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

Abstract

To provide an optical module with three or more color fluorescent light sources and methods for use thereof.SOLUTION: An imaging apparatus is provided to facilitate epifluorescent imaging of three (or more) color channels and to perform phase contrast and / or bright field imaging of samples without manual adjustment of the imaging apparatus. This allows automated imaging, over extended periods of time, of a plurality of samples by a device located inside an incubator without disturbing the incubator environment to manually adjust the apparatus. Also provided are embodiments to facilitate user swapping of removable optical modules and / or transillumination modules to allow the imaging apparatus to be adapted to different combinations of assays and / or fluorescent indicators so as to increase the variety of experiments and / or fluorescent dyes that can be imaged using the imaging apparatus.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Non-Provisional Patent Application No. 16 / 854,756, filed April 21, 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Live cell biological samples can be microscopically imaged in a variety of ways to assess the growth, metabolism, morphology, or other characteristics of the sample 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 excitation wavelength of the fluorophore, causing them to fluorescently emit light at the emission wavelength of the fluorophore. In epifluorescence imaging, the excitation light is provided through the same objective lens used to collect the emission light.

[0003] Achieving multi-channel fluorescence imaging often requires moving a different filter set (and possibly an excitation light source) to the appropriate position each time a fluorescence image is acquired for a particular emission wavelength. However, such an arrangement results in a larger, slower, more costly, and less reliable system due to the need to physically move components. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] PCT / US19 / 21171 Summary of the Invention [Means for solving the problem]

[0005] In a first aspect, an exemplary optical module for imaging fluorophores in live cell biological samples is disclosed. The optical module includes: (a) a first light source configured to emit first light at a first band of excitation wavelengths; (b) a first filter disposed in a first optical path of the first light source, the first filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths; (c) a second light source configured to emit second light at a second band of excitation wavelengths; (d) a second filter disposed in a second optical path of the second light source, the second filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths; (e) a third light source configured to emit third light at a third band of excitation wavelengths; and (f) a third filter disposed in a third optical path of the third light source. (g) an emission filter disposed in a primary emission optical path for light emitted by fluorophores in the live cell biological sample, the primary emission optical path being configured to terminate at an imaging sensor; and (h) an emission filter disposed in a primary emission optical path for light emitted by fluorophores in the live cell biological sample, the primary emission optical path being configured to terminate at an imaging sensor; the emission filter configured to pass light at emission wavelengths of the first band, the second band, and the third band, and to reflect light at excitation wavelengths of the first band, the second band, and the third band.

[0006] In a second aspect, an exemplary system for assaying a live cell biological sample is disclosed, the system including: (a) an optical module according to the first aspect of the present disclosure; (b) a fluorescence microscope removably coupled to the optical module, the fluorescence microscope having at least one objective lens; (c) an imaging sensor disposed in an emission path for light emitted by a fluorophore in the live cell biological sample from the objective lens; and (d) a phase lamp removably coupled to the fluorescence microscope, the phase lamp disposed at a terminal end of the primary transmission optical path.

[0007] In a third aspect, an exemplary method for imaging fluorophores in live cell biological samples is disclosed. The method includes: (i) aligning a first biological sample and a fluorescence microscope, wherein the first biological sample is positioned within a field of view of the fluorescence microscope, the first biological sample including (a) a first fluorophore that emits light at a first band of emission wavelengths in response to illumination with light at a first band of excitation wavelengths, (b) a second fluorophore that emits light at a second band of emission wavelengths in response to illumination with light at a second band of excitation wavelengths, and (c) a third fluorophore that emits light at a third band of emission wavelengths in response to illumination with light at a third band of excitation wavelengths; (ii) acquiring a set of images of the first biological sample using the fluorescence microscope, wherein the images in the set differ with respect to focus settings; and (iii) determining first, second, and third in-focus settings for the first, second, and third bands of emission wavelengths based on the set of images. (iv) illuminating a first biological sample with light of a first band of excitation wavelengths using a first light source during a first period of time and operating the fluorescence microscope according to a first in-focus setting to acquire a first image of light of the first band of emission wavelengths via an image sensor of the fluorescence microscope; (v) illuminating the first biological sample with light of a second band of excitation wavelengths using a second light source during a second period of time and operating the fluorescence microscope according to a second in-focus setting to acquire a second image of light of the second band of emission wavelengths via an image sensor; and (vi) illuminating the first biological sample with light of a third band of excitation wavelengths using a third light source during a third period of time and operating the fluorescence microscope according to a third in-focus setting to acquire a third image of light of the third band of emission wavelengths via an image sensor.

[0008] In a fourth aspect, an exemplary non-transitory computer-readable medium is disclosed having stored thereon program instructions that, when executed by a processor, cause the implementation of the method of the third aspect.

[0009] The features, functions, and advantages that have been discussed may be realized independently in various examples or may be combined in yet other examples, further details of which may be seen with reference to the following description and drawings. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a functional block diagram of a system according to one exemplary implementation. [Figure 2] FIG. 1 is a block diagram of a computing device and a computer network, according to an example implementation. [Figure 3] FIG. 1 is a functional block diagram of a system for assaying live cell biological samples, including an optical module, according to an exemplary implementation. [Figure 4] FIG. 1 is a functional block diagram of a system for assaying live cell biological samples, including an optical module, according to an exemplary implementation. [Figure 5] FIG. 1 is a functional block diagram of a system for assaying live cell biological samples, including an optical module, according to an exemplary implementation. [Figure 6] FIG. 2 is a front view of an optical module according to an exemplary implementation. [Figure 7] FIG. 7 is a rear view of the optical module according to the example implementation of FIG. 6. [Figure 8] 7 is a side view of an optical module according to the exemplary implementation of FIG. 6. [Figure 9] 7 is a front cross-sectional view of an optical module according to the exemplary implementation of FIG. 6. [Figure 10] 7 is a cross-sectional side view of an optical module according to the exemplary implementation of FIG. 6. [Figure 11] 7 is a cross-sectional side view of an optical module according to the exemplary implementation of FIG. 6. [Figure 12] FIG. 12 is a detailed view of the shaft of the optical module according to the example implementation of FIG. 11. [Figure 13] FIG. 1 is a functional block diagram of a system for assaying live cell biological samples, including an optical module, according to an exemplary implementation. [Figure 14] FIG. 1 is a perspective view of a phase lamp according to an exemplary implementation. [Figure 15] 1 is a cross-sectional view of a phase lamp coupled to a fluorescence microscope according to an exemplary implementation. [Figure 16] FIG. 1 illustrates a flowchart of a method according to an exemplary implementation. DETAILED DESCRIPTION OF THE INVENTION

[0011] The drawings are for purposes of illustrating examples, it being understood, however, that the invention is not limited to the arrangements and instrumentality shown in the drawings.

[0012] I. Overview Microscopic imaging of live cell samples can provide information about the health, growth, and activity of a population of cells under various experimental conditions. This information can include the number of cells in a sample over time, information about the morphology or other structural characteristics of the cells, the internal contents or structure of the cells (e.g., contents related to aspects of mitosis or other metabolic processes of the cells), or other information about the cells. This information can be used to evaluate the behavior of the cells under "normal" conditions and / or under various applied experimental conditions. For example, microscopic imaging of cells can be used to evaluate the response of cells to experimental drugs or other added substances, the effects of genetic modification of cells, the effects on cells of added cancer cells, other added cell types, and / or added bacteria, fungi, viruses, or other microorganisms, or the effect of some other applied experimental condition on a sample of live cells.

[0013] To reduce the cost of such imaging, to reduce the size of the equipment (e.g., incubator, imaging device) used to perform the imaging, to reduce the impact on the stability of the conditions applied to the cell samples, and / or to provide other benefits, microscopic imaging of multiple live cell samples can be performed by an automated imaging device configured to reside with the live cell samples inside the incubator. Such automated imaging device can include a gantry or other actuator configured to move the imaging device and / or the live cell samples (e.g., a multi-well plate or other multi-sample container) to facilitate automated imaging of multiple live cell samples within the incubator. Such live cell samples can include multiple live cell samples that may differ with respect to the identity or mix of live cell contents, the identity or amount of added pharmaceuticals, microorganisms, cancer cells, or other added substances, the type of genetic modification applied to the live cell contents, or some other experimental condition.

[0014] The microscopic imaging device of such a system can include mirrors, filters, or other elements to fold the optical path of the imaging device, thereby reducing the size of the imaging device so that the imaging device can fit inside an incubator. Additionally or alternatively, elements of the imaging device can be separated into individual subassemblies to facilitate various microscopic imaging modalities. For example, a phase lamp and / or other transmitted illumination source can be provided in a module that is separate from the image sensor-containing module and on the opposite side of the live-cell sample container from the image sensor-containing module to facilitate bright-field imaging, phase-contrast imaging, or other microscopic imaging modes.

[0015] Fluorescent dyes, non-fluorescent dyes or pigments, nanorods or other conductive elements that exhibit 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 contents of the sample 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 in the sample. For example, imaging agents can be functionalized to specifically bind to or otherwise interact with proteins, specific cell surface markers, specific sequences of DNA / RNA, or some other substance or element of interest in a biological sample. Such functionalization can facilitate imaging of specific substances in a sample, such as the presence, amount, distribution, or other information about proteins or other substances of interest in a sample. Optically distinguishable substances can be added by being introduced into a live cell sample as an exogenous substance (e.g., by adding a specific amount of a fluorophore conjugated to an antibody specific to a particular type of cell or receptor into each well of a multi-well sample plate). Additionally or alternatively, optically distinguishable substances may be added by genetically modifying living cells in the sample to express the optically distinguishable substances (e.g., by adding a gene for green fluorescent protein to living cells in the sample). Additionally or alternatively, such optically distinguishable substances may be naturally present in the living cells and / or in substances secreted thereby (e.g., autofluorescent proteins naturally expressed by a population of living cells).

[0016] Fluorophores or other substances (e.g., Raman dyes) that emit light in an emission band in response to excitation with light in an excitation band different from the emission band are particularly useful in imaging the contents of a sample. This is due in part to the ability to distinguish the excitation light from the responsively emitted emission light and to control the magnitude and timing of the emission light by controlling the excitation light. These properties allow fluorescent dyes to be imaged with greater fidelity than other substances (e.g., dyes that scatter light within a predetermined wavelength range so that the wavelength of the scattered light is substantially the same as the wavelength of the illuminating light). Additionally, optically distinguishable fluorophores can be used to facilitate independent imaging of multiple different fluorophores. These different fluorophores can differ in excitation spectrum, emission spectrum, or other properties (e.g., fluorescence lifetime) to facilitate such independent imaging. Such imaging can be achieved by providing light at different excitation wavelengths for each of the different fluorophores at different times. When excitation light is delivered to the sample through the same objective (or objective system) used to collect and image emission light from the sample, the process may be referred to as "epifluorescence imaging."

[0017] To image different fluorophores (or other optically distinguishable sample contents) at different times, a fluorescence imager (e.g., an epifluorescence imager) can 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 selectively receiving and imaging light in different emission wavelength bands. However, such imagers can be more mechanically complex, more costly, larger, less reliable, or exhibit some other undesirable performance characteristics. Instead, an imager can include a static set of dichroic mirrors, optical filters, or other elements configured to allow different light sources to emit light in the different excitation bands of each different fluorophore, while allowing light in the emission bands of each different fluorophore to be passed to and imaged by the image sensor.

[0018] For example, an optics module for assaying live cell biological samples using epi-fluorescence capabilities in a fluorescence microscope system can include two light sources and associated filters (e.g., dichroic mirrors). The wavelengths associated with these light sources and the wavelengths associated with the pass / stop / reflection bands of the filters in the optics module are selected to work with one or more sets of fluorophores according to the color of light by which the fluorophores are excited and in which they responsively emit, respectively.

[0019] Such imaging systems can be configured and operated to independently excite (and detect responsive fluorescently emitted light from) two different fluorophores that differ at least with respect to their respective excitation bands. For example, such systems can detect green and red fluorophores in a first configuration and detect orange and near-infrared ("NIR") fluorophores in a second configuration (e.g., achieved by swapping optical modules containing light sources, dichroic mirrors, filters, or other optical components). Different configurations of such two-color imaging systems (e.g., different swappable optical modules of the system) can be configured to excite pairs of fluorophores associated with a particular assay (e.g., a fluorescent ubiquitination cell cycle indicator ("FUCCI") assay, a genetically encoded two-color (red and green) indicator that allows for the observation of cell division within a cell population). When the system is set to a particular configuration, independent images can be collected only for fluorophores that are compatible with the particular configuration.

[0020] Such two-fluorophore optical systems are limited in the number of distinct fluorophores they can image independently. They are therefore limited in the type of information they can generate within a single sample or across a population of different samples in an automated imaging scenario (e.g., different wells of a multi-well sample container differ in the fluorophores present in the wells). This can include being limited in the types of fluorescent assays that can be performed within a single sample (i.e., assays involving no more than two fluorescent indicators). One example of such an assay is the fluorescent ubiquitination-based cell cycle indicator ("FUCCI"), a genetically encoded two-color (red and green) indicator that allows for the observation of 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 their DNA in their nuclei), G2 phase (i.e., the second gap phase; when cells grow more, make proteins and organelles, and begin to reorganize their contents in preparation for mitosis), and mitosis (M) phase (i.e., when cells separate their DNA into two sets, divide their cytoplasm, and form two new cells). There is also an achromatic phase at the M / G1 transition, which makes 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 expression of TagGFP2, which can be imaged using the 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 gap phase; when cells physically become larger, copy organelles, and create molecular building blocks for later stages of growth) and during the S phase transition, cells emit far-red fluorescence via expression of mKate, which can also be imaged using a two-color optical module. However, such a two-color optical module would not allow the use of additional fluorescent indicators to identify additional phases or subphases in the cell division process.

[0021] The capabilities of such two-fluorophore imaging systems can be expanded by swapping optical modules containing light sources, filters, mirrors, or other optical elements associated with the excitation and emission bands of the two fluorophores. However, such manual swapping can be difficult to perform while an automated imaging experiment is running and would require the environment of the live-cell sample to be severely perturbed by opening the incubator so that modules can be swapped.

[0022] In various applications, it would be beneficial to be able to use a fluorescence imaging device to independently image three (or more) fluorescence channels (without swapping optical modules or performing any other manual processes that could result in perturbations to the incubation environment). Such a system can enable the increased use of more complex assays (e.g., assays involving three or more fluorescent indicators, such as a three-color FUCCI assay that monitors the complete cell cycle), the identification of more cell types in a single sample while also evaluating metabolic or other fluorescent indicators (e.g., two or more fluorescent indicators to tag different cell types, while a third fluorescent indicator in the sample represents metabolism, cell death, or some other process of interest), the use of more types of assays / individual fluorescent indicators in individual samples within an incubator, and / or the use of more types of assays / individual fluorescent indicators in different samples within an incubator. These benefits can reduce costs by reducing the time and incubator space required to perform a particular number of experiments / assays, by allowing multiplexing of multiple different assays of a single experiment in a single sample, and / or by allowing different assays of different experiments to be performed in different wells of a sample plate in a single incubator.

[0023] Embodiments herein provide methods and systems relating to such three- (or more) channel microscopic fluorescence imaging processes in a manner compatible with automated multi-sample imaging in an incubator. These embodiments provide a solution to the increased complexity associated with fitting a three- (or more) channel fluorescence imaging device into a limited volume / dimension while allowing the imaging device to also be used for bright-field and / or phase-contrast microscopy. These embodiments also provide a solution to the complex problem of identifying bifurcated optical paths that route excitation light in three (or more) excitation bands to the sample, while routing light from the sample in three (or more) emission bands to an image sensor, while rejecting light in the excitation band. Some of these embodiments include providing a phase lamp (or other trans-illumination light source) that is part of a removable module that is paired with a corresponding three (or more) channel fluorescence imaging module. Such pairing may be necessary to ensure that light from the phase lamp includes wavelengths that can pass through the paired fluorescence imaging module. Such illumination modules and paired optical filter modules may include bar codes, on-board memory, or other features to facilitate automated module detection and identification, and to warn the user before running an experiment if the modules are mismatched.

[0024] Additionally, the embodiments provided herein include improvements to devices used to manually swap optical modules (e.g., phase lamp modules, light sources, and filter modules) that improve the seating and alignment of such modules within an imaging device and increase the ease with which a user can perform such manual swapping. In previous systems, separate tools were required to connect and disconnect various optical modules to and from the system. The tools were difficult to align with corresponding screws through small holes in the optical modules. Additionally, due in part to the placement and configuration of the electrical connectors used to electrically connect the light sources in the modules to the controller and power source of the rest of the system, the force required to connect or disconnect optical modules was difficult for many end users to generate. As a result, many end users needed assistance to swap optical modules.

[0025] The flexible interchangeability of the optical module allows the system to be configured to enable different combinations of methods for detecting fluorophores, including, but not limited to, (i) activating three light sources at three different bands of excitation wavelengths to direct excitation light to a sample and detecting responsively emitted emission light from three individual fluorophores (e.g., green, orange, and near-infrared ("NIR")); (ii) activating three light sources at three different bands of excitation wavelengths, with two of the excitation wavelengths directed to a Förster resonance energy transfer ("FRET")-based measurement (e.g., ATP) and with the third band of excitation wavelengths identifying an independent fluorophore (e.g., a nuclear label); and (iii) using only two light sources of the optical module at two different bands of excitation wavelengths and detecting responsively emitted emission light from two individual fluorophores (e.g., (a) green and red, or (b) orange and NIR).

[0026] Additionally, phase lamps matched to filters in the three (or more) band optical modules may also be included in the system to advantageously enable phase and brightfield imaging to be performed (e.g., to enhance fluorescence imaging information and / or provide independent image information, further process and refine images to identify fluorophores, measure FRET, or provide some other benefit). One advantage of the phase lamp modules (or other trans-illumination light source modules) of the present disclosure is the system's ability to directly identify that a particular phase lamp module has been installed. Detecting the identity of the phase lamp advantageously enables the system to determine when an invalid configuration exists (in which the phase lamp is not properly matched for a given optical module (which may, for example, result in light from the phase lamp being totally or partially blocked from being transmitted through the optical module to be imaged)) and to warn the user before an experiment (e.g., an experiment involving performing one or more assays) is performed.

[0027] II. Exemplary Architecture FIG. 1 is a block diagram illustrating an operating environment 100 that includes or requires, for example, a system 105 for assaying live cell biological samples, which includes a fluorescence microscope 115 in electrical communication with a computing device 200a. The fluorescence microscope 115 is positioned within an incubator 108, which is configured to control temperature, humidity, and / or other environmental parameters and promote the cultivation of live cell samples, which can be imaged by the fluorescence microscope 115 in an automated manner. By being positioned within the incubator 180, the fluorescence microscope 115 is able to image the samples without requiring them to be removed from the incubator 180 (a process that could perturb the sample and modify its growth / response to applied experimental conditions). Method 300 of FIG. 16, described below, illustrates an implementation of a method that can be implemented within this operating environment 100.

[0028] The fluorescence microscope 115 includes an optical module 110, which, in combination with an imaging sensor 120, can be used 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 at three (or more) respective bands of excitation wavelengths corresponding to respective fluorophores in the sample (e.g., a fluorescent indicator including a fluorophore conjugated to an antibody or other structure to promote selective binding to a protein or other substance of interest). The optical module 110 additionally includes dichroic mirrors, filters, and / or other elements configured to provide diverged optical paths so that light from the three (or more) light sources is delivered to the sample via an objective lens. The objective lens can be part of the optical module 110 or can be separate from it. The optical module 110 is also configured to deliver responsively emitted fluorescent light collected through the objective lens at emission wavelengths in each of the three (or more) bands to the imaging sensor 120 to facilitate epifluorescence imaging of the three different fluorophores in the sample.

[0029] The ability to independently epifluorescently image three different fluorophores using a single optical module 110 provides various benefits. It can facilitate the use of more complex three-color (or more) assays. It can facilitate imaging multiple assays or other fluorescent indicators in a single sample (e.g., a two-color FUCCI assay and independent fluorescent indicators that selectively bind to specific types of cells, allowing both the identity and cell division phase of the cells in the sample to be determined). It can facilitate fluorophore / assay selection by relaxing the requirement that all indicators / assays match only two sets of excitation / emission bands (e.g., instead of selecting a suboptimal indicator that matches one of the two available excitation / emission bands of a two-color optical module, a more optimal fluorescent indicator can be selected for a particular use). It can facilitate imaging different sets of assays / fluorescent indicators in different samples contained within the same incubator, saving time and other costs by allowing space within a single instrument / incubator to be used more efficiently. For example, first and second different experiments (having respective first and second sets of fluorescent indicators / assays, which may overlap in terms of emission / excitation wavelengths) may be performed in respective sets of wells in the same incubator. Additionally or alternatively, a single experiment may be performed with multiple different sets of assays / fluorescent indicators present in a subset of the wells used to perform a single experiment, allowing additional data about the experiment to be generated simultaneously using the same incubator. The ability to independently epifluorescently image three (or more) different fluorophores using a single optical module 110 may provide additional or alternative benefits, or combinations of benefits.

[0030] The fluorescence microscope 115 also includes a phase lamp 125 (or other trans-illumination light source) configured to provide light for phase contrast, brightfield, or other forms of imaging. The optical module 110 is configured to transmit at least some of the light emitted from the phase lamp 125. In some examples, this can include the phase lamp 125 being a narrowband light source (e.g., laser, LED) and the optical module being configured to transmit light over a narrow band of wavelengths emitted by the narrowband light source. Details of the optical module 110 and the phase lamp 125 are provided elsewhere herein (e.g., with respect to Figures 3-15).

[0031] The optical module 110 may be user-swappable (e.g., according to embodiments described elsewhere herein) to use the fluorescence microscope 115 to image different fluorescent indicators / assays for different time periods. This may include an optical module having pins, slots, or other alignment features to facilitate aligning the optical module 110 with other imaging components of the fluorescence microscope 115 (e.g., with the imaging sensor 120). This may also include an optical module 110 with one or more electrical connectors to facilitate powering and controlling the three (or more) light sources or providing some other functionality. For example, the optical module 110 may include memory or other electrical components to enable a computing device (e.g., 200, 200a) to identify the optical module 110 and / or determine the bands of wavelengths of light that can be emitted from and / or imaged using the optical module 110.

[0032] The phase lamp 125 (or other trans-illumination light source) may also be user-swappable. This may be due to different swappable optical modules 110 having different passbands (i.e., the wavelengths of light from the sample that can be passed through the optical module 110 and onto the imaging sensor 120) that may not be compatible with all possible phase lamps 125. For example, a first phase lamp 125 may be "optimal" in some sense (e.g., for phase-contrast imaging of a particular type of sample) but may produce light in a band of wavelengths that does not significantly overlap with any passband of the optical module 110 selected for performing the experiment (e.g., to facilitate imaging a particular assay of interest). Thus, a first phase lamp 125 module may be swapped 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.

[0033] Such swappable phase lamp 125 modules can include memory or other electrical components to enable a computing device (e.g., 200, 200a) to identify the phase lamp 125 module and / or determine the band of wavelengths of light that can be emitted from the phase lamp 125 module. This identity / information can be automatically compared with similar information / identity of the optical module 110 installed in the fluorescence microscope 115 to ensure that the installed modules are compatible (e.g., ensure that the installed optical module 110 is capable of passing the wavelengths of light emitted from the installed phase lamp 125 module, such that the combination can be used to image a sample via phase contrast, brightfield, or some other imaging modality using the phase lamp 125 module). If an incompatibility is detected, a user can be warned before initiating an automated imaging study or other experiment using the system 105.

[0034] FIG. 2 is a block diagram illustrating an example of a computing device 200 according to an exemplary implementation, which is configured to interface, either directly or indirectly, with operating environment 100. Computing device 200 may be used to perform the functions of the method shown in FIG. 16 and described below. In particular, computing device 200 may be configured to perform one or more functions, including, but not limited to, using a single optical module to acquire images of three or more fluorescent colors in a single biological sample in a single container or in multiple containers. The acquired images may then be used to perform additional analyses on the imaged biological sample related to properties of the fluorophores and / or materials conjugated thereto that correspond to the three or more fluorescent colors. Functions may also include using a light source that is not part of the single optical module (e.g., a phase lamp) to acquire bright-field, phase-contrast, or some other image of the sample using the signal optical module and another light source.

[0035] The availability of three or more colors imaged within a single sample facilitates more complex multicolor analyses or assays (e.g., a three-color FUCCI assay to observe the complete cell cycle), performing multiple different assays within a single sample or within different samples (e.g., a two-color FUCCI assay (green / orange) combined with an Annexin NIR assay for cell apoptosis), the use of one or more fluorescent indicators in combination with each other and / or in combination with one or more multicolor assays (e.g., two fluorescent reporters to distinguish each cell type and an Annexin NIR assay for cell apoptosis that may be used as part of a three-color immune cell killing assay), the use of different sets of indicators / assays in different samples located within the same incubator, or other examples. The availability of three or more colors can also relax requirements on the indicators / assays selected, allowing for greater flexibility. For example, if a particular assay is only available in a particular color, that color may be reserved for the assay, while other colors may be used for cell type-specific indicators or other uses (e.g., color channels of an assay with more color options available).

[0036] Computing device 200 has a processor 202, a communication interface 204, data storage 206, an output interface 208, and a display 210, each connected to a communication bus 212. Computing device 200 may also include hardware to enable communication within computing device 200 and between computing device 200 and other devices (e.g., not shown). The hardware may include, for example, transmitters, receivers, and antennas.

[0037] The communication interface 204 can be a wireless interface and / or one or more wired interfaces, which enable both short-range and long-range communication to one or more networks 214 or to one or more remote computing devices 216 (e.g., a tablet 216a, a personal computer 216b, a laptop computer 216c, and a mobile computing device 216d). Such wireless interfaces can provide communication under one or more wireless communication protocols (e.g., Bluetooth, Wi-Fi (e.g., the Institute of Electrical and Electronics Engineers (IEEE) 802.11 protocol), Long-Term Evolution (LTE), cellular communication, Near Field Communication (NFC), etc., and / or other wireless communication protocols). Such wired interfaces can include an Ethernet interface, a Universal Serial Bus (USB) interface, or similar interface, and can communicate to a wired network via a wire, a twisted pair of wire, a coaxial cable, an optical link, a fiber optic link, or other physical connection. Thus, communication interface 204 may be configured to receive input data from one or more devices and may be configured to send output data to other devices.

[0038] The communications interface 204 may also include user input devices such as a keyboard, keypad, touch screen, touchpad, computer mouse, trackball, and / or other similar devices.

[0039] Data storage 206 may include or take the form of one or more computer-readable storage media that can be read or accessed by processor 202. The computer-readable storage media may include volatile and / or non-volatile storage components (e.g., optical, magnetic, organic, or other memory or disk storage, etc.), which may be integral, in whole or in part, with processor 202. Data storage 206 is considered a non-transitory computer-readable medium. In some examples, data storage 206 may be implemented using a single physical device (e.g., one optical, magnetic, organic, or other memory or disk storage unit), while in other examples, data storage 206 may be implemented using two or more physical devices.

[0040] Thus, data storage 206 is a non-transitory computer-readable storage medium having executable instructions 218 stored thereon. Instructions 218 include computer-executable code. When instructions 218 are executed by processor 202, processor 202 is caused to perform functions, including, but not limited to, using a single optical module to acquire images of three or more fluorescent colors of a single biological sample in a single container or in multiple containers, using a phase lamp or other light source in addition to a single optical module in combination with a single optical module to acquire bright-field, phase-contrast, or some other image of a biological sample, and / or performing analysis based on the acquired images.

[0041] Processor 202 can be a general-purpose processor or a special-purpose processor (e.g., a digital signal processor, an application-specific integrated circuit, etc.). Processor 202 can receive input from communication interface 204, process the input, and generate output, which is stored in data storage 206 and output to display 210. Processor 202 can be configured to execute executable instructions 218 (e.g., computer-readable program instructions), which are stored in data storage 206 and are executable to provide the functionality of computing device 200 described herein.

[0042] The output interface 208 outputs information to a display 210 or similarly to another component. Thus, the output interface 208 can be similar to the communication interface 204 and can similarly be a wireless interface (e.g., a transmitter) or a wired interface. The output interface 208 can, for example, send commands to one or more controllable devices.

[0043] 2 may represent, for example, a local computing device 200a (FIG. 1) in operating environment 100 that is in communication with system 105. This local computing device 200a may perform one or more of the steps of method 300 described below, may receive input from a user, and / or may send image data and user input to computing device 200 to perform all or some of the steps of method 300.

[0044] FIG. 16 shows a flow chart of an exemplary method 300 for imaging fluorophores in live cell biological samples. The method 300 includes aligning (305) a first biological sample and a fluorescence microscope, where the first biological sample is positioned within a field of view of the fluorescence microscope, the first biological sample including (i) a first fluorophore that emits light at a first band of emission wavelengths in response to illumination with light at a first band of excitation wavelengths, (ii) a second fluorophore that emits light at a second band of emission wavelengths in response to illumination with light at a second band of excitation wavelengths, and (iii) a third fluorophore that emits light at a third band of emission wavelengths in response to illumination with light at a third band of excitation wavelengths; acquiring (310) a set of images of the first biological sample using the fluorescence microscope, where the images in the set differ with respect to focus settings; and determining first, second, and third in-focus settings for the first, second, and third bands of emission wavelengths based on the set of images. determining (315) a focus setting for each of the first and second biological samples; illuminating a first biological sample with light of a first band of excitation wavelengths using a first light source for a first period of time and operating the fluorescence microscope according to a first in-focus setting to acquire a first image of light of the first band of emission wavelengths via an image sensor of the fluorescence microscope (320); illuminating the first biological sample with light of a second band of excitation wavelengths using a second light source for a second period of time and operating the fluorescence microscope according to a second in-focus setting to acquire a second image of light of the second band of emission wavelengths via an image sensor (325); and illuminating the first biological sample with light of a third band of excitation wavelengths using a third light source for a third period of time and operating the fluorescence microscope according to a third in-focus setting to acquire a third image of light of a third band of emission wavelengths via an image sensor (330). The method 300 shown in FIG. 16 represents an example of a method that may be used, for example, with the computing device 200 of FIG.In some cases, components of a system may be configured to perform a function, such that the components are configured and structured with hardware and / or software to enable such performance. Components of a system may be arranged to be adapted to perform a function, capable of performing a function, or suitable for performing a function, e.g., when operated in a particular manner. Method 300 may include one or more operations, functions, or acts, as illustrated by one or more of blocks 305-330. While the blocks are illustrated in sequential order, some of these blocks may be performed in parallel and / or in a different order than described herein. Also, various blocks may be combined into fewer blocks, divided into additional blocks, and / or eliminated based on the desired implementation.

[0045] With regard to this and other processes and methods disclosed herein, it should be understood that the flowchart illustrates the functionality and operation of one possible implementation of the example. In this regard, each block may represent a module, segment, or portion of program code, which comprises one or more instructions executable by a processor to implement specific logical functions or steps in the process. The program code may be stored on any type of computer-readable medium or data storage (e.g., storage devices including disks or hard drives). Furthermore, the program code may be encoded on a computer-readable storage medium in a machine-readable format or on other non-transitory media or articles of manufacture. The computer-readable medium may include non-transitory computer-readable media or memory, such as computer-readable media that store data for short periods of time, such as register memory, processor cache, and random access memory (RAM). The computer-readable medium may also include non-transitory media, such as secondary or permanent long-term storage, such as a read-only memory (ROM), an optical or magnetic disk, a compact disk read-only memory (CD-ROM), etc. The computer-readable medium may also be any other volatile or non-volatile storage system. The computer-readable medium may be considered, for example, to be a tangible computer-readable storage medium.

[0046] 16, as well as in other processes and methods disclosed herein, may represent circuitry that is hardwired to perform specific logical functions in the process. As would be reasonably understood by one of ordinary skill in the art, alternative implementations in which functions may be performed in a different order from that shown or discussed, including substantially concurrently or in reverse order, depending on the functionality involved, are included within the scope of examples of the present disclosure.

[0047] III. Exemplary Optical Modules 3-5 and 13 show simplified diagrams of 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, emitters, and other elements configured to provide independently controllable excitation light in at least three distinct bands of excitation wavelengths and to pass light in at least three corresponding bands of emission wavelengths (FIGS. 3, 4, and 5 show a three-color configuration, while FIG. 13 shows a four-color configuration). The optical module 110 is also configured to pass light from a phase lamp 125 (or other trans-illumination source) that has passed through and / or been scattered by the sample 130 to be imaged by the image sensor 120.

[0048] The optical module 110 includes a first light source 135 configured to emit first light at a first band of excitation wavelengths. A first filter 136 is disposed in a first optical path 137 of the first light source 135. The first filter 136 is configured to pass light of one or more wavelengths 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 at a second band of excitation wavelengths. A second filter 141 is disposed in a second optical path 142 of the second light source 140. The second filter 141 is configured to pass light of one or more wavelengths and reflect light of one or more other wavelengths. The optical module 110 also includes a third light source 145 configured to emit third light at a third band of excitation wavelengths. A third filter 146 is disposed in a third optical path 147 of the third light source 145. The third filter 146 is configured to pass light of one or more wavelengths and reflect light of one or more other wavelengths. The direction of excitation light transmitted to the sample 130 and / or the direction of image light (e.g., fluorescence emission light, bright field, phase contrast, or other scattered and / or transmitted image light) passed through the optics module 110 to the image sensor 120 is indicated in the diagram by arrows above the optical paths.

[0049] Filters 136, 141, 146 can include a variety of materials or components configured in various ways to promote reflection and / or absorption of certain bands of wavelengths and transmission of certain other bands of wavelengths. For example, 136, 141, 146 can be dichroic mirrors, which are composed of many alternating layers of materials whose compositions, thicknesses, and order can be specified to provide desired pass-bands, stop-bands, reflection bands, or other wavelength-selective optical behavior.

[0050] In the optical module 110, the first optical path 137, the second optical path 142, and the third optical path 147 converge along a primary transmission optical path 150 that is configured to be directed toward the live cell biological sample 130 via an objective lens 165. The module also includes an emission filter 155 positioned in a primary emission optical path 156 that is configured to pass light emitted by fluorophores in the live cell biological sample 130 in response to being illuminated by the light sources 134, 140, 145, and to pass at least some of the light emitted from the phase lamp 125. The primary emission optical path 156 terminates at the imaging sensor 120. Emission filter 155 is configured to pass light of at least a first band, a second band, and a third band of emission wavelengths corresponding to the first, second, and third excitation wavelengths emitted by light sources 134, 140, 145 via the respective first, second, and third fluorophores in sample 130. Emission filter 155 is also configured to pass at least some of the wavelengths of light emitted from phase lamp 125. In practice, this may involve matching the wavelength of the phase lamp light source to one or more of the first, second, or third band of emission wavelengths.

[0051] Emission filter 155 may also be configured to reject (e.g., reflect, absorb) light at excitation wavelengths in the first, second, and third bands. Alternatively, the reflective action of other filters 136, 141, 146 may be relied upon to prevent excitation light from light sources 135, 140, 145 from being received by image sensor 120. Furthermore, emission filter 155 may also be configured to reflect or otherwise reject artificial autoluminescence from biological sample 130 and / or reflect light from other fluorescent dyes that may be present in biological sample 130.

[0052] It should be noted that optical module 110 can include one or both of objective lens 165 or image sensor 120. Alternatively, optical module 110 can be configured to be removably coupled to one or both of objective lens 165 or image sensor 120. This may be done, for example, to reduce the cost of individual swappable optical modules.

[0053] 3 , the first filter 136 is configured to pass light of the first band of excitation wavelengths and reflect light of the second and third bands of excitation wavelengths, as well as light of the first, second, and third bands of emission wavelengths, and light of a predetermined band of wavelengths emitted by the phase lamp 125 (which may overlap with one or more of the first, second, or third bands of emission wavelengths). The second filter 141 is configured to pass light of the third band of excitation wavelengths, as well as light of the first, second, and third bands of emission wavelengths and phase lamp wavelengths, and reflect light of the second band of excitation wavelengths. The third filter 146 is configured to pass light of the first, second, and third bands of emission wavelengths and phase lamp wavelengths, and reflect light of the third band of excitation wavelengths.

[0054] As shown in FIG. 3 , first light source 135, second light source 140, and third light source 145 are arranged in series in the same plane. The arrangement of the elements in FIG. 3 results in optical module 110 that is fairly long in one dimension (horizontally in FIG. 3 ) while having smaller dimensions in the other two dimensions (vertically and into and out of the plane of FIG. 3 ). This may be desirable in some applications. However, in some applications, it may be beneficial to reduce the maximum dimension of optical module 110 and / or to conform the shape and size of optical module 110 to a particular shape and / or size (e.g., to fit the module into an incubator or onto the gantry of an automated imaging system). Thus, the arrangement of the elements of optical module 110 may be modified, for example, to fold, nest, and / or diverge the various optical paths and / or to change the direction and / or order of the various optical paths.

[0055] In another exemplary implementation shown in FIG. 4 , the first filter 136 is configured to pass light of the excitation wavelengths in the first and second bands and reflect light of the excitation wavelengths in the third band and light of the emission wavelengths in the first, second, and third bands. The second filter 141 is configured to pass light of the excitation wavelengths in the second band and reflect light of the excitation wavelengths in the first band. And the third filter 146 is configured to pass light of the emission wavelengths in the first, second, and third bands and reflect light of the excitation wavelengths in the third band. Thus, in the implementation shown in FIG. 4 , the first light source 135 is positioned such that the first optical path 137 begins at the first light source 135, reflects off the second filter 141, passes through the first filter 136, and exits the optical module 110 along the primary transmission optical path 150. The second light source 140 is positioned 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 a primary transmission optical path 150. The third light source 145 is positioned such that a third optical path 147 begins at the third light source 145, reflects off the third filter 146 to the first filter 136, reflects off the first filter 136, and exits the optical module 110 along a primary transmission optical path 150. A primary emission optical path 156 for light emitted by fluorophores in the live cell biological sample 130 reflects off the first filter 136, passes through the third filter 146, passes through the emission filter 155, and exits the optical module 110.

[0056] 4, the second light source 140 and the third light source 145 are positioned parallel to one another, and the first light source 135 is positioned 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 that includes three light sources within the housing 111.

[0057] In yet another optional implementation shown in FIG. 5 , first filter 136 is configured to pass light of excitation wavelengths in the first band. First filter 136 is configured to reflect light of excitation wavelengths in the second and third bands and light of emission wavelengths in the first, second, and third bands. Second filter 141 is configured to pass light of emission wavelengths in the first, second, and third bands. Second filter 141 is configured to reflect light of excitation wavelengths in the second and third bands. Third filter 146 is configured to pass light of excitation wavelengths in the third band. Third filter 146 is configured to reflect light of excitation wavelengths in the second band.

[0058] 5 , the first light source 135 is positioned such that a first optical path 137 begins at the first light source 135, passes through the first filter 136, and exits the optical module 110 along a primary transmitted optical path 150. The second light source 140 is positioned such that a second optical path 142 begins at the second light source 140, reflects at the third filter 146 to the second filter 141, reflects at the second filter 141 to the first filter 136, reflects at the first filter 136, and exits the optical module 110 along the primary transmitted optical path 150. The third light source 145 is positioned such that a third optical path 147 begins at the third light source 145, passes through the third filter 146 to the second filter 141, reflects off the second filter 141 to the first filter 136, reflects off the first filter 136, and exits the optical module 110 along a primary transmission optical path 150. Additionally, a primary emission optical path 156, in which light is emitted by fluorophores in the live cell biological sample 130, reflects off the first filter 136, passes through the second filter 141, passes through the emission filter 155, and exits the optical module 110.

[0059] 5, 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 that includes three light sources within the housing 111.

[0060] It should be noted that while the exemplary implementations shown in Figures 3-5 and elsewhere herein depict optical modules with light source optical paths that are all entirely in the same plane (i.e., the plane of the figure), other embodiments are possible, e.g., to reduce the overall size of the optical module and / or to match the shape and size of the optical module to available space (e.g., within a gantry and / or automated in-incubator imaging device).

[0061] For example, the second light source 140 and third filter 146 of the implementation shown in FIG. 5 may be rotated 90 degrees (or some other angle) into (or out of) the plane of FIG. 5. Such an implementation, and additional details thereof, are further illustrated in FIGS. 6-11. As illustrated in FIGS. 6-11, the first, second, and third light sources 135, 140, 145, the first, second, and third filters 136, 141, 146, and the emission filter 155 are all contained within a housing 111. The housing 111 includes a first opening 112 that is positioned to allow the primary transmitted optical path 150 to pass therethrough to illuminate the biological sample 130. The housing 111 also includes a second opening 113 positioned to allow a primary emission optical path 156 to pass through toward the imaging sensor 120. The first and second openings 112, 113 can include optical devices (e.g., lenses, filters, mirrors, etc.) and / or a sensor surface. In one optional example, an emission filter 155 can be disposed within the second opening 113. In the exemplary optical module 110 shown in FIGS. 3-8, the housing 111 includes a main body portion 111a extending vertically and a cantilevered extension portion 111b extending horizontally therefrom. The main body portion 111a of the housing 111 includes the first light source 135 and the third light source 145 in the same plane. And, cantilevered extension 111b of housing 111 contains second light source 140, which is positioned at a 90 degree angle relative to first light source 135 and third light source 145.

[0062] In other words, the second light source 140 and the third filter 146 are rotated 90 degrees around 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 and third light sources 135, 145, with the second optical path 142 directed toward the third filter 146, where the light is reflected upward. Such an arrangement can be particularly beneficial in space-limited applications. For example, the aforementioned arrangement allows the optical module to remain compact for integration into a combined epifluorescence and brightfield / phase-contrast imaging system within an automated incubator, as described below, while also allowing the optical module to be easily swappable by the user, thus extending the utility and reconfigurability of the imaging system.

[0063] The light sources 135, 140, 145 discussed herein can each include any device and / or assembly capable of delivering 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 (e.g., halogen or tungsten filament) lamps, arc (e.g., mercury, mercury-xenon, or xenon) lamps, light-emitting diodes ("LEDs"), and / or lasers, among others. The associated optics ("source optics") can include fiber optics and / or liquid light guides, one or more lenses, filters (e.g., polarization or wavelength-based filters, etc.), diffraction gratings, mirrors, and / or masks, among others. The associated optics can select / adjust the intensity, wavelength, polarization, phase, direction, and / or shape of the light directed to the sample. In one 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., for collimating the light output from the light source and / or for matching the focus of the output light to the infinity focus or other focus of the objective lens), and a single bandpass dichroic filter.

[0064] The specific boundaries of the first, second, third, and / or additional bands of excitation wavelengths emitted from the light sources of the optical module can be determined according to the application (e.g., according to 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 optional implementation, the excitation wavelengths of the first band are in the range of 453 nm to 485 nm, corresponding largely to blue light that causes the corresponding fluorophores to emit green (or longer wavelength) light. The excitation wavelengths of the second band are in the range of 546 nm to 568 nm, corresponding largely to lime light that causes the corresponding fluorophores to emit orange (or longer wavelength) light. And, the excitation wavelengths of the third band are in the range of 648 nm to 674 nm, corresponding largely to red light that causes the corresponding fluorophores to emit near-infrared (NIR) light. In further implementations, the aforementioned range boundaries of this optional embodiment can vary by + / - 3 nm.

[0065] In another optional implementation, the emission wavelength of the first band is in the range of 494 nm to 533 nm and corresponds largely to green light, the emission wavelength of the second band is in the range of 576 nm to 639 nm and corresponds largely to orange light, and the emission wavelength of the third band is in the range of 686 nm to 756 nm and corresponds largely to NIR light. In further implementations, the boundaries of the aforementioned ranges can vary by + / - 3 nm.

[0066] In some examples, the optical module 110 can include a fourth light source 160 configured to emit fourth light at a fourth band of excitation wavelengths. An exemplary implementation of such an optical module 110 is shown in FIG. 13 . In this implementation, the optical module 110 also includes a fourth filter 161 disposed in a fourth optical path 162 of the fourth light source 160. The fourth filter 161 is configured to pass light of one or more wavelengths and reflect light of one or more wavelengths. And, the emission filter 155 is further configured to pass light of the fourth band of emission wavelengths and reflect light of the fourth band of excitation wavelengths. The addition of the fourth light source 160 improves the ability to view a fourth fluorophore in the biological sample 130 and to perform an even greater number of assays without relying on a separate optical module with different light sources and filters and corresponding configurations.

[0067] 13 , the first filter 136 is configured to pass light of the excitation wavelengths in the first band and reflect light of the excitation wavelengths in the fourth band. The second filter 141 is configured to pass light of the emission wavelengths in the first, second, third, and fourth bands and reflect light of the excitation wavelengths in the second and third bands. The third filter 146 is configured to pass light of the excitation wavelengths in the third band and reflect light of the excitation wavelengths in the second band. And the fourth filter 161 is configured to pass light of the excitation wavelengths in the first and fourth bands and reflect light of the excitation wavelengths in the second and third bands and light of the emission wavelengths in the first, second, third, and fourth bands.

[0068] 13 , first light source 135 is positioned such that first optical path 137 begins at first light source 135, passes through first filter 136, then passes through fourth filter 161, and exits optical module 110 along primary transmitted optical path 150. Second light source 140 is positioned such that second optical path 142 begins at second light source 140, reflects at third filter 146 to second filter 141, reflects at second filter 141 to fourth filter 161, reflects at fourth filter 161, and exits optical module 110 along primary transmitted optical path 150. The third light source 145 is positioned such that a third optical path 147 begins at the third light source 145, passes through the third filter 146 to the second filter 141, reflects off the second filter 141 to the fourth filter 161, reflects off the fourth filter 161, and exits the optical module 110 along a primary transmission optical path 150. The fourth light source 160 is positioned such that a fourth optical path 162 begins at the fourth light source 160, reflects off the first filter 136, passes through the fourth filter 161, and exits the optical module 110 along a primary transmission optical path 150. And a primary emission optical path 156 for light emitted by fluorophores in the live cell biological sample 130 reflects off the fourth filter 161, passes through the second filter 141, passes through the emission filter 155, and exits the optical module 110. 13, the first light source 135 and the third light source 145 are arranged parallel to one another, and the second light source 140 and the fourth light source 160 are arranged at a 90 degree angle relative to the first light source 135 and the third light source 145, respectively. This arrangement allows for a compact optical module 110 that includes four light sources within the housing 111.

[0069] The emission wavelengths of such a fourth band may include wavelengths shorter than 453 nm and may correspond largely to violet light, and the emission wavelengths of a corresponding fourth band may correspond largely to blue light.

[0070] IV. Exemplary Systems In a second embodiment of the present disclosure shown in Figures 1, 3, 4, 5, and 13, a system 105 for assaying a live cell biological sample 130 is provided. The system 105 includes an optical module 110 according to the first embodiment of the present disclosure. The system 105 also includes a fluorescence microscope 115, which includes the optical module 110, which is removably coupled to the fluorescence microscope 115. The fluorescence microscope 115 has at least one objective lens 165. The system 105 further includes an imaging sensor 120 disposed in a primary emission optical path for light emitted by fluorophores in the live cell biological sample 130 and / or for light from a phase lamp 125 (or other transmitted illumination source, e.g., a light source configured to provide illumination for bright-field imaging but not for phase-contrast imaging) transmitted through and / or scattered by the sample 130 from the objective lens 165. And, the system 105 includes a phase lamp 125 that is removably coupled to the fluorescence microscope 115 and positioned at the terminal end of the primary transmitted light path 150 .

[0071] As used herein, a fluorescence microscope 115 is any optical device that magnifies images of small objects (e.g., cells, organelles, tissues, small organisms, particles, etc.). Exemplary modes of microscopy implemented 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 light (e.g., IR, NIR, UV) microscopy). An objective lens 165 is positioned between the optical module 110 and the biological sample 130 such that the primary transmission optical path 150 and the primary emission optical path 156 pass through the objective lens 165.

[0072] The 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-generating device. The imaging sensor 120 may be configured or operated to detect light in a predetermined range of wavelengths. For example, the imaging sensor 120 may be configured or operated to detect light in multiple wavelengths / wavelength ranges corresponding to the emission spectra of fluorescent dyes or other fluorophores in the biological sample 130. For example, these wavelengths may correspond to peaks in the emission spectra of multiple fluorophores in the sample and / or extend over a wide wavelength range. This may include an imaging sensor 120 that is a monochrome imaging sensor, sensitive to wavelengths of light in each of the emission spectra and / or the wavelengths of light emitted by a phase lamp or other transmitted illumination source. Additionally, the imaging sensor 120 may be configured to measure any suitable photoluminescence, including, among others, fluorescence intensity (FLINT), fluorescence resonance energy transfer (FRET), fluorescence lifetime (FLT), fluorescence correlation coefficient (FCS), fluorescence recovery after photobleaching (FRAP), and phosphorescence and other analogs thereof.

[0073] In one optional implementation shown in FIGS. 6-8 and 11-12, the system 105 includes a shaft 170 extending through the optical module 110. Here, the fluorescence microscope 115 has a receiver (not shown) configured to receive the shaft 170 in a first orientation. The shaft 170 is then configured to rotate to a second orientation under the application of force, thereby locking the optical module 110 to the rest of the fluorescence microscope 115 (e.g., to a housing or other element of the fluorescence microscope 115). For example, the shaft 170 can have a flip tab 171 coupled to a first end 172 and a protrusion 173 coupled to a second end 174, thereby forming a T-shape. The fluorescence microscope 115 can have a corresponding slot (not shown) and receiver configured to receive the T-shaped protrusion 173 in the first orientation. When the shaft rotates to a second orientation under the application of force to the flip tab 171, the T-shaped protrusion 173 rotates within the receiver so that the T-shaped protrusion 173 opposes the slot, thereby locking the optical module 110 to the fluorescence microscope 115. When the optical module 110 is coupled to the rest of the fluorescence microscope 115, the flip tab 171 can be folded flat against the housing 111.

[0074] In a further implementation, once shaft 170 is inserted, rotation of shaft 170 in the locking direction causes protrusion 173 to ride along the ramp, pulling them, shaft 170 and optics module 110, onto the mount for the rest of system 105. A flexible element (e.g., spring 189) can be disposed between shaft 170 and housing 111 of optics module 110 to control the force that pulls optics module 110 onto the mount. Additionally, detents can provide tactile feedback when shaft 170 is in the locked and unlocked positions.

[0075] In another optional implementation shown in FIG. 4 , the system 105 includes a first electrical connector 175 coupled to the optical module 110. The system 105 includes a second electrical connector (not shown) coupled to the remainder of the fluorescence microscope 115. The second electrical connector is reciprocal with the first electrical connector 175. The system 105 then includes a processor 202 in electrical communication with the second electrical connector. The processor 202 is configured to identify the optical module 110 coupled to the remainder of the fluorescence microscope 115. The first and second electrical connectors 175 may be selected to require less than a certain force to connect and disconnect, for example, to make it easier for a user to swap different optical modules in the fluorescence microscope 115.

[0076] The swappable phase lamp module 125 includes a housing 126, a trans-illumination light source 127 (e.g., a halogen lamp, an LED), and at least one condenser lens that focuses light from the phase lamp module 125 onto the biological sample 130 from above. In yet another optional implementation shown in FIGS. 14-15 , the phase lamp module 125 has a third electrical connector 177 that corresponds to a fourth electrical connector (not shown) that is coupled to the rest of the fluorescence microscope 115 (e.g., to the same housing to which the second electrical connector is coupled). The third electrical connector 177 is reciprocal with the fourth electrical connector. The processor 202 is configured to determine whether the optical module 110 and the phase lamp module 125 are compatible and, in response to determining that they are incompatible, to display a warning. Such a determination may be made by performing a lookup in a database containing records of valid correspondences between available optical modules 110 and available phase ramp modules 125. Additionally or alternatively, such a determination may be made by comparing a set of wavelengths of light emitted by phase ramp module 125 with a set of wavelengths of light that optical module 110 is configured to pass from sample 130 to imaging sensor 120.

[0077] The housing 126 of the phase lamp module 125 includes a protrusion 128 that is shaped to be received in a corresponding receptacle 186 in a phase lamp mount 185 of the system 105. The protrusion 128 and receptacle 186 are shaped so that the phase lamp module 125 can only be installed in a single orientation. Rather than being secured by a screw, the phase lamp module 125 is held in place by a detent 187. The detent 187 is in the form of a spring-loaded ball 188 in the phase lamp mount 185 that is configured to mate with a groove 129 in the protrusion 128 of the housing 126.

[0078] In one optional implementation shown in FIG. 1 , the system 105 includes an incubator 180 configured to maintain a live cell biological sample 130 at a temperature ranging from 30° C. to 42° C. and a relative humidity ranging from 80% to 100%. In this implementation, a fluorescence microscope 115 according to a first aspect of the present disclosure is coupled to a chamber of the incubator 180. In another implementation, the fluorescence microscope 115 may be partially or entirely contained within the incubator 180. For example, the fluorescence microscope 115 may be entirely disposed within a standard CO incubator for time-lapse examination of the biological sample 130 during continuous culture (e.g., over a specific period of hours, days, or weeks). Due to the length of the incubation time, the fluorescence microscope 115 may remain within the incubator during cell culture to avoid adversely affecting the biological sample 130. Additionally, the compact profile of the fluorescence microscope 115 maintains the functionality of the incubator 180 for placing other biological samples 130 in the open space around the fluorescence microscope 115. The compact profile of the fluorescence microscope 115 also reduces airflow restrictions that can have detrimental effects on the fluorescence microscope 115 or the biological samples 130 in the form of condensation and improper ventilation.

[0079] The third light source 145 (and fourth light source 160) allows for multiplexed assays on the biological samples 130, running multiple assays and / or fluorescent indicators within individual samples (e.g., sample wells) within the incubator 180 and / or within sets of different samples within the same incubator 180.

[0080] V. Exemplary Methods 16, a method 300 is illustrated that can utilize the optical module 110 and system 105 of FIGS. 3-15 and the computing device 200 of FIGS. 1-2 to image fluorophores in a live cell biological sample. The method 300 includes, at block 305, aligning a first biological sample and a fluorescence microscope such that the first biological sample is positioned within a field of view of the fluorescence microscope, the first biological sample including (i) a first fluorophore that emits light at a first band of emission wavelengths in response to illumination with light at a first band of excitation wavelengths, (ii) a second fluorophore that emits light at a second band of emission wavelengths in response to illumination with light at a second band of excitation wavelengths, and (iii) a third fluorophore that emits light at a third band of emission wavelengths in response to illumination with light at a third band of excitation wavelengths. Then, in block 310, the method includes acquiring a set of images of a first biological sample using a fluorescence microscope, where the images in the set differ with respect to focus settings. Next, in block 315, the method 300 includes determining first, second, and third in-focus settings for the first, second, and third bands of emission wavelengths, respectively, based on the set of images. And, in block 320, the method includes illuminating the first biological sample with light of the first band of excitation wavelengths using a first light source for a first period of time, operating the fluorescence microscope according to the first in-focus setting, and acquiring a first image of light of the first band of emission wavelengths via an image sensor of the fluorescence microscope. The method 300 also includes, at block 325, illuminating the first biological sample with light of a second band of excitation wavelengths using a second light source for a second period of time, and operating the fluorescence microscope according to a second in-focus setting to acquire a second image of light of the second band of emission wavelengths via the image sensor.Method 300 also includes illuminating the first biological sample with light at a third band of excitation wavelengths using a third light source for a third time period, and operating the fluorescence microscope according to a third in-focus setting to acquire a third image of light at the third band of emission wavelengths via the image sensor, at block 330. All of the foregoing steps may be performed automatically by processor 202.

[0081] Acquiring a specific fluorescent image of a specific 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 enable the synthetic generation of high dynamic range images. This can be done for fluorophores / samples / assays that exhibit very high variation in the intensity of fluorescent emission over the range of fluorophore concentrations / activities of interest.

[0082] Method 300 can additionally include acquiring one or more brightfield, phase-contrast, or other non-fluorescent images by operating a phase lamp (e.g., 125) or other transmitted illumination source. Such non-fluorescent image information can then be used in combination with the fluorescent images (e.g., using the phase-contrast images to identify the location, shape, size, and / or extent of cells in the sample, regardless of cell type, and then using one or more fluorescent images to determine cell type, aspect of cell division, cell health, cell metabolic activity, or other information about the cells identified using the phase-contrast images) or by itself.

[0083] Additionally, method 300 can include acquiring a set of brightfield, phase-contrast, or other non-fluorescent images over a range of different focus settings (e.g., over a range of different objective-sample distances) to determine in-focus settings for the three (or more) fluorescent images acquired using method 300. This can include determining an in-focus setting for the wavelength of illumination used to generate the non-fluorescent images and then applying a known offset (e.g., a distance offset) from that in-focus setting for each of the fluorescent emission wavelengths being imaged to determine the in-focus settings for those emission wavelengths. If the wavelength of illumination used to generate the non-fluorescent images is the same or substantially the same as one of the fluorescent emission wavelengths, the offset can be zero. Brightfield or other non-fluorescent images often contain significantly more image data for the same exposure time when compared to fluorescent images; therefore, this method of determining in-focus settings can advantageously reduce the time required to generate such in-focus settings. This can also reduce the amount of photobleaching the sample experiences to generate such in-focus settings.

[0084] In practice, images of fluorophores excited by shorter wavelengths may contain artifacts related to light emitted from fluorophores excited by longer wavelengths, and vice versa. Also, as one example, a fluorophore corresponding to a first band of wavelengths may be excited to some extent by the second and third band wavelengths (especially if the second and third band wavelengths include wavelengths shorter than the first band wavelengths). Similarly, a fluorophore corresponding to a second band of wavelengths may also be excited to some extent by the first and third band wavelengths, while a fluorophore corresponding to a third band of wavelengths may also be excited to some extent by the first and second band wavelengths.

[0085] To address the artifact problem, in one optional implementation, method 300 further includes a processor 202 in electrical communication with imaging sensor 120 (or some other computing device) that generates a first image of light emitted by the first fluorophore based on the first set of images to reduce artifacts from the light emitted by the second fluorophore and the third fluorophore. Processor 202 additionally generates a second image of light emitted by the second fluorophore based on the second set of images to reduce artifacts from the light emitted by the first fluorophore and the third fluorophore. Furthermore, processor 202 can generate a third image of light emitted by the third fluorophore based on the third set of images to reduce artifacts from the light emitted by the first fluorophore and the second fluorophore. This spectral unmixing process is described in more detail for two excitation wavelengths in U.S. Patent Application No. 16 / 264,819, filed February 1, 2019, which is incorporated herein by reference.

[0086] In one non-limiting example, when the biological sample 130 is positioned at a first in-focus setting such that light emitted from the green fluorophore is in focus, a first image can be acquired of the green fluorophore by illuminating the biological sample 130 with light at a blue excitation wavelength corresponding to the green fluorophore. Specifically, the "in-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 green fluorophore emission light is imaged in focus. This first image can also include light emitted from orange fluorophores (primarily excited by light at a lime excitation wavelength but also to some extent by blue light) and NIR fluorophores (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 fluorophores and NIR fluorophores will be out of focus in the first image. This is due to chromatic aberration caused by elements along the primary emission optical path 156 between the sample 130 and the imaging sensor 120 (e.g., the objective lens, the tube lens, the optical properties of the sample, and / or the optical properties of the container containing the sample).

[0087] An artifact image can then be removed from the first image to remove artifact light from the orange and NIR fluorophores. As described above, one artifact image can be obtained by illuminating the biological sample 130 with light at a lime excitation wavelength corresponding to the orange fluorophore at a first in-focus setting used to acquire the first image. Another artifact image can be obtained by illuminating the biological sample 130 with light at a red excitation wavelength corresponding to the red fluorophore at a first in-focus setting used to acquire the first image. Alternatively, the artifact image can be obtained by blurring the in-focus images of the orange and red fluorophores, or by applying some image processing technique to simulate the effect of the focus setting used to acquire the first image on an image taken using another focus setting. For example, an image taken using an in-focus setting such that light emitted from the orange and NIR fluorophores is imaged separately in focus.

[0088] In one optional implementation, method 300 further includes an incubator including or otherwise coupled to a fluorescence microscope that maintains at least the first biological sample at a temperature ranging from 30° C. to 42° C. and at a relative humidity ranging from 80% to 100% when acquiring the first set of image data, the second set of image data, and the third set of image data. This data can be acquired in a standard CO2 incubator for time-lapse testing of biological sample 130 during continuous incubation (e.g., over a period of minutes, hours, days, or weeks, depending on the experiment of interest). For example, images can be taken over time throughout the incubation, which can be longer than 14 days or longer than or equal to 30 days.

[0089] Method 300 may include performing some additional analysis on the fluorescent and / or other images acquired using the system (e.g., bright-field, phase-contrast). For example, if one (or more) of the images corresponds to fluorophore colors characteristic of one or more particular types of cells in a particular sample, method 300 may include analyzing the images to determine the number, shape, size, distribution, interconnection pattern, or other information about one or more particular types of cells present in the sample. Such identification may be enhanced by the use of phase-contrast or other non-fluorescent images to identify the location, shape, and extent of individual cells within the sample, regardless of type. Additionally or alternatively, if one (or more) of the images corresponds to fluorescent indicators of a particular assay (e.g., Annexin V NIR assay, two-color or three-color FUCCI cell division phase assay), method 300 may include analyzing the images to generate an output of the particular assay to, for example, determine the health, cell division phase, or other metabolic state or status of one or more cells in the sample. The results of different analyses of different (or overlapping) images can be combined; for example, a first image analysis (corresponding to a cell-specific fluorophore) can be used to identify cells of a cell type of interest, and the 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 analyses can be the same for all samples imaged by the imaging system in the incubator (e.g., due to all of the samples containing the same assay / fluorescent indicator / dye), or can be different for each sample in the incubator.

[0090] In one optional implementation, method 300 further includes processor 202 identifying a first cell type in the first biological sample based on the first set of images. Processor 202 then identifies a second cell type in the first biological sample based on the second set of images. Processor 202 then identifies cell death or some other metabolic process or characteristic in the first biological sample based on a third set of images. This has the technical effect of allowing complex assays and / or multiple assays to be performed in a single vessel in system 105 without changing the configuration of optical module 110.

[0091] In one optional implementation, phase contrast, bright field, or other non-fluorescent images may be acquired at the first, second, and third in-focus settings, and these images may be used to remove further artifacts from the first, second, and third images (e.g., to remove artifacts due to autoluminescence) or to otherwise improve the first, second, and third images (e.g., by providing additional high spatial frequency image data to enhance the fluorescent image).

[0092] In one optional implementation, the method 300 also includes the processor receiving compatibility information regarding the optical module 110 and the phase lamp module. The processor 202 then determines whether the optical module 110 and the phase lamp 125 are compatible based on the compatibility information. In response to determining that the optical module 110 and the phase lamp 125 are incompatible, the processor 202 then causes a warning to be displayed with an indication of the incompatibility. Such a compatibility determination may be made by performing a lookup in a database containing records of valid correspondences between available optical modules 110 and available phase lamp modules 125. Additionally or alternatively, such a determination may be made by comparing a set of wavelengths of light emitted by the phase lamp module 125 with a set of wavelengths of light that the optical module 110 is configured to pass from the sample 130 to the imaging sensor 120.

[0093] In one optional implementation, method 300 includes extending shaft 170 in a first orientation through optical module 110 to a receiver in fluorescence microscope 115. Shaft 170 is then rotated under the application of a 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.

[0094] The foregoing method 300 has the technical effect of enabling increased variability in assays that can be performed in individual vessels or across multiple vessels in system 105 without changing the configuration of optical module 110. Method 300 may be implemented by or in combination with any of the embodiments of a fluorescent imager, optical module, phase lamp module, incubator, automated imaging system, or other system, device, or component described herein. Thus, a variety of assays, fluorescent indicators, and combinations thereof in individual samples within an incubator and / or across different samples within an incubator are enabled by the embodiments described herein. Several examples of such applications are provided below. These applications are intended as illustrative embodiments and are not intended to be limiting. Additional or alternative applications are contemplated, as are additional or alternative combinations of such applications and / or the applications described below.

[0095] As discussed above, a non-transitory computer-readable medium may have stored thereon program instructions that, when executed by the processor 202, may be utilized to cause performance of any of the methods described herein.

[0096] VI. Exemplary Biological Uses The embodiments described above enable a variety of applications in fluorescence imaging by providing independent imaging of three different fluorescent channels. Advantages of additional fluorescent channels can include improving the throughput of a single imaging device by allowing additional assays to be performed simultaneously using a single device (e.g., in a single incubator), by providing flexibility in experimental readout across different samples, and / or by allowing additional assays to be performed from individual samples. The availability of three-color (or more) imaging can also allow for increased flexibility in reagent selection (e.g., the ability to use multiple reagents to obtain additional information while monitoring cells expressing a green fluorescent protein-based reporter).

[0097] Additionally or alternatively, simultaneous three-color (or more) imaging can enable information to be generated that could not be generated using only two colors. For example, information from a three-color reporter (e.g., a three-color FUCCI assay) can be generated. In another example, information about the proliferation and interaction of multiple cell types, as well as metabolic or cell death information across cell populations, can be generated. Such information can enable additional insight into the impact of experimental conditions on the activity of effector cells in destroying target cells, or, for example, metabolic exchanges between cancer cells and stromal cells.

[0098] Furthermore, three-color (or higher) imaging can enable the generation of experimental data in a more reliable manner. For example, multiplexing multiple readouts in a single sample can provide increased confidence that observed differences between readouts are scientifically valid and not due to experimental variation (e.g., cell plating) between assays run in parallel.

[0099] Example 1: Two-color FUCCI + cell death Various implementations of the optical module 110, system 105, and method 300 described herein can be advantageously used to perform two-color cell cycle (e.g., green / orange FUCCI) observation and cell death analysis in a single sample. Cell cycle and apoptosis readouts can demonstrate, for example, the concentration-difference-dependent and time-dependent effects of a compound 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 immune cell killing of targeted cancer cells on cancer cells and / or on some other population of cells in the sample. Multiplexing these two readouts in a single sample increases throughput and also provides confidence that differences between the two readouts are scientifically valid and not due to experimental variation between parallel assays.

[0100] With respect to two-color FUCCI analysis, the three-color optical module of the present disclosure enables differentiation between various cell cycle phases based on the phase-dependent expression of two different fluorescent proteins. For example, during S, G2, and M phases, cells can emit green fluorescence through the expression of TagGFP2 (a green fluorophore), which can be detected using the three-color optical module. During G1 phase and during the S phase transition, cells can emit orange fluorescence through the expression of TagRFP (an orange fluorescent protein with bright fluorescence having excitation / emission maxima at 555 nm and 584 nm, respectively), which can be detected using the three-color optical module. In this two-color FUCCI assay, cells transition through a colorless period immediately following mitosis. As a result, cell cycle phases have fluorescent footprints (e.g., S, G2, and M phases: green; G1 phase: orange; G1 / S transition: both orange and green; M / G1 transition: colorless (no fluorescence)). A third color can then be used to image a fluorescent cell death readout (eg, Annexin V NIR apoptosis indicator).

[0101] Example 2: 3-color FUCCI Various implementations of the optical module 110, system 105, and method 300 described herein can be advantageously used to perform three-color FUCCI assays that independently observe additional phases in the cell cycle. Unlike previously described two-color cell cycle assays, three-color assays allow for differentiation between S and G2 phases and, advantageously, do not result in a colorless phase due to appropriate gene expression of a gene for a fluorescent protein fused to a targeted ubiquitination domain or other cell-phase-related target. For example, during S, G2, and M phases, cells can emit green fluorescence based on expression of TagGFP2, which can be detected using the three-color optical module, and dimmer fluorescence is observed during S phase. Different fluorescent markers can then be used to identify other phases of the cell cycle, including, but not limited to, TagRFP and iRFP713. During G1 and S phases, cells expressing TagRFP emit orange fluorescence, which can be detected using the three-color optical module. iRFP713 is a near-infrared fluorescent protein with fluorescence having excitation / emission maxima at 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. As a result, cell cycle phases have fluorescent footprints (e.g., G2 and M phases: green and NIR; G1 phase: orange and NIR; S phase: orange and green), and no achromatic phase exists.

[0102] Example 3: Three-color immune cell killing Various implementations 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 to provide cell death readouts across both cell types. This offers the benefit of being able to independently measure target and effector cell proliferation and interactions, along with cell death across both populations. In this way, the effectiveness of immune cell killing of target cells can be directly correlated with changes in the effector population (e.g., associated with activation) and interactions between two cell populations within the same sample, measured by determining the overlap between the fluorescence of the effector and target cell labels.

[0103] Generally, immune cell recognition and killing of unwanted target cells (e.g., emerging tumor cells) are important components of human host defense mechanisms. 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 (e.g., natural killer (NK) cells or cytotoxic T cells (CTLs)), which then actively lyse target cells. The disclosed systems and methods enable the observation of interactions between immune cells and cancer cells, potentially providing information leading to the development of diagnostics and therapies ("cancer immunotherapy" or "immuno-oncology") to restore and promote the immune system's ability to fight and eliminate tumors.

[0104] Example 4: ATP+cell death Various implementations of the optical module 110, system 105, and method 300 described herein can be advantageously used to perform two-color Förster resonance energy transfer (FRET)-based measurements of ATP multiplexed with cell death analysis to investigate possible differences in the time- and concentration-dependent effects of compounds on cancer cell metabolism and mortality. In operation, three light sources in the optical module can be activated at three different bands of excitation wavelengths, two of which can be used to measure metabolic information via a single emission band via the FRET mechanism. The third excitation wavelength can then be used to monitor an independent readout related to cell death (e.g., Annexin V NIR). The ATP measurement process is described in more detail in PCT / US19 / 21171, filed March 7, 2019, entitled "Methods and Compositions for Live Cell Analysis of Intracellular ATP," which is incorporated herein by reference in its entirety.

[0105] Example 5: Live cell immunocytochemistry Various implementations of the optical module 110, system 105, and method 300 described herein can be used to measure surface protein expression by live-cell immunocytochemistry (ICC) using IncuCyte® FabFluor Antibody Labeling Reagent (or any other fluorescently labeled antibody reagent). This method can be used to track changes in cell subpopulations following experimental treatment (e.g., addition of a test compound or immune cell activation), to monitor changes in differentiation markers over time, or to otherwise assess surface protein expression. Three-color (or more) imaging offers advantages in flexibility of antibody selection and allows for monitoring of additional proteins or subpopulations of interest within a single sample.

[0106] The description of different advantageous arrangements is presented for purposes of illustration and description and is not intended to be exhaustive or limited to the disclosed exemplary forms. Many modifications and variations will become apparent to those skilled in the art. The selected exemplary example or exemplary embodiments have been chosen and described to best explain the principles, practical applications, and to enable those skilled in the art to understand the present disclosure in terms of various exemplary embodiments with various modifications as may be suitable for the particular use contemplated. [Explanation of symbols]

[0107] 100 Environment 105 System 110 Optical Module 111 Housing 111a Main body part 111b Cantilever extension 112 First Opening 113 Second Opening 115 Fluorescence Microscope 120 imaging sensor 125 Phase Ramp 126 Housing 127 Transmitted illumination light source 128 Protrusion 129 Groove 130 samples 135 First Light Source 136 First Filter 137 First Optical Path 140 Second Light Source 141 Second Filter 142 Second Optical Path 145 The Third Light Source 146 Third Filter 147 Third Optical Path 150 Primary Transmission Optical Path 155 Emission Filter 156 Primary Emission Optical Path 160 The Fourth Light Source 161 The Fourth Filter 162 Fourth Optical Path 165 objective lens 170 shaft 171 Flip Tab 172 first end 173 Protrusion 174 Second End 175 First Electrical Connector 177 Third Electrical Connector 180 Incubator 185 Phase Lamp Mount 186 Receptor 187 Detent 188 Spring-loaded ball 189 Spring 200 computing devices 200a Computing Devices 202 processors 204 Communication Interface 206 Data Storage 208 Output Interface 210 Display 212 communication bus 214 Network 216a Tablet 216b Personal Computer 216c laptop computer 216d Mobile Computing Devices 218 executable instructions

Claims

1. 1. An optical module for imaging fluorophores in a live cell biological sample, said optical module comprising: a first light source configured to emit first light at a first band of excitation wavelengths; a first filter disposed in a first optical path of the first light source, the first filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths; and a second light source configured to emit second light at a second band of excitation wavelengths; a second filter disposed in a second optical path of the second light source, the second filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths; and a third light source configured to emit third light at a third band of excitation wavelengths; a third filter disposed in a third optical path of the third light source, the third filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths, the first optical path, the second optical path, and the third optical path converging along a primary transmission optical path configured to be directed toward the live cell biological sample; an emission filter disposed in a primary emission optical path for light emitted by the fluorophores in the live cell biological sample, the primary emission optical path being configured to terminate at an imaging sensor, the emission filter being configured to pass light at emission wavelengths of a first band, a second band, and a third band, and to reflect light at excitation wavelengths of the first band, the second band, and the third band; an optical module including:

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

3. The optical module of claim 1 , 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 first filter is configured to pass light at excitation wavelengths in the first band and reflect light at excitation wavelengths in the second and third bands and light at emission wavelengths in the first, second, and third bands; the second filter is configured to pass light at emission wavelengths in the first band, the second band, and the third band and to reflect light at excitation wavelengths in the second band and the third band; 4. The optical module of claim 1, wherein the third filter is configured to pass light in the third band of excitation wavelengths and to reflect light in the second band of excitation wavelengths.

5. the first light source is positioned such that the first optical path begins at the first light source, passes through the first filter, and exits the optical module along the primary transmitted optical path; the second light source is positioned such that the second optical path begins at the second light source, reflects at the third filter to the second filter, reflects at the second filter to the first filter, reflects at the first filter, and exits the optical module along the primary transmitted optical path; the third light source is positioned such that the third optical path begins at the third light source, passes through the third filter to the second filter, reflects at the second filter to the first filter, reflects at the first filter, and exits the optical module along the primary transmitted optical path; 5. The optical module of claim 4, wherein the primary emission optical path for light emitted by the fluorophores in the live cell biological sample reflects at the first filter, passes through the second filter, passes through the emission filter, and exits the optical module.

6. the first filter is configured to pass light at excitation wavelengths in the first band and the second band and to reflect light at excitation wavelengths in the third band and light at emission wavelengths in the first band, the second band, and the third band; the second filter is configured to pass light at emission wavelengths in the second band and reflect light at excitation wavelengths in the first band; 6. The optical module of claim 1, wherein the third filter is configured to pass light at emission wavelengths in the first band, the second band, and the third band and to reflect light at excitation wavelengths in the third band.

7. the first light source is positioned such that the first optical path begins at the first light source, reflects off the second filter, passes through the first filter, and exits the optical module along the primary transmitted optical path; the second light source is positioned 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 primary transmitted optical path; the third light source is positioned such that the third optical path begins at the third light source, reflects at the third filter to the first filter, reflects at the first filter, and exits the optical module along the primary transmitted optical path; 7. The optical module of claim 6, wherein the primary emission optical path for light emitted by the fluorophores in the live cell biological sample reflects at the first filter, passes through the third filter, passes through the emission filter, and exits the optical module.

8. 8. The optical module of claim 1, wherein the excitation wavelength of the first band is in the range of 453 nm to 485 nm, the excitation wavelength of the second band is in the range of 546 nm to 568 nm, and the excitation wavelength of the third band is in the range of 648 nm to 674 nm.

9. 8. The optical module according to claim 1, wherein the emission wavelength of the first band is in the range of 494 nm to 533 nm, the emission wavelength of the second band is in the range of 576 nm to 639 nm, and the emission wavelength of the third band is in the range of 686 nm to 756 nm.

10. a fourth light source configured to emit fourth light at a fourth band of excitation wavelengths; a fourth filter disposed in a fourth optical path of the fourth light source, the fourth filter configured to pass light at one or more wavelengths and reflect light at one or more wavelengths, the emission filter further configured to pass light at a fourth band of emission wavelengths and reflect light at an excitation wavelength in the fourth band; and The optical module according to claim 1 , further comprising:

11. the first filter is configured to pass light of the first band of excitation wavelengths and reflect light of the fourth band of excitation wavelengths; the second filter is configured to pass light at emission wavelengths in the first band, the second band, and the third band and to reflect light at excitation wavelengths in the second band and the third band; the third filter is configured to pass light of the third band of excitation wavelengths and to reflect light of the second band of excitation wavelengths; 11. The optical module of claim 10, wherein the fourth filter is configured to pass light at excitation wavelengths in the first band and the fourth band and to reflect light at excitation wavelengths in the second band and the third band and light at emission wavelengths in the first band, the second band, the third band, and the fourth band.

12. 12. The optical module according to claim 10 or 11, wherein the emission wavelength of the fourth band is smaller than 453 nm and the excitation wavelength of the fourth band is smaller than the emission wavelength of the fourth band.

13. the first light source is positioned such that the first optical path begins at the first light source, passes through the first filter, then passes through a fourth filter, and exits the optical module along the primary transmitted optical path; the second light source is positioned such that the second optical path begins at the second light source, reflects at the third filter to the second filter, reflects at the second filter to the fourth filter, reflects at the fourth filter, and exits the optical module along the primary transmitted optical path; the third light source is positioned such that the third optical path begins at the third light source, passes through the third filter to the second filter, reflects at the second filter to the fourth filter, reflects at the fourth filter, and exits the optical module along the primary transmitted optical path; a fourth light source positioned such that a fourth optical path begins at the fourth light source, reflects off the first filter, passes through the fourth filter, and exits the optical module along the primary transmitted optical path; 13. The optical module of claim 1, wherein the primary emission optical path for light emitted by the fluorophore in the live cell biological sample reflects at the fourth filter, passes through the second filter, passes through the emission filter, and exits the optical module.

14. 1. A system for assaying a live cell biological sample, said system comprising: an optical module according to any one of claims 1 to 13; a fluorescence microscope removably coupled to the optical module, the fluorescence microscope having at least one objective lens; the imaging sensor positioned in an emission path for light emitted by the fluorophores in the live cell biological sample from the objective lens; a phase lamp removably coupled to the fluorescence microscope, the phase lamp being positioned at a terminal end of the primary transmission optical path; Including, the system.

15. a shaft extending through the optical module, the fluorescence microscope having a receiver configured to receive the shaft in a first orientation, the shaft configured to rotate to a second orientation under application of a force, thereby locking the optical module to the fluorescence microscope; The system of claim 14 further comprising:

16. a first electrical connector coupled to the optical module; a second electrical connector coupled to the fluorescence microscope, the second electrical connector being reciprocal to the first electrical connector; a processor in electrical communication with at least one of the first electrical connector and the second electrical connector, the processor configured to identify the optical module coupled to the fluorescence microscope; and 16. The system of claim 14 or 15, further comprising:

17. the phase lamp having a third electrical connector; a fourth electrical connector coupled to the fluorescence microscope, the third electrical connector being reciprocal with the fourth electrical connector, and the processor being configured to determine whether the optical module and the phase lamp are compatible and to display a warning in response to the determination; and The system of claim 16 further comprising:

18. an incubator configured to maintain the live cell biological sample at a temperature ranging from 30°C to 42°C and at a relative humidity ranging from 80% to 100%, wherein the optical module is coupled to a chamber of the incubator.

18. The system of claim 14, further comprising:

19. 1. A method for imaging a fluorophore in a live cell biological sample, the method comprising: aligning a first biological sample and a fluorescence microscope, wherein the first biological sample is positioned within a field of view of the fluorescence microscope, the first biological sample comprising (i) a first fluorophore that emits light at a first band of emission wavelengths in response to illumination with light at a first band of excitation wavelengths, (ii) a second fluorophore that emits light at a second band of emission wavelengths in response to illumination with light at a second band of excitation wavelengths, and (iii) a third fluorophore that emits light at a third band of emission wavelengths in response to illumination with light at a third band of excitation wavelengths; acquiring a set of images of the first biological sample using the fluorescence microscope, wherein images in the set differ with respect to a focus setting; determining first, second, and third in-focus settings for the first, second, and third bands of emission wavelengths, respectively, based on the set of images; illuminating the first biological sample with light at the first band of excitation wavelengths using a first light source for a first period of time; operating the fluorescence microscope according to the first in-focus setting to acquire a first image of light at the first band of emission wavelengths via an image sensor of the fluorescence microscope; illuminating the first biological sample with light at the second band of excitation wavelengths using a second light source for a second period of time; and operating the fluorescence microscope according to the second in-focus setting to acquire a second image of light at the second band of emission wavelengths via the image sensor. illuminating the first biological sample with light at the third band of excitation wavelengths using a third light source for a third time period; and operating the fluorescence microscope according to the third in-focus setting to acquire a third image of light at the third band of emission wavelengths via the image sensor. A method comprising:

20. generating, via a processor in electrical communication with an imaging sensor, a first corrected image of light emitted by the first fluorophore based on the first, second, and third images to reduce artifacts from light emitted by the second fluorophore and the third fluorophore; generating, via the processor, a second corrected image of light emitted by the second fluorophore based on the first, second, and third images to reduce artifacts from light emitted by the first fluorophore and the third fluorophore; generating, via the processor, a third corrected image of light emitted by the third fluorophore based on the first, second, and third images to reduce artifacts from light emitted by the first fluorophore and the second fluorophore; 20. The method of claim 19, further comprising:

21. maintaining at least the first biological sample via an incubator coupled to the fluorescence microscope at a temperature ranging from 30° C. to 42° C. and at a relative humidity ranging from 80% to 100% when acquiring the first, second, and third images.

21. The method of claim 19 or 20, further comprising:

22. 22. The method of any one of claims 19 to 21, further comprising generating a first corrected image based on a phase or bright field image acquired when the first biological sample is at the first in-focus setting and the first image.

23. receiving, via a processor, compatibility information regarding an optical module and a phase lamp module of the fluorescence microscope; determining, via the processor, whether the optical module and the phase lamp are compatible based on the compatibility information; in response to determining that the optical module and the phase lamp are incompatible, displaying, via the processor, a warning with an indication of the incompatibility; 23. The method of any one of claims 19 to 22, further comprising:

24. extending a shaft in a first orientation through an optical module of the fluorescence microscope to a receiver of the fluorescence microscope; rotating the shaft under application of a force such that the shaft moves to a second orientation, thereby coupling the optical module to the fluorescence microscope; 24. The method of any one of claims 19 to 23, further comprising:

25. 14. A non-transitory computer readable medium having stored thereon program instructions that, when executed by a processor in electromechanical communication with an optical module according to any one of claims 1 to 13, cause performance of a set of actions, the actions including: aligning a first biological sample and a fluorescence microscope, wherein the first biological sample is positioned within a field of view of the fluorescence microscope, the first biological sample comprising (i) a first fluorophore that emits light at a first band of emission wavelengths in response to illumination with light at a first band of excitation wavelengths, (ii) a second fluorophore that emits light at a second band of emission wavelengths in response to illumination with light at a second band of excitation wavelengths, and (iii) a third fluorophore that emits light at a third band of emission wavelengths in response to illumination with light at a third band of excitation wavelengths; acquiring, with the imaging sensor, a first set of images of the first biological sample using the fluorescence microscope, wherein images in the set differ with respect to a focus setting; determining first, second, and third in-focus settings for the first, second, and third bands of emission wavelengths, respectively, based on the set of images; illuminating the first biological sample with light at the first band of excitation wavelengths using a first light source for a first period of time; operating the fluorescence microscope according to the first in-focus setting to acquire a first image of light at the first band of emission wavelengths via an image sensor of the fluorescence microscope; illuminating the first biological sample with light at the second band of excitation wavelengths using a second light source for a second period of time; and operating the fluorescence microscope according to the second in-focus setting to acquire a second image of light at the second band of emission wavelengths via the image sensor. illuminating the first biological sample with light at the third band of excitation wavelengths using a third light source for a third time period; and operating the fluorescence microscope according to the third in-focus setting to acquire a third image of light at the third band of emission wavelengths via the image sensor. a non-transitory computer-readable medium,

Images