowchart of a method according to an example implementation is shown.

[0027] The accompanying drawings are for illustrative purposes, but it should be understood that the invention is not limited to the arrangements and tools shown in the drawings. Detailed Implementation

[0028] I. Overview

[0029] Microscopic imaging of live cell samples can provide information about the health, growth, and viability of cell populations under various experimental conditions. This information may include: information about the number of cells in the sample over time, cell morphology or other structural features, internal contents or structures of the cells (e.g., contents associated with mitosis or other metabolic processes), or other information about the cells. This information can be used to assess cell behavior under “normal” conditions and / or under various applied experimental conditions. For example, microscopic imaging of cells can be used to assess the effects of cell responses to experimental drugs or other additives, the effects of cell genetic modifications, the addition of cancer cells, other added cell types and / or the effects of added bacteria, fungi, viruses, or other microorganisms on cells, or the effects of other applied experimental conditions on live cell samples.

[0030] To reduce the cost of such imaging, to decrease the size of the apparatus used to perform the imaging (e.g., incubator, imaging device), to reduce the impact on the stability of conditions applied to the cell samples, and / or to provide other benefits, the microscopic imaging of multiple live cell samples can be performed by an automated imaging device configured to coexist with the live cell samples within the incubator. Such an automated imaging device may include a gantry or other actuator configured to move the imaging device and / or the live cell samples (e.g., multiwell plates or other multi-sample containers) to facilitate automated imaging of multiple live cell samples within the incubator. Such live cell samples may include multiple live cell samples, which may differ in the identity or mixture of the live cell contents, the identity or amount of added drugs, microorganisms, cancer cells, or other added substances, the type of genetic modification applied to the live cell contents, or some other experimental conditions.

[0031] Such a system's microscopic imaging apparatus may include mirrors, filters, or other elements to fold the optical path of the imaging apparatus, thereby reducing its size so that it can be mounted within an incubator. Additionally or alternatively, the elements of the imaging apparatus may be divided into discrete sub-components to facilitate various microscopic imaging modalities. For example, a phase lamp and / or other transmission light source may be provided in a module separate from the module containing the image sensor and opposite the live cell sample container to facilitate bright-field imaging, phase-contrast imaging, or other microscopic imaging modalities.

[0032] Fluorescent dyes, non-fluorescent dyes or pigments, nanorods, or other conductive elements exhibiting surface plasmon resonance at appropriate wavelengths, Raman dyes, or other optically distinguishable substances can be added to live cell samples to facilitate imaging of the sample's contents and / or its processes or contents. Optically distinguishable substances can be functionalized (e.g., with antibodies) to specifically bind to or otherwise interact with substances of interest within the sample. For example, contrast agents can be functionalized to specifically bind to proteins, surface markers of specific cells, specific sequences of DNA / RNA, etc., or other substances or elements of interest within a biological sample, or to otherwise interact with such substances. This functionalization can facilitate imaging of specific substances within the sample, such as the presence, amount, distribution, or other information of proteins or other substances of interest in the sample. Optically distinguishable substances can be added as extrinsic substances to live cell samples (e.g., by adding a specific amount of fluorophore to each well of a multi-well sample plate, which is conjugated to an antibody specific to a particular cell type or receptor). Additionally or alternatively, optically distinguishable substances can be added by genetically modifying the live cells in the sample to express the optically distinguishable substance (e.g., by adding a gene for green fluorescent protein to the live cells in the sample). Additionally or alternatively, such optically distinguishable substances can be naturally present in live cells and / or substances secreted by them (e.g., autofluorescent proteins naturally expressed by a population of live cells).

[0033] Phosphors or other substances (e.g., Raman dyes) that emit light in their emission bands in response to excitation from light in an excitation band different from the emission band are particularly useful for imaging inclusions in a sample. This is partly due to the ability to distinguish the excitation light from the emitted light that is emitted in response, and the ability to control the magnitude and timing of the emitted light by controlling the excitation light. These properties allow fluorescent dyes to be imaged with higher fidelity than other substances (e.g., dyes that scatter light over a range of wavelengths such that the wavelength of the scattered light is substantially the same as the wavelength of the irradiating light). Furthermore, optically distinguishable fluorophores can be used to facilitate independent imaging of multiple different fluorophores. These different fluorophores can differ in their excitation spectra, emission spectra, or other properties (e.g., fluorescence lifetime) to facilitate such independent imaging. Such imaging can be achieved by providing light at correspondingly different excitation wavelengths for different fluorophores at correspondingly different time points. This process can be called “epitaphofluorescence imaging” when the excitation light is delivered to the sample via the same objective lens (or objective system) used to collect and image the emitted light from the sample.

[0034] To image different fluorophores (or other optically distinguishable sample inclusions) at different time points, a fluorescence imager (e.g., an epifluorescence imager) may mechanically move one or more wavelength-selective filters, mirrors, or other wavelength-selective optical elements into and out of the imager's optical path to facilitate illumination of the sample with light in different excitation wavelength bands and / or to facilitate selective reception and imaging of light in different emission wavelength bands. However, such an imager may be mechanically more complex, more expensive, larger, less reliable, or may exhibit other undesirable performance characteristics. Instead, the imager may include: a set of static dichroic mirrors, optical filters, or other elements configured to allow different light sources to emit light in corresponding different excitation bands of different fluorophores while also allowing light in corresponding emission bands of different fluorophores to pass through an image sensor and be imaged by the image sensor.

[0035] For example, an optical module for determining the epifluorescence capability of live cellular biological samples in a fluorescence microscopy system may include two light sources and associated filters (e.g., dichroic mirrors). Wavelengths associated with these light sources and with the passband / stopband / reflection band of the filters within the optical module are selected to work with one or more groups of fluorophores according to the color of the light they are excited and responsively emitted.

[0036] Such imaging systems can be configured and operated to independently excite two distinct fluorophores with at least their respective excitation bands different, and to detect light emitted responsively from the two distinct fluorophores with at least their respective excitation bands different. For example, such a system can detect a green and a red fluorophore in a first configuration and an orange and a near-infrared (“NIR”) fluorophore in a second configuration (e.g., by changing the optical module containing a light source, dichroic mirror, filter, or other optical components). Different configurations of such dual-color imaging systems (e.g., different replaceable optical modules of the system) can be configured to excite fluorophore pairs associated with a specific assay (e.g., a fluorescence ubiquitination cell cycle indicator (FUCCI) assay, which is a two-color (red and green) indicator encoded by genes that allows observation of cell division within a cell population). When the system is set in a specific configuration, independent images can be collected only for fluorophores compatible with that specific configuration.

[0037] This dual-fluorophore optical system is limited in the number of different fluorophores that can be imaged independently. Therefore, in automated imaging scenarios (e.g., where different pores of a porous sample container differ in the presence of fluorophores within them), there is a limitation on the type of information that can be generated in a single sample or across different sample populations. This can include limitations on the types of fluorescence assays that can be performed in a single sample (i.e., assays involving two or fewer fluorescent indicators). One example of such an assay is based on a fluorescent ubiquitinated cell cycle indicator (“FUCCI”), a two-color (red and green) indicator that allows observation of gene-encoded cell division within a cell population. However, the two-color optical module used to perform this assay cannot distinguish between S phase (i.e., when cells synthesize a complete copy of DNA in their nucleus), G2 phase (i.e., the second interphase, when cells grow further, produce proteins and organelles, and begin reorganizing their contents in preparation for mitosis), and mitotic (M) phase (i.e., when cells split their DNA into two groups and separate their cytoplasm to form two new cells). A colorless phase exists during the M / G1 transition, making cells indistinguishable from non-expressing cells. TagGFP2 is a protein with bright green fluorescence, with excitation / emission maxima at 483 nm and 506 nm, respectively. During S, G2, and M phases, cells emit green fluorescence via TagGFP2 expression, which can be imaged using a two-color optical module. mKate is a far-red fluorescent protein with excitation / emission maxima at 588 nm and 633 nm, respectively. During G1 phase (i.e., the first interphase, when cells physically grow larger, replicate organelles, and produce molecular building blocks for subsequent growth phases) and during the transition to S phase, cells emit far-red fluorescence via mKate expression, which can also be imaged using a two-color optical module. However, this two-color optical module cannot use additional fluorescent indicators to identify additional phases or sub-phases in cell division.

[0038] The capabilities of this dual-fluorophore imaging system can be expanded by replacing the optical module, which includes a light source, filters, mirrors, or other optical elements associated with the excitation and emission bands of the two fluorophores. However, such manual replacement may be difficult to perform once automated imaging experiments are complete, and requires severely disrupting the environment of live cell samples by opening the incubator to allow the module to be replaced.

[0039] Advantages across various applications include the ability to independently image three (or more) fluorescence channels using a fluorescence imaging device (without replacing optical modules or performing additional manual procedures that could disturb the culture environment). Such a system allows for the use of more complex assays (e.g., assays involving three or more fluorescent indicators, such as a three-color FUCCI assay for observing the complete cell cycle), identification of more cell types in a single sample, and evaluation of metabolic or other fluorescent indicators (e.g., labeling two or more fluorescent indicators corresponding to different cell types, while a third fluorescent indicator in the sample represents metabolism, cell death, or some other process of interest), and the use of more types of assays / individual fluorescent indicators in a single sample in the incubator and / or more types of assays / individual fluorescent indicators in different samples in the incubator. These benefits can reduce costs by decreasing the time and incubator space required to perform a specified number of experiments / assays, achieved by allowing the reuse of multiple different assays for a single experiment in a single sample and / or by allowing different assays for different experiments in different wells of a sample plate in a single incubator.

[0040] The embodiments described herein provide methods and systems for such three (or more) channel fluorescence microscopy processes in a manner compatible with automated multi-sample imaging within an incubator. These embodiments offer solutions to the increased complexity associated with adapting a three (or more) channel fluorescence imaging device to a limited volume / size, while also allowing the imaging device to be used for bright-field and / or phase-contrast microscopy. These embodiments also provide solutions to the complex problem of designated branched optical paths that route excitation light in three (or more) excitation bands to the sample, while also routing light from the sample to the image sensor in three (or more) emission bands, while simultaneously rejecting light in the excitation bands. Some of these embodiments include providing a phase lamp (or other illumination source) as part of a detachable module paired with a corresponding three (or more) channel fluorescence imaging module. Such pairing may be necessary to ensure that the light from the phase lamp includes wavelengths capable of passing through the paired fluorescence imaging module. Such illumination modules and paired optical filter modules may include barcodes, onboard memory, or other features to facilitate automated module detection and identification, alerting the user before running experiments whether these modules are incompatible.

[0041] The embodiments provided herein also include improvements to the apparatus for manually replacing optical modules (e.g., phase lamp modules, light source modules, and filter modules), which improve the seating and alignment of such modules within the imaging device and increase the ease with which the user can perform such manual replacements. In existing systems, separate tools are required to couple and decouple various optical modules from the system. These tools are difficult to align with the corresponding screws through the small holes in the optical modules. Furthermore, the force required to couple or decouple optical modules is difficult for many end users to generate, partly due to the arrangement and configuration of the electrical connectors used to electrically couple the light source within the module to the controller and power supply of the rest of the system. Therefore, many end users require assistance to replace optical modules.

[0042] The flexible interchangeability of the optical modules allows the system to be configured to allow different combinations of methods for detecting fluorophores, including but not limited to: (i) activating three light sources in three different excitation wavelength bands to direct excitation light to the sample and detecting emission light responsively emitted from three different fluorophores (e.g., green, orange, and near-infrared (“NIR”)); (ii) activating three light sources in three different excitation wavelength bands to identify independent fluorophores (e.g., nuclear labels) using two and a third excitation wavelength bands for measurements based on Forster resonance energy transfer (“FRET”) (e.g., ATP); and (iii) using only two light sources in two different excitation wavelength bands of the optical modules and detecting emission light responsively emitted from two different fluorophores (e.g., (a) green and red, or (b) orange and NIR).

[0043] Furthermore, phase lamps that match the filters in three (or more) optical modules can also be included in the system to advantageously allow for phase and bright-field imaging (e.g., to enhance fluorescence imaging information and / or provide independent image information, further process and refine the image to identify fluorophores, measure FRET, or provide some other benefit). One advantage of the phase lamp module (or other light source module) disclosed herein is that the system can directly identify a specific phase lamp module that has been installed. Detecting the identity of the phase lamp advantageously allows the system to determine when an invalid configuration exists, in which the phase lamp is not properly matched to a given optical module (which may result in, for example, light from the phase lamp being completely or partially blocked from transmission through the optical module for imaging) and warns the user before running experiments (e.g., experiments involving the performance of one or more measurements).

[0044] II. Example Architecture

[0045] Figure 1This is a block diagram illustrating an operating environment 100, which, for example, includes or relates to a system 105 for measuring live cellular biological samples. This system 105 includes a fluorescence microscope 115 in electrical communication with a computing device 200a. The fluorescence microscope 115 is located within an incubator 108, which is configured to control temperature, humidity, and / or other environmental parameters to promote the culture of live cellular samples that can be automatically imaged by the fluorescence microscope 115. By placing the fluorescence microscope 115 within an incubator 180, the fluorescence microscope 115 can image the sample without removing the sample from the incubator 180, a process that could disturb the sample and alter its growth / response to applied experimental conditions. The following description... Figure 16 Method 300 illustrates an implementation of the method that can be carried out within the operating environment 100.

[0046] The fluorescence microscope 115 includes an optical module 110 that can be used in combination with an imaging sensor 120 to image a sample in an incubator 180 using epifluorescence imaging. The optical module 110 includes three (or more) light sources configured to provide illumination in three (or more) corresponding excitation wavelength bands corresponding to a specific fluorophore in the sample (e.g., a fluorescent indicator comprising a fluorophore conjugated to an antibody or other structure to facilitate selective binding to a protein or other substance of interest). The optical module 110 also includes a dichroic mirror, a filter, and / or other elements configured to provide branching optical paths, such that light from the three (or more) light sources is delivered to the sample via an objective lens. The objective lens may be part of the optical module 110 or may be separate from the optical module 110. The optical module 110 is also configured to deliver responsively emitted fluorescence from the three (or more) corresponding emission wavelength bands collected by the objective lens to the imaging sensor 120 to facilitate epifluorescence imaging of three different fluorophores in the sample.

[0047] The ability to independently image three different fluorophores using a single optical module 110 offers several benefits. It facilitates the use of more complex three (or more) color assays. It facilitates imaging of multiple assays or other fluorescent indicators in a single sample (e.g., a dual-color FUCCI assay and an independent fluorescent indicator that selectively binds to a specific cell type, allowing for the determination of both cell identity and cell division phase in the sample). It promotes fluorophore / assay selection by relaxing the requirement that all indicators / assays only meet two sets of excitation / emission bands (e.g., selecting a more optimized fluorescent indicator for a specific purpose instead of a less optimized indicator that matches one of the two available excitation / emission bands of the dual-color optical module). It facilitates imaging of different sets of assays / fluorescent indicators in different samples contained in the same incubator by allowing for more efficient use of space within a single instrument / incubator, saving time and other costs. For example, first and second different experiments (possibly overlapping with emission / excitation wavelengths) with corresponding first and second sets of fluorescent indicators / assays can be performed in the corresponding sets of wells within the same incubator. Additionally or alternatively, a single experiment can be performed using multiple different sets of assay / fluorescence indicators present in a subset of wells used for a single experiment, thereby allowing the simultaneous generation of additional data on that experiment using the same incubator. The ability to independently image three (or more) different fluorophores using a single optical module 110 can provide additional or alternative benefits or combinations of benefits.

[0048] The fluorescence microscope 115 also includes a phase lamp 125 (or other light source) configured to provide light for phase contrast, bright field, or other forms of imaging. An optical module 110 is configured to allow at least some of the light emitted from the phase lamp 125 to pass through. In some examples, this may include the phase lamp 125 being a narrowband light source (e.g., a laser, LED), and the optical module being configured to allow light to pass through a narrowband wavelength emitted by the narrowband light source. Details of the optical module 110 and the phase lamp 125 are described elsewhere in this document (e.g., regarding...). Figures 3-15 )supply.

[0049] (For example, according to embodiments described elsewhere herein) The optical module 110 may be user-replaceable to image different fluorescence indicators / measurements using the fluorescence microscope 115 at different time periods. This may include an optical module with needles, slots, or other alignment features to facilitate alignment of the optical module 110 with other imaging components of the fluorescence microscope 115 (e.g., with the imaging sensor 120). It may also include an optical module 110 with one or more electrical connectors to facilitate power supply and control of three (or more) light sources or to provide some other functionality. For example, the optical module 110 may include memory or other electrical components to allow a computing device (e.g., 200, 200a) to identify the optical module 110 and / or determine the wavelength band of light that can be emitted from the optical module 110 and / or the wavelength band of light imaged using the optical module 110.

[0050] The phase lamp 125 (or other illumination source) can also be user-replaceable. This is likely because different replaceable optical modules 110 have different passbands (i.e., the band through which the wavelength of light from the sample can pass from the optical module 110 to the imaging sensor 120), and these different passbands may not be compatible with every possible phase lamp 125. For example, a first phase lamp 125 may be “optimal” in some sense (e.g., relative to phase-contrast imaging of a particular type of sample), but may produce light in a wavelength band that does not significantly overlap with any passband of the optical module 110 that has been selected for the experiment (e.g., to facilitate imaging of a specific measurement of interest). Thus, the first phase lamp 125 module can be replaced with a second phase lamp 125 module that emits light at wavelengths that are entirely or substantially within the passband of the selected optical module 110.

[0051] Such replaceable phase lamp 125 modules may include memory or other electrical components to allow computing devices (e.g., 200, 200a) to identify the phase lamp 125 module and / or determine the wavelength band of light that can be emitted from the phase lamp 125 module. This identity / information can be automatically compared with similar information / identity of optical modules 110 mounted in the fluorescence microscope 115 to ensure that the mounted modules are compatible (e.g., to ensure that the mounted optical modules 110 can transmit light at the wavelengths emitted from the mounted phase lamp 125 module, thus enabling the combination to be used to image samples using the phase lamp 125 module via phase contrast, bright field, or some other imaging modality). If incompatibility is detected, the user can be warned before starting automated imaging trials or other experiments using system 105.

[0052] Figure 2This is a block diagram illustrating an example of a computing device 200 configured to interface directly or indirectly with an operating environment 100 according to an example embodiment. The computing device 200 can be used to perform... Figure 16 The functions of the methods shown and described below are as follows. Specifically, computing device 200 can be configured to perform one or more functions, including, but not limited to, using a single optical module to acquire an image of three or more fluorescent colors in a single biological sample in a single or multiple dishes. The acquired image can then be used to perform additional analysis of the imaged biological sample in relation to the properties of fluorophores corresponding to the three or more fluorescent colors and / or substances conjugated thereto. Functions may also include using a light source (e.g., a phase lamp) that is not part of the single optical module to acquire a bright-field image, phase-contrast image, or some other image of the sample using a signal optical module and other light sources.

[0053] The availability of three or more colors for imaging in a single sample facilitates more complex multicolor analyses or assays (e.g., a three-color FUCCI assay for observing the complete cell cycle), multiple different assays in a single sample or in different samples (e.g., a two-color FUCCI assay (green / orange) combined with an annexin NIR assay for apoptosis), the use of one or more fluorescent indicators in combination with each other and / or with one or more multicolor assays (e.g., two fluorescent reporters for identifying corresponding cell types and an annexin NIR assay for apoptosis, which can be used as part of a three-color immune cell killing assay), the use of different sets of indicators / assays in different samples located in the same incubator, or other examples. The availability of three or more colors also relaxes the requirements for the selected indicators / assays, allowing for greater flexibility. For example, if a particular assay is only available in a specific color, that color can be reserved for the assay, while other colors can be used for cell type-specific indicators or other applications (e.g., color channels for assays with more color options available).

[0054] The computing device 200 includes a processor 202 connected to a communication bus 212, a communication interface 204, a data storage device 206, an output interface 208, and a display 210. The computing device 200 may also include hardware to enable communication within the computing device 200 and between the computing device 200 and other devices (e.g., not shown). The hardware may include, for example, a transmitter, a receiver, and an antenna.

[0055] Communication interface 204 can be a wireless interface and / or one or more wired interfaces, which allow both short-range and long-range communication with one or more networks 214 or with one or more remote computing devices 216 (e.g., tablet device 216a, personal computer 216b, laptop computer 216c, and mobile computing device 216d). Such wireless interfaces can provide communication under one or more wireless communication protocols, such as Bluetooth, Wi-Fi (e.g., IEEE 802.11), Long-Term Evolution (LTE), cellular communication, Near Field Communication (NFC), and / or other wireless communication protocols. Such wired interfaces can include Ethernet interfaces, Universal Serial Bus (USB) interfaces, or similar interfaces for communication via cables, twisted pairs, coaxial cables, optical links, fiber optic links, or other physical connections to wired networks. Therefore, communication interface 204 can be configured to receive input data from one or more devices and can also be configured to send output data to other devices.

[0056] The communication interface 204 may also include user input devices, such as a keyboard, keypad, touchscreen, touchpad, computer mouse, trackball and / or other similar devices.

[0057] Data storage device 206 may include or take the form of one or more computer-readable storage media that can be read or accessed by processor 202. Computer-readable storage media may include volatile and / or non-volatile storage components that can be integrated integrally or partially with processor 202, such as optical, magnetic, organic, or other memory or disk storage devices. Data storage device 206 is considered a non-transitory computer-readable medium. In some examples, data storage device 206 may be implemented using a single physical device (e.g., a single optical, magnetic, organic, or other memory or disk storage unit), while in other examples, data storage device 206 may be implemented using two or more physical devices.

[0058] Therefore, data storage device 206 is a non-transitory computer-readable storage medium on which executable instructions 218 are stored. Instructions 218 include computer-executable code. When instructions 218 are executed by processor 202, processor 202 performs functions. Such functions include, but are not limited to: using a single optical module to obtain an image of three or more fluorescent colors in a single biological sample in a single or multiple dishes; using a phase lamp or other light source other than the single optical module in combination with the single optical module to obtain a bright-field image, phase-contrast image, or some other image of the biological sample; and / or performing analysis based on the obtained images.

[0059] Processor 202 may be a general-purpose processor or a special-purpose processor (e.g., a digital signal processor, an application-specific integrated circuit, etc.). Processor 202 may receive input from communication interface 204 and process the input to generate output that is stored in data storage device 206 and output to display 210. Processor 202 may be configured to execute executable instructions 218 (e.g., computer-readable program instructions) that are stored in data storage device 206 and can be executed to provide the functionality of computing device 200 described herein.

[0060] Output interface 208 outputs information to display 210 or other components. Therefore, output interface 208 can be similar to communication interface 204, or it can be a wireless interface (e.g., a transmitter) or a wired interface. For example, output interface 208 can send commands to one or more controllable devices.

[0061] Figure 2 The computing device 200 shown can also represent, for example, a local computing device 200a in an operating environment 100 that communicates with system 105. Figure 1 The local computing device 200a can perform one or more of the steps of the method 300 described below, and can receive input from the user and / or send image data and user input to the computing device 200 to perform all or some of the steps of method 300.

[0062] Figure 16A flowchart of an example method 300 for imaging fluorophores in a live cellular biological sample is shown. Method 300 includes: aligning a first biological sample and a fluorescence microscope such that the first biological sample is within the field of view of the fluorescence microscope, wherein the first biological sample contains (i) a first fluorophore that emits light in a first emission wavelength band in response to illumination by light in a first excitation wavelength band, (ii) a second fluorophore that emits light in a second emission wavelength band in response to illumination by light in a second excitation wavelength band, and (iii) a third fluorophore that emits light in a third emission wavelength band in response to illumination by light in a third excitation wavelength band (305); obtaining an image set of the first biological sample using the fluorescence microscope, wherein the images in the image set differ in terms of focus settings (310); and identifying the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band based on the image set. A first focus setting, a second focus setting, and a third focus setting are determined (315); during a first time period, a first biological sample is illuminated with light in a first excitation wavelength band using a first light source and the fluorescence microscope is operated according to the first focus setting to obtain a first image of light in a first emission wavelength band via the image sensor of the fluorescence microscope (320); during a second time period, the first biological sample is illuminated with light in a second excitation wavelength band using a second light source and the fluorescence microscope is operated according to the second focus setting to obtain a second image of light in a second emission wavelength band via the image sensor (325); during a third time period, the first biological sample is illuminated with light in a third excitation wavelength band using a third light source and the fluorescence microscope is operated according to the third focus setting to obtain a third image of light in a third emission wavelength band via the image sensor (330). Figure 16 The method 300 shown presents, for example, methods that can be used with... Figure 2 Examples of methods used with computing device 200. In some instances, components of the system can be configured to perform functions such that the components are configured and constructed with hardware and / or software to achieve such performance. For example, when operated in a particular manner, components of the system can be arranged to be suitable, capable of, or adapted to perform functions. Method 300 may include one or more operations, functions, or actions as shown by one or more of blocks 305-330. Although the blocks are shown in sequential order, some of these blocks may also be performed in parallel and / or in an order different from that described herein. Furthermore, the individual blocks may be combined into fewer blocks, divided into additional blocks, and / or removed based on desired implementation.

[0063] It should be understood that, for the processes and methods disclosed herein, the flowchart illustrates the function and operation of one possible implementation of this example. In this regard, each block may represent a module, segment, or portion of program code, which includes one or more instructions executable by a processor to implement a specific logical function or step in the process. The program code may be stored on any type of computer-readable medium or data storage device, such as storage devices including disks or hard disk drives. Furthermore, the program code can be encoded in a machine-readable format on a computer-readable storage medium or on other non-transitory media or articles of art. Computer-readable media may include non-transitory computer-readable media or memories, such as computer-readable media storing data for short periods, such as register memory, processor cache, and random access memory (RAM). Computer-readable media may also include non-transitory media, such as secondary or persistent long-term storage devices, such as read-only memory (ROM), optical disks or magnetic disks, and optical disc read-only memory (CD-ROM). Computer-readable media may also be any other volatile or non-volatile storage system. For example, a computer-readable medium may be considered a tangible computer-readable storage medium.

[0064] also, Figure 16 Each box in the document, as well as other processes and methods disclosed herein, may represent circuitry connected to perform a specific logical function within that process. As those skilled in the art will understand, alternative implementations are included within the scope of the examples in this disclosure, wherein, depending on the function involved, the function may be performed in a different order than that shown or discussed (including substantially simultaneously or in reverse order).

[0065] III. Example Optical Module

[0066] Figures 3-5 and Figure 13 The simplified schematic diagram illustrates various configurations and embodiments of an optical module 110 for imaging fluorophores in a live-cell biological sample 130 using an image sensor 120. The optical module 110 includes filters, a light emitter, and other elements, and is configured to provide excitation light that is independently controllable in at least three different excitation wavelength bands and to allow light from at least three corresponding emission wavelength bands to pass through (…). Figure 3 , Figure 4 and Figure 5 The three-color configuration is shown. Figure 13 (A four-color configuration is shown). The optical module 110 is also configured to allow light from the phase lamp 125 (or other light source) that has passed through and / or been scattered by the sample 130 to pass through for imaging by the image sensor 120.

[0067] The optical module 110 includes: a first light source 135 configured to emit first light in a first excitation wavelength band; a first filter 136 disposed in a first optical path 137 of the first light source 135; the first filter 136 configured to allow light of one or more wavelengths to pass through and reflect light of one or more other wavelengths; the optical module 110 further includes a second light source 140 configured to emit second light in a second excitation wavelength band; a second filter 141 disposed in a second optical path 142 of the second light source 140; the second filter 141 configured to allow light of one or more wavelengths to pass through and reflect light of one or more other wavelengths; and the optical module 110 further includes a third light source 145 configured to emit third light in a third excitation wavelength band; a third filter 146 disposed in a third optical path 147 of the third light source 145; the third filter 146 configured to allow light of one or more wavelengths to pass through and reflect light of one or more other wavelengths. The directions of the excitation light transmitted to sample 130 and / or the image light (e.g., fluorescence emission, bright field, phase contrast, or other scattered and / or transmitted image light) passing through optical module 110 to image sensor 120 are indicated by arrows in the optical path in the figure.

[0068] Filters 136, 141, and 146 may include a variety of materials or components configured in a variety of ways to facilitate reflection and / or absorption in certain wavelength bands and transmission in certain other wavelength bands. For example, 136, 141, and 146 may be dichroic mirrors comprising a number of alternating layers of material, the composition, thickness, and order of which may be specified to provide desired passband, stopband, reflection band, or other wavelength-selective optical behavior.

[0069] In the optical module 110, a first optical path 137, a second optical path 142, and a third optical path 147 converge along a main transmission optical path 150, which is configured to guide the light towards the live-cell biological sample 130 via an objective lens 165. The module also includes an emission filter 155 located in the main emission optical path 156 and configured to allow light emitted from fluorophores in the live-cell biological sample 130 to pass through in response to illumination by light sources 134, 140, and 145, and to allow at least some of the light emitted from the phase lamp 125 to pass through. The main emission optical path 156 terminates at the imaging sensor 120. The emission filter 155 is configured to allow light from at least a first emission wavelength band, a second emission wavelength band, and a third emission wavelength band to pass through, the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band corresponding to a first fluorophore, a second fluorophore, and a third fluorophore in the sample 130, which correspond to a first excitation wavelength, a second excitation wavelength, and a third excitation wavelength emitted by light sources 134, 140, and 145. The emission filter 155 is also configured to allow at least some wavelengths of light emitted from the phase lamp 125 to pass through. In practice, this may include matching the wavelength of the phase lamp's light source to one or more of the first emission wavelength band, the second emission wavelength band, or the third emission wavelength band.

[0070] The emission filter 155 can also be configured to suppress (e.g., reflect, absorb) light in the first, second, and third excitation wavelength bands. Alternatively, the reflection of other filters 136, 141, 146 can be relied upon to prevent excitation light from the light sources 135, 140, 145 from being received by the image sensor 120. Furthermore, the emission filter 155 can also be configured to reflect or otherwise suppress artificial autofluorescence from the biological sample 130, and / or reflect light from other fluorescent dyes that may be present in the biological sample 130.

[0071] Note that the optical module 110 may include one or both of the objective lens 165 or the image sensor 120. Alternatively, the optical module 110 may be configured to be detachably coupled to one or both of the objective lens 165 or the image sensor 120. This can be done, for example, to reduce the cost of a single replaceable optical module.

[0072] In the example implementation, such as Figure 3As shown, the first filter 136 is configured to allow light in the first excitation wavelength band to pass through, and to reflect light in the second and third excitation wavelength bands, as well as light in the first, second, and third emission wavelength bands, and the wavelength band emitted by the phase lamp 125 (the wavelength band emitted by the phase lamp 125 may overlap with one or more of the first, second, or third emission wavelength bands). The second filter 141 is configured to allow light in the third excitation wavelength band, as well as light in the first, second, and third emission wavelength bands and the phase lamp wavelength band, to pass through, and to reflect light in the second excitation wavelength band. The third filter 146 is configured to allow light in the first, second, and third emission wavelength bands and the phase lamp wavelength band to pass through, and to reflect light in the third excitation wavelength band.

[0073] like Figure 3 As shown, the first light source 135, the second light source 140, and the third light source 145 are arranged in series in the same plane. Figure 3 The arrangement of the components results in the optical module 110 in one dimension (in Figure 3 It is quite long in the horizontal direction, while in the other two dimensions (vertical and in and out) it is quite long. Figure 3 The optical module 110 has a smaller size within a plane. This may be desirable in some applications. However, in some applications, it may be beneficial to reduce the maximum size of the optical module 110 and / or to make the shape and size of the optical module 110 conform to a specified shape and / or size (e.g., to fit the module within an incubator or onto a gantry of an automated imaging system). Consequently, the arrangement of the elements of the optical module 110 can be modified, for example, to fold, nest, and / or branch the individual optical paths and / or change the direction and / or order of the individual optical paths.

[0074] In another example implementation, such as Figure 4 As shown, the first filter 136 is configured to allow light in the first excitation wavelength band and the second excitation wavelength band to pass through and to reflect light in the third excitation wavelength band, as well as light in the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band. The second filter 141 is configured to allow light in the second excitation wavelength band to pass through and to reflect light in the first excitation wavelength band. The third filter 146 is configured to allow light in the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band to pass through and to reflect light in the third excitation wavelength band. Therefore, in Figure 4In the illustrated embodiment, the first light source 135 is arranged such that a first optical path 137 begins at the first light source 135, is reflected from the second filter 141, passes through the first filter 136, and exits the optical module 110 along the main transmission optical path 150. The second light source 140 is arranged such that a second optical path 142 begins at the second light source 140, passes through the second filter 141, then passes through the first filter 136, and exits the optical module 110 along the main transmission optical path 150. The third light source 145 is arranged such that a third optical path 147 begins at the third light source 145, is reflected from the third filter 146 to the first filter 136, is reflected from the first filter 136, and exits the optical module 110 along the main transmission optical path 150. The main emission optical path 156 of the light emitted by the fluorophore in the live cell biological sample 130 is reflected from the first filter 136, passes through the third filter 146, passes through the emission filter 155, and exits the optical module 110.

[0075] exist Figure 4 In the example embodiment, the second light source 140 and the third light source 145 are arranged parallel to each other, and the first light source 135 is arranged at a 90-degree angle relative to the second light source 140 and the third light source 145. This arrangement allows for a more compact optical module 110, which includes three light sources within a housing 111.

[0076] exist Figure 5 In another alternative embodiment shown, the first filter 136 is configured to allow light in the first excitation wavelength band to pass through. The first filter 136 is also configured to reflect light in the second and third excitation wavelength bands, as well as light in the first, second, and third emission wavelength bands. The second filter 141 is configured to allow light in the first, second, and third emission wavelength bands to pass through. The second filter 141 is also configured to reflect light in the second and third excitation wavelength bands. The third filter 146 is configured to allow light in the third excitation wavelength band to pass through. The third filter 146 is also configured to reflect light in the second excitation wavelength band.

[0077] Therefore, in Figure 5In the illustrated embodiment, a first light source 135 is arranged such that a first optical path 137 begins at the first light source 135, passes through a first filter 136, and exits the optical module 110 along the main transmission optical path 150. A second light source 140 is arranged such that a second optical path 142 begins at the second light source 140, is reflected from a third filter 146 to a second filter 141, is reflected from the second filter 141 to the first filter 136, is reflected from the first filter 136, and exits the optical module 110 along the main transmission optical path 150. A third light source 145 is arranged such that a third optical path 147 begins at the third light source 145, passes through a third filter 146 to the second filter 141, is reflected from the second filter 141 to the first filter 136, is reflected from the first filter 136, and exits the optical module 110 along the main transmission optical path 150. Furthermore, the main emission optical path 156 of the light emitted by the fluorophore in the live cell biological sample 130 is reflected from the first filter 136, passes through the second filter 141, passes through the emission filter 155, and exits the optical module 110.

[0078] like Figure 5 As shown, the first light source 135 and the third light source 145 are arranged in parallel, and the second light source 140 is arranged at a 90-degree angle relative to the first light source 135 and the third light source 145. This arrangement allows for a more compact optical module 110, which includes three light sources within a housing 111.

[0079] Note that, although Figure 3-5 The exemplary embodiments shown elsewhere in this document depict an optical module having a light source path that is entirely in the same plane (i.e., the plane of the figures), but other embodiments are possible, for example, to reduce the overall size of the optical module and / or to make the shape and size of the optical module conform to available space (e.g., within an imaging device in a bench and / or automated incubator).

[0080] For example, Figure 5 The second light source 140 and the third filter 146 in the illustrated embodiment can be rotated 90 degrees (or some other angle) to enter (or exit) them. Figure 5 The plane. Figures 6-11 This implementation method and its additional details are further illustrated. For example... Figures 6-11As shown, a first light source 135, a second light source 140, and a third light source 145, a first filter 136, a second filter 141, and a third filter 146, and an emission filter 155 are all contained within a housing 111. The housing 111 includes a first opening 112 arranged to allow a main transmission light path 150 to pass through and illuminate the biological sample 130. The housing 111 also includes a second opening 113 arranged to allow a main emission light path 156 to pass through and reach the imaging sensor 120. Both the first opening 112 and the second opening 113 can include optical devices (such as lenses, filters, mirrors, etc.) and / or sensor surfaces. In an optional example, the emission filter 155 may be disposed within the second opening 113. Figures 3-8 In the example optical module 110 shown, the housing 111 includes a vertically extending main body 111a and a cantilevered extension 111b extending horizontally from the housing 111. The main body 111a of the housing 111 includes a first light source 135 and a third light source 145 in the same plane. The cantilevered extension 111b of the housing 111 further includes a second light source 140, which is arranged at a 90-degree angle relative to the first light source 135 and the second light source 140.

[0081] In other words, the second light source 140 and the third filter 146 have been rotated 90 degrees about a vertical axis passing through the center of the third light source 145. This arrangement places the second light source 140 behind the plane of the first light source 135 and the third light source 145, such that the second optical path 142 is guided to the third filter 146, where the light is reflected upwards. Such an arrangement can be particularly advantageous in space-constrained applications. For example, the aforementioned arrangement allows the optical module to remain compact for integration into an automated incubator-assembled epifluorescence and bright-field / phase-contrast imaging system, as described below, while also allowing the optical module to be easily replaced by the user, thus expanding the practicality and reconfigurability of the imaging system.

[0082] The light sources 135, 140, and 145 discussed herein can all include any device and / or component capable of sending light to or illuminating the biological sample 130. Exemplary light sources can include one or more lamps and associated optics. Exemplary lamps can include incandescent lamps (e.g., halogen or tungsten filament lamps), arc lamps (e.g., mercury lamps, mercury-xenon lamps, or xenon lamps), light-emitting diodes (“LEDs”), and / or lasers, etc. Associated optics (“source optics”) can include optical fibers and / or liquid light guides, one or more lenses, filters (such as polarization-based or wavelength-based filters), diffraction gratings, mirrors, masks, etc. The associated optics can select / adjust the intensity, wavelength, polarization, phase, direction, and / or shape of the light directed to the sample. In an optional implementation, the first light source 135, the second light source 140, and the third light source 145 each include: an LED, at least two lenses (e.g., to collimate the light output from the light source and / or to match the focal point of the output light with the infinity focal point or another focal point of the objective lens), and a single bandpass dichroic filter.

[0083] Specific boundaries of a first, second, third, and / or additional excitation wavelength bands emitted from the light source of the optical module can be specified depending on the application (e.g., based on the available fluorophores or excitation spectra of interest, the availability of suitable LEDs or other light-emitting elements, and / or associated filters, mirrors, lenses, objectives, or other optical elements). In one alternative embodiment, the first excitation wavelength band ranges from 453 nm to 485 nm and generally corresponds to blue light, which causes the corresponding fluorophore to emit green (or longer wavelength) light. The second excitation wavelength band ranges from 546 nm to 568 nm and generally corresponds to yellow-green light, which causes the corresponding fluorophore to emit orange (or longer wavelength) light. The third excitation wavelength band ranges from 648 nm to 674 nm and generally corresponds to red light, which causes the corresponding fluorophore to emit near-infrared (NIR) light. In a further embodiment, the boundaries of the above ranges in this alternative embodiment can vary by + / - 3 nm.

[0084] In another alternative embodiment, the first emission wavelength band ranges from 494 nm to 533 nm and generally corresponds to green light. The second emission wavelength band ranges from 576 nm to 639 nm and generally corresponds to orange light. The third emission wavelength band ranges from 686 nm to 756 nm and generally corresponds to NIR light. In a further embodiment, the boundaries of the above ranges can vary by + / - 3 nm.

[0085] In some examples, the optical module 110 may include a fourth light source 160 configured to emit a fourth light in a fourth excitation wavelength band. Figure 13 An example embodiment of this optical module 110 is shown. The optical module 110 in this embodiment further includes a fourth filter 161 disposed in the fourth optical path 162 of the fourth light source 160. The fourth filter 161 is configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths. The emission filter 155 is further configured to allow light in a fourth emission wavelength band to pass through and reflect light in a fourth excitation wavelength band. Adding the fourth light source 160 increases the ability to view a fourth fluorophore in the biological sample 130 and perform even more measurements without relying on separate optical modules with different light sources and filters and their corresponding configurations.

[0086] exist Figure 13 In the illustrated example embodiment, the first filter 136 is configured to allow light in a first excitation wavelength band to pass through and reflect light in a fourth excitation wavelength band. The second filter 141 is configured to allow light in a first emission wavelength band, a second emission wavelength band, a third emission wavelength band, and a fourth emission wavelength band to pass through, and to reflect light in a second excitation wavelength band and a third excitation wavelength band. The third filter 146 is configured to allow light in a third excitation wavelength band to pass through and reflect light in a second excitation wavelength band. The fourth filter 161 is configured to allow light in the first excitation wavelength band and a fourth excitation wavelength band to pass through, and to reflect light in the second excitation wavelength band, the third excitation wavelength band, and the first emission wavelength band, the second emission wavelength band, the third emission wavelength band, and the fourth emission wavelength band.

[0087] exist Figure 13In the illustrated example embodiment, a first light source 135 is arranged such that a first optical path 137 begins at the first light source 135, passes through a first filter 136, then through a fourth filter 161, and exits the optical module 110 along the main transmission optical path 150. A second light source 140 is arranged such that a second optical path 142 begins at the second light source 140, is reflected from a third filter 146 to a second filter 141, is reflected from the second filter 141 to a fourth filter 161, is reflected from the fourth filter 161, and exits the optical module 110 along the main transmission optical path 150. A third light source 145 is arranged such that a third optical path 147 begins at the third light source 145, passes through a third filter 146 to a second filter 141, is reflected from the second filter 141 to a fourth filter 161, is reflected from the fourth filter 161, and exits the optical module 110 along the main transmission optical path 150. A fourth light source 160 is arranged such that a fourth optical path 162 begins at the fourth light source 160, is reflected from the first filter 136, passes through the fourth filter 161, and exits the optical module 110 along the main transmission optical path 150. The main emission optical path 156 of the light emitted by the fluorophore in the live-cell biological sample 130 is reflected from the fourth filter 161, passes through the second filter 141, passes through the emission filter 155, and exits the optical module 110. (As...) Figure 13 As shown, the first light source 135 and the third light source 145 are arranged parallel to each other, and the second light source 140 and the fourth light source 160 are each arranged at a 90-degree angle relative to the first light source 135 and the third light source 145. This arrangement allows for a compact optical module 110, which includes four light sources within a housing 111.

[0088] This fourth emission wavelength band can include wavelengths shorter than 453 nm and can generally correspond to violet light, while the corresponding fourth emission wavelength band can generally correspond to blue light.

[0089] IV. Example System

[0090] In the second aspect of this disclosure, such as Figure 1 , Figure 3 , Figure 4 , Figure 5 ,and Figure 13As shown, a system 105 for measuring a live cellular biological sample 130 is provided. System 105 includes an optical module 110 according to a first aspect of this disclosure. System 105 also includes a fluorescence microscope 115 surrounding the optical module 110, the optical module 110 being detachably coupled to the fluorescence microscope 115. The fluorescence microscope 115 has at least one objective lens 165. System 105 further includes an imaging sensor 120 arranged in the main emission path of light emitted by fluorophores in the live cellular biological sample 130 and / or light transmitted and / or scattered from the objective lens 165 by a phase lamp 125 (or other illumination source, e.g., a light source configured to provide illumination for bright-field imaging but not for phase-contrast imaging). System 105 includes a phase lamp 125, the phase lamp 125 being detachably coupled to the fluorescence microscope 115 and arranged at the end of the main transmission optical path 150.

[0091] The fluorescence microscope 115 used herein is any optical device that magnifies images of small objects, such as cells, organelles, tissues, small organisms, particles, etc. Exemplary microscopy modes that can be performed by the detection mechanism include: optical microscopy (e.g., bright-field, dark-field, phase-contrast, differential interference contrast (e.g., Nomarski, DIC, and Hoffman Modulation Contrast), fluorescence, and / or other forms of visible and / or invisible (e.g., IR, NIR, ultraviolet) light microscopy). Objective 165 is arranged between optical module 110 and biological sample 130 such that main transmission light path 150 and main emission light path 156 pass through objective 165.

[0092] Imaging sensor 120 is configured to detect light and may include a camera, a multi-channel photodetector, a planar Fourier capture array, a single-pixel imager, or some other image generation device. Imaging sensor 120 may be configured or operated to detect light across a range of wavelengths. For example, imaging sensor 120 may be configured or operated to detect light across multiple wavelengths / wavelength ranges corresponding to the emission spectrum of a fluorescent dye or other fluorophore in biological sample 130. For example, these wavelengths may correspond to peaks and / or extensions across a wide range of wavelengths in the emission spectra of multiple fluorophores in the sample. This may include imaging sensor 120 being a monochromatic imaging sensor that is sensitive to the wavelength of light in each emission spectrum emitted by a phase lamp or other light source and / or the wavelength of the light. Furthermore, the imaging sensor 120 can be configured to measure any suitable photoluminescence, including fluorescence intensity (FLINT), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), fluorescence correlation (FCS), fluorescence recovery after photobleaching (FRAP), and their phosphorescence and other similar phenomena.

[0093] exist Figures 6-8 and Figures 11-12 In one alternative embodiment shown, system 105 includes a shaft 170 extending through optical module 110. Here, fluorescence microscope 115 has a receptacle (not shown) configured to receive shaft 170 in a first orientation. Shaft 170 is configured to rotate under a force applied to a second orientation, thereby locking optical module 110 to the remainder of fluorescence microscope 115 (e.g., to the housing or other element of fluorescence microscope 115). For example, shaft 170 may have a flip tab 171 coupled to a first end 172 and may have a protrusion 173 coupled to a second end 174, thereby forming a T-shape. Fluorescence microscope 115 may have a corresponding slot (not shown) and a receptacle configured to receive the T-shaped protrusion 173 in the first orientation. When shaft rotates to the second orientation under a force applied to flip tab 171, T-shaped protrusion 173 rotates in the receptacle such that T-shaped protrusion 173 aligns with the slot, thereby locking optical module 110 to fluorescence microscope 115. Once the optical module 110 is coupled to the remainder of the fluorescence microscope 115, the flip-up tab 171 can be folded flat against the housing 111.

[0094] In another embodiment, once the shaft 170 is inserted, rotation of the shaft 170 in the locking direction causes the protrusion 173 to travel along the ramp, thereby pulling the protrusion 173, the shaft 170, and the optical module 110 onto the mount of the remainder of the system 105. A flexible element (e.g., a spring 189) may be arranged between the shaft 170 and the housing 111 of the optical module 110 to control the force pulling the optical module 110 onto the mount. Furthermore, a stop may provide tactile feedback when the shaft 170 is in the locked and unlocked positions.

[0095] exist Figure 4 In another alternative embodiment shown, system 105 includes a first electrical connector 175 coupled to optical module 110. System 105 includes a second electrical connector (not shown) coupled to the remainder of fluorescence microscope 115. The second electrical connector is the reverse of the first electrical connector 175. System 105 includes a processor 202 in electrical communication with the second electrical connector. Processor 202 is configured to identify optical module 110 coupled to the remainder of fluorescence microscope 115. The first electrical connector 175 and the second electrical connector can be selected to require less than a specified force to connect and disconnect, for example, to make it easier for the user to replace different optical modules of fluorescence microscope 115.

[0096] The replaceable phase lamp module 125 includes a housing 126, a light source 127 (e.g., a halogen lamp, LED), and at least one focusing lens that focuses light from the phase lamp module 125 onto the biological sample 130 from above. In another alternative embodiment, such as Figures 14-15 As shown, the phase lamp module 125 has a third electrical connector 177, which corresponds to a fourth electrical connector (not shown) coupled to the remainder of the fluorescence microscope 115 (e.g., to the same housing to which the second electrical connector is coupled). The third electrical connector 177 and the fourth electrical connector are inverses of each other. The processor 202 is configured to determine whether the optical module 110 and the phase lamp module 125 are compatible, and to display an alarm in response to determining that the optical module 110 and the phase lamp module 125 are incompatible. This determination can be made by searching a database containing records of valid correspondences between available optical modules 110 and available phase lamp modules 125. Additionally or alternatively, this determination can be made by comparing the wavelengths of a set of light emitted by the phase lamp module 125 with the wavelengths of a set of light that the optical module 110 is configured to transmit from the sample 130 to the imaging sensor 120.

[0097] The housing 126 of the phase lamp module 125 includes a protrusion 128 formed to be received into a corresponding receptor 186 in the phase lamp holder 185 of the system 105. The protrusion 128 and the receptor 186 are formed such that the phase lamp module 125 can only be mounted in a single orientation. The phase lamp module 125 is held in place by a stop 187, rather than by screws. The stop 187 is in the form of a spring-loaded ball 188 in the phase lamp holder 185, which is configured to align with a recess 129 in the protrusion 128 of the housing 126.

[0098] In one alternative implementation, such as Figure 1 As shown, system 105 includes an incubator 180 configured to maintain a live cellular biological sample 130 in a temperature range of 30°C to 42°C and a relative humidity range of 80% to 100%. In this embodiment, a fluorescence microscope 115 according to a first aspect of the present disclosure is coupled to a chamber of the incubator 180. In another embodiment, the fluorescence microscope 115 may be partially or completely contained within the incubator 180. For example, the fluorescence microscope 115 may be completely housed within a standard CO2 incubator for periodic examination of the biological sample 130 during continuous culture (e.g., over specified time periods of several hours, days, or weeks). Due to the length of the culture time, the fluorescence microscope 115 can be kept in the incubator during cell culture to avoid adverse effects on the biological sample 130. Furthermore, the compact profile of the fluorescence microscope 115 maintains the functionality of the incubator 180 for placing other biological samples 130 within the open space surrounding the fluorescence microscope 115. The compact profile of the fluorescence microscope 115 also reduces airflow limitation, which can have detrimental effects on the fluorescence microscope 115 or the biological sample 130 in the form of condensation and inadequate ventilation.

[0099] The third light source 145 (and the fourth light source 160) enables reuse assays on biological sample 130 to perform multiple assays and / or fluorescence indicators in a single sample (e.g., sample well) in incubator 180 and / or in a group of different samples in the same incubator 180.

[0100] V. Example Method

[0101] Now for reference Figure 16 This shows that it can be utilized Figures 3-15 The optical module 110 and system 105 and Figures 1-2The computing device 200 is used for a method 300 to image fluorophores in a live cellular biological sample. In block 305, method 300 includes aligning a first biological sample and a fluorescence microscope such that the first biological sample is within the field of view of the fluorescence microscope, wherein the first biological sample comprises: (i) a first fluorophore that emits light in a first emission wavelength band in response to illumination by light in a first excitation wavelength band; (ii) a second fluorophore that emits light in a second emission wavelength band in response to illumination by light in a second excitation wavelength band; and (iii) a third fluorophore that emits light in a third emission wavelength band in response to illumination by light in a third excitation wavelength band. Then, in block 310, the method includes obtaining an image set of the first biological sample using the fluorescence microscope, wherein the images in the image set differ in terms of focus settings. Next, in block 315, method 300 includes determining a first focus setting, a second focus setting, and a third focus setting, respectively, for the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band, based on the image set. In block 320, the method includes: during a first time period, illuminating a first biological sample with light in a first excitation wavelength band using a first light source, and operating a fluorescence microscope according to a first focusing setting to obtain a first image of light in a first emission wavelength band via an image sensor of the fluorescence microscope. In block 325, the method 300 further includes: during a second time period, illuminating the first biological sample with light in a second excitation wavelength band using a second light source, and operating a fluorescence microscope according to a second focusing setting to obtain a second image of light in a second emission wavelength band via an image sensor. In block 330, the method 300 further includes: during a third time period, illuminating the first biological sample with light in a third excitation wavelength band using a third light source, and operating a fluorescence microscope according to a third focusing setting to obtain a third image of light in a third emission wavelength band via an image sensor. All the foregoing steps can be performed automatically by processor 202.

[0102] Obtaining a specific fluorescence image of a particular color can include operating the imaging system to generate multiple different images of the specific color using different exposure times. This can be done to allow the synthesis of high dynamic range images. This can be done on a fluorophore / sample / measurement that exhibits very high variations in fluorescence emission intensity within the concentration / activity range of the fluorophore of interest.

[0103] Method 300 may additionally include obtaining one or more bright-field, phase-contrast, or other non-fluorescent images by operating a phase lamp (e.g., 125) or other light source. Such non-fluorescent image information can then be combined with fluorescent images (e.g., using phase-contrast images to identify the location, shape, size, and / or extent of cells in a sample, regardless of cell type, and then using one or more fluorescent images to determine cell type, cell division phase, cell health, cell metabolic activity, or other information about cells identified using the phase-contrast images) or used on its own.

[0104] Furthermore, method 300 may include: obtaining a set of bright-field, phase-contrast, or other non-fluorescent images spanning a range of different focal settings (e.g., a range of different objective-sample distances) to determine the focus settings for three (or more) fluorescence images obtained using method 300. This may include: determining the focus settings for the illumination wavelengths used to generate the non-fluorescent images, and then applying a known offset (e.g., a distance offset) from that focus setting for each of the fluorescence emission wavelengths being imaged to determine the focus settings for those emission wavelengths. The offset may be zero if the wavelength of the illumination used to generate the non-fluorescent images is the same as or substantially the same as one of the fluorescence emission wavelengths. Bright-field images or other non-fluorescent images typically contain significantly more image data in the same exposure time when compared to fluorescence images, therefore this method of determining focus settings can advantageously reduce the time required to generate such focus settings. This can also reduce the amount of photobleaching the sample undergoes in order to generate such focus settings.

[0105] In practice, images of fluorophores excited by shorter wavelengths may include artifacts associated with light emitted from fluorophores excited by longer wavelengths, and vice versa. As an example, a fluorophore corresponding to a first wavelength band can also be excited to some extent by a second and third wavelength band (especially if both the second and third wavelength bands include wavelengths shorter than the first wavelength band). Similarly, a fluorophore corresponding to a second wavelength band can also be excited to some extent by the first and third wavelength bands, and a fluorophore corresponding to a third wavelength band can also be excited to some extent by the first and second wavelength bands.

[0106] To address the artifact problem, in an alternative embodiment, method 300 further includes a processor 202 electrically communicating with imaging sensor 120 (or some other computing device) to generate a first image of light emitted by a first fluorophore based on a first image set, in order to reduce artifacts from light emitted by a second and third fluorophore. The processor 202 additionally generates a second image of light emitted by a second fluorophore based on a second image set, in order to reduce artifacts from light emitted by the first and third fluorophores. Furthermore, the processor 202 may generate a third image of light emitted by a third fluorophore based on a third image set, in order to reduce artifacts from light emitted by the first and second fluorophores. This spectral unmixing process is described in more detail with respect to the two excitation wavelengths in U.S. Patent Application No. 16 / 264819, filed February 1, 2019, which is incorporated herein by reference.

[0107] In a non-limiting example, when the biological sample 130 is positioned at a first focusing setting such that the light emitted from the green fluorophore is focused, a first image of the green fluorophore can be obtained by illuminating the biological sample 130 with light corresponding to the blue excitation wavelength of the green fluorophore. Specifically, the "focus setting" occurs by setting the distance between the biological sample 130 and the objective lens 165 of the fluorescence microscope 115 such that the light emitted from the green fluorophore is focused and imaged. This first image may also include light emitted from the orange fluorophore (primarily excited by light at a yellow-green excitation wavelength, but also to some extent by blue light) and the NIR fluorophore (primarily excited by light at a red excitation wavelength, but also to some extent by blue light) in the biological sample 130. Note that the light emitted from the orange fluorophore and the NIR fluorophore will be out of focus in the first image. This is due to chromatic aberration caused by elements along the main emission light path 156 between the sample 130 and the imaging sensor 120 (e.g., objective lens, tube lens, sample, and / or the optical properties of the container holding the sample).

[0108] The artifact image can then be removed from the first image to remove artifact light from the orange fluorophore and NIR fluorophore. As described above, an artifact image can be obtained by illuminating the biological sample 130 with light corresponding to the yellow-green excitation wavelength of the orange fluorophore at the first focusing setting used to obtain the first image. Another artifact image can be obtained by illuminating the biological sample 130 with light corresponding to the red excitation wavelength of the red fluorophore at the first focusing setting used to obtain the first image. Alternatively, an artifact image can be obtained by blurring the focusing images of the orange and red fluorophores or otherwise applying some image processing techniques to simulate the effect of the focusing setting used to obtain the first image in an image taken using a different focusing setting. For example, an image taken using a focusing setting allows the light emitted from the orange fluorophore and NIR fluorophore to be focused and imaged separately.

[0109] In an optional implementation, method 300 further includes an incubator that includes or is otherwise coupled to a fluorescence microscope, wherein the incubator maintains the first biological sample at least within a temperature range of 30°C to 42°C and a relative humidity range of 80% to 100% when the first image set data, the second image set data, and the third image set data are acquired. This data can be obtained in a standard CO2 incubator for timed examination of the biological sample 130 during continuous incubation (e.g., within timeframes of minutes, hours, days, or weeks, depending on the experiment of interest). For example, images can be taken over time throughout the incubation, which may exceed 14 days, or up to 30 days or more.

[0110] Method 300 may include performing additional analyses on the fluorescence image and / or other images obtained using the system (e.g., bright-field images, phase-contrast images). For example, in a particular sample, if one (or more) in the image corresponds to the color of a fluorophore specific to one or more particular types of cells, method 300 may include analyzing the image to determine the number, shape, size, distribution, interconnection pattern, or other information about the presence of one or more particular types of cells in the sample. Such identification can be enhanced by using phase-contrast or other non-fluorescent images to identify the location, shape, and extent of individual cells in the sample, regardless of type. Additionally or alternatively, if one (or more) in the image corresponds to a fluorescent indicator for a particular assay (e.g., annexin VNIR assay, two-color or three-color FUCCI cell division phase assay), method 300 may include analyzing the image to generate the output of that particular assay, for example, to determine the health, cell division phase, or other metabolic status or state of one or more cells in the sample. The results of different (or overlapping) analyses of images can be combined. For example, a first image analysis corresponding to a cell-specific fluorophore can be used to identify cells of the cell type of interest, and combined second and third image analyses can determine the output of a two-color assay (e.g., a two-color FUCCI assay) for the identified cells. The analysis can be identical for each sample imaged by the imaging system in the incubator (e.g., since all samples contain the same assay / fluorescent indicator / dye), or it can differ between samples within the incubator.

[0111] In an alternative implementation, method 300 further includes: processor 202 identifying a first cell type in a first biological sample based on a first image set. Then, processor 202 identifies a second cell type in the first biological sample based on a second image set. Next, processor 202 identifies cell death or some other metabolic process or characteristic in the first biological sample based on a third image set. This has the technical effect of allowing complex assays and / or multiple assays to be performed in a single dish in system 105 without altering the configuration of the optical module 110.

[0112] In an alternative implementation, phase-contrast, bright-field, or other non-fluorescent images can be obtained at the first, second, and third focus settings. These images can be used to remove further artifacts from the first, second, and third images (e.g., remove artifacts caused by autofluorescence) or to otherwise improve the first, second, and third images (e.g., enhance the fluorescent image by providing additional high spatial frequency image data).

[0113] In an alternative implementation, method 300 further includes: the processor receiving compatibility information between the optical module 110 and the phase lamp module. The processor 202 then determines, based on the compatibility information, whether the optical module 110 and the phase lamp 125 are compatible. Next, in response to determining that the optical module 110 and the phase lamp 125 are incompatible, the processor 202 displays an alarm indicating incompatibility. This compatibility determination can be performed by searching a database containing records of valid correspondences between available optical modules 110 and available phase lamp modules 125. Additionally or alternatively, this determination can be performed by comparing the wavelengths of a set of light emitted by the phase lamp module 125 with the wavelengths of a set of light that the optical module 110 is configured to transmit from the sample 130 to the imaging sensor 120.

[0114] In an alternative embodiment, method 300 includes extending shaft 170 in a first orientation through optical module 110 to a base in fluorescence microscope 115. Then, shaft 170 is rotated under applied force such that shaft 170 moves to a second orientation, thereby coupling optical module 110 to fluorescence microscope 115, as described in detail above with respect to system 105.

[0115] The aforementioned method 300 has the following technical effects: it allows for increased variability in the determination without changing the configuration of the optical module 110, which can be performed in a single vessel or across multiple vessels in system 105. Method 300 can be performed by any embodiment of a fluorescence imager, optical module, phase lamp module, incubator, automated imaging system, or other systems, devices, or components described herein, or can be performed in combination with such an embodiment. Therefore, through the embodiments described herein, multiple determinations, fluorescence indicators, and combinations thereof are possible for a single sample within an incubator and / or between different samples within an incubator. Several examples of such applications are provided below. These applications are intended as illustrative examples and are not intended to be limiting. Additional or alternative applications are expected, as are additional or alternative combinations of such applications and / or applications described below.

[0116] As discussed above, a non-transitory computer-readable medium stores program instructions that, when executed by processor 202, can be used to perform any of the methods described herein.

[0117] VI. Example Biological Applications

[0118] By providing independent imaging with three different fluorescence channels, the embodiments described above enable a variety of applications in fluorescence imaging. The advantages of additional fluorescence channels can include: improved throughput of a single imaging device by allowing simultaneous additional assays using a single device (e.g., in a single incubator), by providing flexibility in experimental readings across different samples, and / or by allowing additional assays from individual samples. The availability of three (or more) color imaging also allows for increased flexibility in reagent selection, such as the ability to use multiple reagents to obtain additional information while monitoring cells expressing a green fluorescent protein-based reporter.

[0119] Additionally or alternatively, simultaneous three (or more) color imaging can allow the generation of information that cannot be generated using only two colors. For example, information from trichromatic reporters (e.g., trichromatic FUCCI assays) can be generated. In another example, information about the proliferation and interactions of multiple cell types, as well as metabolic or cell death information across cell populations, can be generated. Such information can enable the addition of insights into the effects of experimental conditions on the activity of effector cells in disrupting metabolic exchange between target cells or, for example, cancer and stromal cells.

[0120] Furthermore, three (or more) color imaging can allow experimental data to be generated with greater confidence. For example, multiplexing multiple readings in a single sample can provide increased confidence that differences observed between readings are scientifically valid, rather than due to experimental variations between parallel-run assays (e.g., cell plane culture).

[0121] Example 1: Two-color FUCCI+ cell death

[0122] The various embodiments of the optical module 110, system 105, and method 300 described herein can be advantageously used for two-color cell cycle (e.g., green / orange FUCCI) observation and cell death analysis in a single sample. For example, cell cycle and apoptosis readings can demonstrate differences in the concentration- and time-dependent effects of compounds in cancer cells. In another example, two-color cell cycle observation and cell death analysis can be performed in a sample containing immune cells and cancer cells to observe the effects of targeted immune cell killing of cancer cells on cancer cells and / or some other cell populations in the sample. Multiplexing these two readings in a single sample increases throughput and provides confidence that the difference between the two readings is scientifically valid rather than due to experimental variation between parallel assays.

[0123] Regarding two-color FUCCI analysis, the three-color optical module disclosed herein allows for differentiation between different cell cycle phases based on the period-dependent expression of two different fluorescent proteins. For example, in S, G2, and M phases, cells can emit green fluorescence via the expression of TagGFP2 (a green fluorophore), which can be detected using this three-color optical module. During G1 phase and transition to S phase, cells can emit orange fluorescence via the expression of TagRFP (an orange fluorescent protein with bright fluorescence excitation / emission maxima at 55 nm and 584 nm, respectively), which can be detected using this three-color optical module. In this two-color FUCCI assay, cells immediately transition through a colorless phase after mitosis. Therefore, cell cycle phases have a fluorescence footprint (e.g., S, G2, and M phases: green; G1 phase: orange; G1 / S transition: both orange and green; M / G1 transition: colorless (no fluorescence)). The third color can then be used for imaging fluorescent cell death readings (e.g., annexin VNIR apoptosis indicator).

[0124] Example 2: Three-color FUCCI

[0125] Different embodiments of the optical module 110, system 105, and method 300 described herein can be used to advantageously perform a tricolor FUCCI assay that independently observes additional phases within the cell cycle. Unlike the previously described two-color cell cycle assay, the tricolor assay allows for differences between the S and G2 phases and also advantageously results in the absence of a colorless phase through appropriate gene expression of fluorescent proteins fused to targets associated with targeted ubiquitin-acylated domains or other cell phases. For example, during the S, G2, and M phases, cells can each emit green fluorescence based on the expression of TagGFP2, which can be detected using the tricolor optical module, with a darker fluorescence observed in the S phase. Different fluorescent markers can be used to identify other phases of the cell cycle, including but not limited to TagRFP and iRFP713. During the G1 and S phases, cells expressing TagRFP emit orange fluorescence, which can be detected using the tricolor optical module. iRFP713 is a near-infrared fluorescent protein with fluorescence maxima at excitation / emission points of 690 nm and 713 nm, respectively. During the G2, M, and G1 phases, cells expressing iRFP713 emit NIR fluorescence, which can be detected using a three-color optical module. Therefore, cell cycle phases will have a fluorescence footprint (e.g., G2 and M phases: green and NIR; G1 phase: orange and NIR; S phase: orange and green), and there is no colorless phase.

[0126] Example 3: Trichromatic immune cell killing

[0127] The various embodiments of the optical module 110, system 105, and method 300 described herein can be used to advantageously monitor labeled target (cancer) cells and effector (immune) cells and provide cell death readings across both cell types. This provides the benefit of being able to independently measure the proliferation and interaction of target and effector cells simultaneously across cell death in both populations. In this way, the efficacy of immune cells in killing target cells can be directly correlated with (e.g., associated with activation) changes in the effector cell population and the interaction between the two cell populations in the same sample, the efficacy of immune cells in killing target cells being measured by determining the overlap between the fluorescence of effector and target cell labels.

[0128] Typically, the recognition and killing of unwanted target cells (such as emergency tumor cells) by immune cells is a key component of the human host defense mechanism. Antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell killing are two mechanisms of cell-mediated immune responses. Each of these processes involves the stimulation of immune cell subpopulations (such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs)), which then actively lyse the target cells. The systems and methods disclosed herein allow for the observation of interactions between immune cells and cancer cells, potentially providing information leading to the development of diagnostics and therapies for restoring and enhancing the immune system's ability to fight and eliminate tumors (“cancer immunotherapy” or “immuno-oncology”).

[0129] Example 4: ATP + cell death

[0130] Different embodiments of the optical module 110, system 105, and method 300 described herein can be used to advantageously perform ATP measurements based on two-color Forster resonance energy transfer (FRET), which can be reused with cell death analysis to investigate potential differences in the time- and concentration-dependent effects of compounds on the metabolism and mortality of cancer cells. In operation, three light sources in the optical module can be activated in three different excitation wavelength bands, with two of the three light sources used to measure metabolic information via a single emission band through the FRET mechanism. This third excitation wavelength can then be used to monitor independent readings associated with cell death (e.g., annexin VNIR). The ATP measurement procedure is described in detail in PCT / US19 / 21171, filed March 7, 2019, entitled “Methods and Compositions for Live Cell Analysis of Intracellular ATP,” the contents of which are incorporated herein by reference in their entirety.

[0131] Example 5: Live-cell immunocytochemistry

[0132] The different embodiments of the optical module 110, system 105, and method 300 described herein can be used to... Live cell immunocytochemistry (ICC) using FabFluor antibody-labeled reagents (or some other fluorescently labeled antibody reagents) is used to measure surface protein expression. This method can be used to track changes in cell subpopulations after experimental treatments (e.g., the addition of test compounds or immune cell activation), monitor changes in differentiation markers over time, or otherwise assess surface protein expression. Three (or more) color imaging offers advantages in terms of antibody selection flexibility and allows for the monitoring of additional proteins or subpopulations of interest in a single sample.

[0133] For illustrative and descriptive purposes, descriptions of various advantageous arrangements have been presented and are not intended to be exhaustive or limited to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. One or more examples have been selected and described in order to best explain the principles of the examples, their practical application, and to enable others skilled in the art to understand the various examples used with various modifications suitable for the particular intended purpose.

Claims

1. An optical module for imaging fluorophores in live cell biological samples, comprising: A first light source, configured to emit first light in a first excitation wavelength band; A first filter is disposed in a first optical path of the first light source, and the first filter is configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths. A second light source, configured to emit second light in a second excitation wavelength band; A second filter is arranged in the second optical path of the second light source, and the second filter is configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths. A third light source, configured to emit third light in a third excitation wavelength band; A third filter, disposed in the third optical path of the third light source, is configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths, wherein the first, second, and third optical paths converge along a main transmission optical path, which is configured to guide toward the live cell biological sample; and An emission filter is arranged in a main emission optical path for light emitted by the fluorophore in the live cell biological sample, wherein the main emission optical path is configured to terminate at an imaging sensor, wherein the emission filter is configured to allow light in a first emission wavelength band, a second emission wavelength band, and a third emission wavelength band to pass through, and wherein the emission filter is configured to reflect light in the first excitation wavelength band, the second excitation wavelength band, and the third excitation wavelength band; The first filter is configured to allow light in the first excitation wavelength band to pass through, and to reflect light in the second excitation wavelength band and the third excitation wavelength band, as well as light in the first emission wavelength band, the second emission wavelength band and the third emission wavelength band. The second filter is configured to allow light from the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band to pass through, and to reflect light from the second excitation wavelength band and the third excitation wavelength band; and The third filter is configured to allow light in the third excitation wavelength band to pass through and to reflect light in the second excitation wavelength band.

2. The optical module according to claim 1, wherein, The first filter, the second filter, and the third filter are all dichroic filters.

3. The optical module according to any one of claims 1-2, wherein, The first light source, the second light source, and the third light source each include: an LED, at least one lens, and a single-bandpass dichroic filter.

4. The optical module according to claim 1, wherein, The first light source is arranged such that the first optical path begins at the first light source, passes through the first filter, and exits the optical module along the main transmission optical path; The second light source is arranged such that the second optical path begins at the second light source, is reflected from the third filter to the second filter, is reflected from the second filter to the first filter, is reflected from the first filter, and leaves the optical module along the main transmission optical path; The third light source is arranged such that the third optical path begins at the third light source, passes through the third filter to the second filter, is reflected from the second filter to the first filter, is reflected from the first filter, and exits the optical module along the main transmission optical path; and The main emission path of the light emitted by the fluorophore in the living cell biological sample is reflected from the first filter, passes through the second filter, passes through the emission filter, and exits the optical module.

5. The optical module according to claim 1, wherein, The first filter is configured to allow light in the first excitation wavelength band and the second excitation wavelength band to pass through, and to reflect light in the third excitation wavelength band and in the first emission wavelength band, the second emission wavelength band and the third emission wavelength band; The second filter is configured to allow light in the second emission wavelength band to pass through and to reflect light in the first excitation wavelength band; and The third filter is configured to allow light in the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band to pass through, and to reflect light in the third excitation wavelength band.

6. The optical module according to claim 5, wherein, The first light source is arranged such that the first optical path begins at the first light source, is reflected from the second filter, passes through the first filter, and exits the optical module along the main transmission optical path; The second light source is arranged such that the second optical path begins at the second light source, passes through the second filter, then passes through the first filter, and exits the optical module along the main transmission optical path; The third light source is arranged such that the third optical path begins at the third light source, reflects from the third filter to the first filter, reflects from the first filter, and exits the optical module along the main transmission optical path; and The main emission path of the light emitted by the fluorophore in the living cell biological sample is reflected from the first filter, passes through the third filter, passes through the emission filter, and exits the optical module.

7. The optical module according to claim 1, wherein, The first excitation wavelength band ranges from 453 nm to 485 nm, the second excitation wavelength band ranges from 546 nm to 568 nm, and the third excitation wavelength band ranges from 648 nm to 674 nm.

8. The optical module according to claim 1, wherein, The first emission wavelength band ranges from 494 nm to 533 nm, the second emission wavelength band ranges from 576 nm to 639 nm, and the third emission wavelength band ranges from 686 nm to 756 nm.

9. The optical module according to claim 1, further comprising: A fourth light source, configured to emit a fourth light in a fourth excitation wavelength band; as well as A fourth filter is arranged in the fourth optical path of the fourth light source. The fourth filter is configured to allow light of one or more wavelengths to pass through and reflect light of one or more wavelengths. The emission filter is further configured to allow light in a fourth emission wavelength band to pass through and reflect light in a fourth excitation wavelength band.

10. The optical module according to claim 9, wherein, The first filter is configured to allow light in the first excitation wavelength band to pass through and to reflect light in the fourth excitation wavelength band; The second filter is configured to allow light in the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band to pass through, and to reflect light in the second excitation wavelength band and the third excitation wavelength band. The third filter is configured to allow light in the third excitation wavelength band to pass through and to reflect light in the second excitation wavelength band; and The fourth filter is configured to allow light in the first excitation wavelength band and the fourth excitation wavelength band to pass through, and to reflect light in the second excitation wavelength band and the third excitation wavelength band, as well as light in the first emission wavelength band, the second emission wavelength band, the third emission wavelength band, and the fourth emission wavelength band.

11. The optical module according to any one of claims 9-10, wherein, The fourth emission wavelength band is less than 453nm, and the fourth excitation wavelength band is less than the fourth emission wavelength band.

12. The optical module according to claim 9, wherein, The first light source is arranged such that the first optical path begins at the first light source, passes through the first filter, then passes through the fourth filter, and exits the optical module along the main transmission optical path; The second light source is arranged such that the second optical path begins at the second light source, is reflected from the third filter to the second filter, is reflected from the second filter to the fourth filter, is reflected from the fourth filter, and leaves the optical module along the main transmission optical path; The third light source is arranged such that the third optical path begins at the third light source, passes through the third filter to the second filter, is reflected from the second filter to the fourth filter, is reflected from the fourth filter, and leaves the optical module along the main transmission optical path; The fourth light source is arranged such that the fourth optical path begins at the fourth light source, is reflected from the first filter, passes through the fourth filter, and exits the optical module along the main transmission optical path; and The main emission path of the light emitted by the fluorophore in the live cell biological sample is reflected from the fourth filter, passes through the second filter, passes through the emission filter, and exits the optical module.

13. A system for determining live cell biological samples, the system comprising: The optical module according to any one of claims 1-12; A fluorescence microscope detachably coupled to the optical module, wherein the fluorescence microscope has at least one objective lens; An imaging sensor, arranged in the emission path for light emitted by a fluorophore from the live-cell biological sample from the objective lens; and A phase lamp, which is detachably coupled to the fluorescence microscope and is positioned at the end of the main transmission optical path.

14. The system of claim 13, further comprising: An axis extending through the optical module, wherein the fluorescence microscope has a base configured to receive the axis in a first orientation, wherein the axis is configured to rotate under a force applied to a second orientation, thereby locking the optical module to the fluorescence microscope.

15. The system of claim 13, further comprising: A first electrical connector is coupled to the optical module; A second electrical connector is coupled to the fluorescence microscope, wherein the second electrical connector is the reverse of the first electrical connector; as well as A processor that is in electrical communication with at least one of the first and second electrical connectors, the processor being configured to identify the optical module coupled to the fluorescence microscope.

16. The system of claim 15, further comprising: The phase lamp having a third electrical connector; as well as A fourth electrical connector is coupled to the fluorescence microscope, wherein the third electrical connector is the reverse of the fourth electrical connector, and wherein the processor is configured to determine whether the optical module and the phase lamp are compatible and to display an alarm in response to the determination.

17. The system of claim 13, further comprising: An incubator configured to maintain the live cell biological sample in a temperature range of 30°C to 42°C and a relative humidity range of 80% to 100%, wherein the optical module is coupled to the chamber of the incubator.

18. A method for imaging fluorophores in live cellular biological samples, wherein the method is implemented by a processor in electromechanical communication with the optical module of any one of claims 1-12, the method comprising: A first biological sample and a fluorescence microscope are aligned such that the first biological sample is within the field of view of the fluorescence microscope, wherein the first biological sample comprises: (i) a first fluorophore that emits light in a first emission wavelength band in response to illumination by light in a first excitation wavelength band; (ii) a second fluorophore that emits light in a second emission wavelength band in response to illumination by light in a second excitation wavelength band; and (iii) a third fluorophore that emits light in a third emission wavelength band in response to illumination by light in a third excitation wavelength band. An image set of the first biological sample was obtained using the fluorescence microscope, wherein the images in the image set differed in terms of focus settings; Based on the image set, a first focus setting, a second focus setting, and a third focus setting are determined for the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band, respectively. During a first time period, the first biological sample is illuminated with light in the first excitation wavelength band using a first light source, and the fluorescence microscope is operated according to the first focusing setting to obtain a first image of the light in the first emission wavelength band via the imaging sensor of the fluorescence microscope. During the second time period, the first biological sample is illuminated with light in the second excitation wavelength band using a second light source, and the fluorescence microscope is operated according to the second focusing setting to obtain a second image of the light in the second emission wavelength band via the imaging sensor; and During the third time period, the first biological sample is illuminated with light in the third excitation wavelength band using a third light source, and the fluorescence microscope is operated according to the third focusing setting to obtain a third image of light in the third emission wavelength band via the imaging sensor.

19. The method of claim 18, further comprising: A first corrected image of the light emitted by the first fluorophore is generated based on the first image, the second image, and the third image via a processor that is in electrical communication with the imaging sensor, in order to reduce artifacts from the light emitted by the second fluorophore and the third fluorophore; The processor generates a second corrected image of the light emitted by the second fluorophore based on the first image, the second image, and the third image, in order to reduce artifacts from the light emitted by the first fluorophore and the third fluorophore; as well as The processor generates a third corrected image of the light emitted by the third fluorophore based on the first image, the second image, and the third image, in order to reduce artifacts from the light emitted by the first fluorophore and the second fluorophore.

20. The method of claim 18, further comprising: When the first image, the second image, and the third image are obtained, the first biological sample is maintained at a temperature range of at least 30°C to 42°C and a relative humidity range of 80% to 100% via an incubator coupled to the fluorescence microscope.

21. The method of claim 18, further comprising: A first corrected image is generated based on the phase image or bright field image obtained when the first biological sample is at the first focus setting and the first image.

22. The method of claim 18, further comprising: The processor receives compatibility information of the optical module and phase lamp module of the fluorescence microscope. The processor determines, based on the compatibility information, whether the optical module is compatible with the phase lamp; and In response to the determination that the optical module and the phase lamp are incompatible, an alarm indicating incompatibility is displayed via the processor.

23. The method of claim 18, further comprising: The axis extends in a first orientation through the optical module of the fluorescence microscope to the base in the fluorescence microscope; as well as The axis is rotated under applied force, causing it to move to a second orientation, thereby coupling the optical module to the fluorescence microscope.

24. A non-transitory computer-readable medium storing program instructions that, when executed by a processor in electromechanical communication with an optical module according to any one of claims 1-12, cause a set of actions to be performed, the set of actions comprising: A first biological sample and a fluorescence microscope are aligned such that the first biological sample is within the field of view of the fluorescence microscope, wherein the first biological sample comprises: (i) a first fluorophore that emits light in a first emission wavelength band in response to illumination by light in a first excitation wavelength band; (ii) a second fluorophore that emits light in a second emission wavelength band in response to illumination by light in a second excitation wavelength band; and (iii) a third fluorophore that emits light in a third emission wavelength band in response to illumination by light in a third excitation wavelength band. The imaging sensor uses the fluorescence microscope to obtain a first set of images of the first biological sample, wherein the images in the set differ in terms of focus settings; Based on the image set, a first focus setting, a second focus setting, and a third focus setting are determined for the first emission wavelength band, the second emission wavelength band, and the third emission wavelength band, respectively. During a first time period, the first biological sample is illuminated with light in the first excitation wavelength band using a first light source, and the fluorescence microscope is operated according to the first focusing setting to obtain a first image of the light in the first emission wavelength band via the imaging sensor of the fluorescence microscope. During the second time period, the first biological sample is illuminated with light in the second excitation wavelength band using a second light source, and the fluorescence microscope is operated according to the second focusing setting to obtain a second image of the light in the second emission wavelength band via the imaging sensor; and During the third time period, the first biological sample is illuminated with light in the third excitation wavelength band using a third light source, and the fluorescence microscope is operated according to the third focusing setting to obtain a third image of light in the third emission wavelength band via the imaging sensor.

Images