Method of capturing microscopic images, control unit, and sample investigation system

Abstract

The present invention relates to systems and methods for increasing z-stack capture on microscopy samples, for example through interleaving single stack slice image capture at multiple wavelengths with z-stack capture rather than performing multiple z-stack capture events to capture multiple color images of the sample.

Description

[0001] METHOD OF CAPTURING MICROSCOPIC IMAGES, CONTROL UNIT, AND SAMPLE INVESTIGATION SYSTEM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This document claims the benefit of priority to US Prov Ser No 63 / 519,314, filed August 14, 2023, the contents of which are hereby incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Field of the Invention: The present invention refers to a method of or for capturing microscopic images of a sample control unit, a sample investigation system, as well as systems and methods of capturing a multicolor z-stack microscopic image set of a sample that are designed for reducing time consumption and reproduction errors in z-stack capture of microscopy samples.

[0006] Nowadays, in the field of microscopy investigations, long-term observations and measurements of biological specimens, e.g. under different and / or varying conditions, become more and more important For detailed investigations and in particular for a 3D reconstruction, plural or multiple images of a sample at different wavelengths have to be captured, the images stemming from different layers or slices of an underlying sample in a height direction of the underlying sample.

[0007] Known attempts are comparable time consuming and prone to repositioning and repeatability errors, as for each color or wavelength a complete stroke has to be applied in order to let the focus or focal plane of the microscopes optics entirely pass through the height of the sample to thereby collect the required multicolor images from all the slices.

[0008] It is an object underlying the present invention to suggest a method of or for capturing microscopic images of a sample, a corresponding control unit, and sample investigation system, which reduce the time required for capturing the image and that can avoid the hitherto repositioning and repeatability errors.

[0009] SUMMARY OF THE INVENTION

[0010] The object underlying the present invention is achieved by a method of or for capturing microscopic images of a sample according to independent claim 1, by a control unit according to independent claim 24, by sample investigation system according to independent claim 25, by a method of capturing a multicolor z-stack microscopic image set of a sample according to independent claim 27, by a sample image according to claim 70, a method of reducing z - section image collection time for a three dimensional sample image of a sample according to claim 92, and a microscopy optics column according to claim 96. Preferred embodiments of the present invention are defined in the respective dependent claims.

[0011] According to a first aspect of the present invention, a method of capturing microscopic images of a sample is proposed. The present invention’s method comprises:

[0012] (A) focusing a microscope on the sample at a first position in a direction Z of a height of the sample as a first z-stack slice,

[0013] (B) exposing the sample and at least the first z-stack slice to electromagnetic radiation and in particular to light of a first illumination and / or excitation wavelength,

[0014] (C) collecting a first emission image from emission of electromagnetic radiation or light by the sample and in particular by the first z-stack slice resulting from exposing the sample and at least the first z-stack slice to the electromagnetic radiation or light of the first illumination and / or excitation wavelength,

[0015] (D) exposing the sample and at least the first z-stack slice to electromagnetic radiation and in particular to light of a second illumination and / or excitation wavelength,

[0016] (E) collecting a second emission image from emission of electromagnetic radiation or light by the sample and in particular by the first z-stack slice resulting from exposing the sample and at least the first z-stack slice to the electromagnetic radiation or light of the second illumination and / or excitation wavelength, and

[0017] (F) after collecting the first emission image and the second emission image, focusing the microscope on the sample at a second position in the direction of height of the sample as a second z-stack slice removed in the direction of height from the first z-stack slice.

[0018] According to the present invention, only a single pass or stroke for moving the focus and / or the focal plane through the underlying sample in the z or height direction and thus only a single movement of the optics and / or of focus in z direction is required. Such a procedure reduces the overall operation time as well as repositioning and repeatability errors and alignment problem in view of the captured images.

[0019] In an embodiment of the present invention’s method, a multicolor z-stack microscopic image set of the sample may be captured.

[0020] In an embodiment of the present invention’s method, the steps A to F may partially or completely be repeated, in particular by starting with an initial position zO in the height direction Z of the sample referring to an initial z-stack slice sO of the sample, by ending with a final position zN in the height direction Z of the sample referring to a final z- stack slice sN of the sample, and by successively incrementing with an increment dz a position of a focus or a focal plane of an underlying illuminating and / or imaging device in the height direction Z of the sample 5 starting initial position zO and ending at the final position zN.

[0021] The following relations I and II may advantageously be fulfilled in the repeated process zj = zO + j • dz, with j = 0, ..., N, and I zN = zO + N • dz, II with zO denoting the initial position zO in the height direction Z of the sample referring to an initial z-stack slice sO of the sample, with zN denoting the final position zN in the height direction Z of the sample referring to an final z-stack slice sN, dz begin the increment in the height direction Z of the sample, and with j is an integer counting index ranging from 0 to N, N being a natural number larger than 0.

[0022] In other words, there may be a one-to-one correspondence between the z-stack slices sj and the positions zj in the height or Z direction.

[0023] Collecting with respect to a the first z-stack slice sj of the sample a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 may be performed concurrently prior to focusing the microscope on a second z-stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj.

[0024] Collecting with respect to a the first z-stack slice sj of the sample a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 are performed sequentially prior to focusing the microscope on a second z-stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj.

[0025] Capturing the multicolor z-stack microscopic image set may be accomplished without focusing the microscope on the first z-stack slice sj more or one than once.

[0026] Capturing the multicolor z-stack microscopic image set may also accomplished without focusing the microscope on the first z-stack slice sj again after collecting the first emission image and the second emission image.

[0027] Capturing the multicolor z-stack microscopic image set may comprise at least one of:

[0028] - collecting a brightfield image of the first z-stack slice sj,

[0029] - collecting a transmission illuminated image of the first z-stack slice sj,

[0030] - collecting a back illuminated image of the first z-stack slice sj, and

[0031] - collecting an unilluminated image of the first z-stack slice sj.

[0032] Capturing the multicolor z-stack microscopic image set is accomplished in at least one of:

[0033] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample,

[0034] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of the sample, and

[0035] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image seton the microscope. Capturing the multicolor z-stack microscopic image set may be accomplished in at least one of a time that is less than 110 % as long as it takes

[0036] - to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample,

[0037] - to collect a monocolor or monochromic z-stack microscopic image set of the sample, and

[0038] - to collect a monocolor or monochromic z-stack microscopic image set on the microscope.

[0039] The multicolor z-stack microscopic image set may comprise or may be based on at least two colors, three colors, four colors, five colors of electromagnetic radiation and in particular of light for exposing the sample and at least a respective z-stack slice sj thereof, wherein a color is defined by a wavelength, a weighted discrete and / or continuous plurality of wavelengths, and / or a spectrum of wavelengths.

[0040] A first emission image and a second emission image of a z-stack slice sj of the sample and in particular of a first z-stack slice sj of the sample may be at least one of:

[0041] - aligned upon image capture,

[0042] - not subjected to post capture image alignment relative to one another, and

[0043] - not offset relative to one another upon image capture.

[0044] Light of a first illumination and / or excitation wavelength XI and light of a second illumination and / or excitation wavelength X2 may pass through a common beam splitter that is in particular fixed relative to the microscope.

[0045] In some embodiments, no mechanical image capture part of the microscope may move or may be moved relative to the sample pursuant to capturing an image of the first z-stack slice sj.

[0046] Light of a first illumination and / or excitation wavelength XI and light of a second illumination and / or excitation wavelength X2 may pass through a common beam splitter that is attached to a turret that moves or is configures to move relatively to the microscope. Each first image of a z-stack slice sj of a sample may be captured though a first camera, in particular with respect to a first color and / or wavelength XI.

[0047] At least one first image of a z-stack slice sj of the sample image may be captured though a first camera, in particular with respect to a first color and / or wavelength XI, and at least another one first image of the z-stack slice sj of the sample may be captured though a second camera, in particular with respect to a second color and / or wavelength X2 .

[0048] An underlying sample may be or may comprise a fluorophore labeled nucleic acid and in particular at least one of an RNA molecule and a DNA molecule.

[0049] An underlying sample may be or may comprise at least one of a fluorophore labeled protein, a flash frozen sample, a fresh frozen sample, an FFPE preserved sample, wherein in particular the FFPE preserved sample is subjected to at least one of a nucleic acid assay and a protein assay.

[0050] An embodiment of the present invention’s method may comprise a step of collecting a bright field image of a distinct region of the sample using a second microscope.

[0051] The bright field image of a distinct region of the sample may be collected concurrently.

[0052] An underlying second microscope may be positioned at least one of:

[0053] - adjacentto a microscope that captures the multicolor z-stack microscopic image set of the sample,

[0054] - on an opposite sample side relative to the microscope that captures the multicolor z-stack microscopic image set of the sample, and

[0055] - on an opposite slide side relative to the microscope that captures the multicolor z-stack microscopic image set of the sample .

[0056] According to another aspect of the present invention, a control unit 300, 400 for controlling a sample and / or sample investigation system is proposed and - according to an embodiment of the present invention - it may be configured to initiate, control, and / or perform a method of capturing microscopic images of a sample according to the present invention and the steps thereof.

[0057] In addition, according to the present invention a sample investigation system according is suggested that is configured to initiate, control, perform and / or to be used a or in a method of capturing microscopic images of a sample according to the present invention and the steps thereof.

[0058] An embodiment of the present invention’s system may comprise:

[0059] - at least one microscope having an objective,

[0060] - at least one light source or an optical coupling to an external light source,

[0061] - a primary specimen carrier configured to receive, carry and or support a sample or specimen,

[0062] - at least one camera, and

[0063] - a control unit, wherein:

[0064] - the control unit is formed according to the present invention, and

[0065] - the control unit is configured to control at least one of the microscope, the light source, the primary specimen carrier and in particular a holder and / or stage therefor, and the camera with respect to their operation and cooperation.

[0066] These and further details, advantages and features of the present invention will be described based on embodiments of the invention and by taking reference to the accompanying figures.

[0067] BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1A is a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system to which the invention’s method can be applied and in particular it depicts a sample configured for capturing a multicolor z- stack microscopic image set

[0069] Figure IB is similar to figure 1A and elucidates also by means of a schematical and cross- sectional side view of an embodiment of the present invention’s sample investigation system the concept of z-stack slice of the underlying sample. Figure 2A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for dual wavelength interleaving with single camera imaging.

[0070] Figure 2B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 2A.

[0071] Figure 3A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for dual wavelength interleaving with dual camera imaging.

[0072] Figure 3B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 3A.

[0073] Figure 4A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for dual wavelength interleaving with dual camera imaging.

[0074] Figure 4B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 4A.

[0075] Figure 5A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for dual wavelength interleaving with dual camera imaging.

[0076] Figure 5B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 5A.

[0077] Figure 6A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for triple wavelength interleaving with single camera imaging.

[0078] Figure 6B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 6A. Figure 7A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation system a system for triple wavelength interleaving with dual camera imaging.

[0079] Figure 7B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 7A.

[0080] Figure 8A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation a system for dual wavelength interleaving and trans-illumination with dual camera imaging.

[0081] Figure 8B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 8A.

[0082] Figure 9A presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation a system for dual wavelength interleaving and back-illumination with dual camera imaging.

[0083] Figure 9B presents a schematical diagram or cartoon of temporal activities pursuant to the system or practice of a method consistent with the system of figure 9A.

[0084] Figure 10 presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation an optical train architecture for optimized throughput and reliability.

[0085] Figure 11 presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation an optical train architecture for optimized throughput and reliability.

[0086] Figure 12 presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation a slide-well configuration with an ITO layer.

[0087] Figure 13 presents by means of a schematical and cross-sectional side view of an embodiment of the present invention’s sample investigation a slide-well configuration. Figure 14 is a schematical timing chart elucidating the timings for imaging a single z-stack slice of the sample when using two illumination sources and two cameras.

[0088] Figure 15 is a schematical timing chart elucidating the timings for imaging a single z-stack slice of the sample when using three illumination sources and two cameras.

[0089] Figure 16 is a schematical block diagram of a first embodiment of the present invention’s control unit.

[0090] Figure 17 is a schematical block diagram of a second embodiment of the present invention’s control unit.

[0091] Figure 18 is a cross-sectional side view of an embodiment of the present invention’s sample investigation system to which the invention’s method can be applied and in particular it depicts capturing the image from above the sample.

[0092] Figure 19 is a cross-sectional side view of another embodiment of the present invention’s sample investigation system to which the invention’s method can be applied and in particular it depicts capturing the image from above the sample, too.

[0093] Figure 20 is a cross-sectional side view of still a further embodiment of the present invention’s sample investigation system to which the invention’s method can be applied and in particular it also depicts capturing the image from above the sample.

[0094] DETAILED DESCRIPTION

[0095] In the following, embodiments and the technical background of the present invention are presented in detail by taking reference to accompanying figures 1 to 20. Identical or equivalent elements and elements which act identically or equivalently are denoted with the same reference signs. Notin each case of their occurrence a detailed description of the elements and components is repeated.

[0096] The depicted and described features and further properties of the invention’s embodiments can arbitrarily be isolated and recombined without leaving the gist of the present invention.

[0097] A method of capturing microscopic images of a sample 5 according to the present invention comprises steps of: (A) focusing a microscope 101 on the sample 5 at a first position zj in a direction Z of a height of the sample 5 as a first z-stack slice sj,

[0098] (B) exposing the sample 5 and at least the first z-stack slice sj to electromagnetic radiation and in particular to light of a first illumination and / or excitation wavelength XI,

[0099] (C) collecting a first emission image from emission of electromagnetic radiation or light by the sample 5 and in particular by the first z-stack slice sj resulting from exposing the sample 5 and at least the first z-stack slice sj to the electromagnetic radiation or light of the first illumination and / or excitation wavelength XI,

[0100] (D) exposing the sample 5 and at least the first z-stack slice sj to electromagnetic radiation and in particular to light of a second illumination and / or excitation wavelength X2,

[0101] (E) collecting a second emission image from emission of electromagnetic radiation or light by the sample 5 and in particular by the first z-stack slice sj resulting from exposing the sample 5 and at least the first z-stack slice sj to the electromagnetic radiation or light of the second illumination and / or excitation wavelength X2, and

[0102] (F) after collecting the first emission image and the second emission image, focusing the microscope 101 on the sample 5 at a second position zk in the direction Z of height of the sample 5 as a second z-stack slice sk removed in the direction Z of height from the first z-stack slice sj.

[0103] In an embodiment of the present invention’s method, a multicolor z-stack microscopic image set of the sample 5 may be captured.

[0104] In an embodiment of the present invention’s method, the steps A to F may partially or completely be repeated, in particular by starting with an initial position zO in the height direction Z of the sample 5 referring to an initial z-stack slice sO of the sample 5, by ending with a final position zN in the height direction Z of the sample 5 referring to a final z- stack slice sN of the sample 5, and by successively incrementing with an increment dz a position of a focus or a focal plane of an underlying illuminating and / or imaging device in the height direction Z of the sample 5 starting initial position zO and ending at the final position zN.

[0105] The following relations I and II are fulfilled in the repeated process zj = zO + j • dz, with j = 0, N, and I zN = zO + N • dz, II with zO denoting the initial position zO in the height direction Z of the sample 5 referring to an initial z-stack slice sO of the sample 5, with zN denoting the final position zN in the height direction Z of the sample 5 referring to an final z-stack slice sN, dz begin the increment in the height direction Z of the sample 5, and with j is an integer counting index ranging from 0 to N, N being a natural number larger than 0.

[0106] In other words, there may be a one-to-one correspondence between the z-stack slices sj and the positions zj in the height or Z direction.

[0107] Collecting with respect to a the first z-stack slice sj of the sample 5 a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 may be performed concurrently prior to focusing the microscope 101 on a second z- stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj.

[0108] Collecting with respect to a the first z-stack slice sj of the sample 5 a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 are performed sequentially prior to focusing the microscope 101 on a second z-stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj.

[0109] Capturing the multicolor z-stack microscopic image set may be accomplished without focusing the microscope 101 on the first z-stack slice sj more or one than once.

[0110] Capturing the multicolor z-stack microscopic image set may also accomplished without focusing the microscope 101 on the first z-stack slice sj again after collecting the first emission image and the second emission image.

[0111] Capturing the multicolor z-stack microscopic image set may comprise at least one of:

[0112] - collecting a brightfield image of the first z-stack slice sj,

[0113] - collecting a transmission illuminated image of the first z-stack slice sj,

[0114] - collecting a back illuminated image of the first z-stack slice sj, and

[0115] - collecting an unilluminated image of the first z-stack slice sj.

[0116] In some embodiments, capturing a multicolor z-stack microscopic image set is accomplished in a time that is less than 200% of a time it takes to collect a monocolor or monochromic (e.g., monochromatic) z-stack image set of a comparable sample.

[0117] Capturing the multicolor z-stack microscopic image set is accomplished in at least one of:

[0118] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample 5,

[0119] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of the sample 5, and

[0120] - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image seton the microscope 101.

[0121] Capturing the multicolor z-stack microscopic image set may be accomplished in at least one of a time that is less than 110% as long as it takes

[0122] - to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample 5,

[0123] - to collect a monocolor or monochromic z-stack microscopic image set of the sample 5, and

[0124] - to collect a monocolor or monochromic z-stack microscopic image set on the microscope 101. The multicolor z-stack microscopic image set may comprise or may be based on at least two colors, three colors, four colors, five colors of electromagnetic radiation and in particular of light for exposing the sample 5 and at least a respective z-stack slice sj thereof, wherein a color is defined by a wavelength, a weighted discrete and / or continuous plurality of wavelengths, and / or a spectrum of wavelengths.

[0125] A first emission image and a second emission image of a z-stack slice sj of the sample and in particular of a first z-stack slice sj of the sample 5 may be at least one of:

[0126] - aligned upon image capture,

[0127] - not subjected to post capture image alignment relative to one another, and

[0128] - not offset relative to one another upon image capture.

[0129] Light of a first illumination and / or excitation wavelength XI and light of a second illumination and / or excitation wavelength X2 may pass through a common beam splitter 106 that is in particular fixed relative to the microscope 101.

[0130] In some embodiments, no mechanical image capture part of the microscope 101 may move or may be moved relative to the sample 5 pursuant to capturing an image of the first z-stack slice sj.

[0131] Light of a first illumination and / or excitation wavelength XI and light of a second illumination and / or excitation wavelength X2 may pass through a common beam splitter 106 that is attached to a turret that moves or is configures to move relatively to the microscope 101.

[0132] Each first image of a z-stack slice sj of a sample 5 may be captured though a first camera 112, in particular with respect to a first color and / or wavelength XI.

[0133] At least one first image of a z-stack slice sj of the sample 5 image may be captured though a first camera 112, in particular with respect to a first color and / or wavelength XI, and at least another one first image of the z-stack slice sj of the sample 5 may be captured though a second camera 116, in particular with respect to a second color and / or wavelength X2 .

[0134] An underlying sample 5 may be or may comprise a fluorophore labeled nucleic acid and in particular at least one of an RNA molecule and a DNA molecule. An underlying sample 5 may be or may comprise at least one of a fluorophore labeled protein, a flash frozen sample, a fresh frozen sample, an FFPE preserved sample, wherein in particular the FFPE preserved sample is subjected to at least one of a nucleic acid assay and a protein assay.

[0135] An embodiment of the present invention’s method may comprise a step of collecting a bright field image of a distinct region of the sample 5 using a second microscope 201.

[0136] The bright field image of a distinct region of the sample 5 may be collected concurrently.

[0137] An underlying second microscope 201 may be positioned at least one of:

[0138] - adjacent to a microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5,

[0139] - on an opposite sample side relative to the microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5, and

[0140] - on an opposite slide side relative to the microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5.

[0141] A control unit 300, 400 for controlling a sample and / or sample investigation system 1 according to an embodiment of the present invention may be configured to initiate, control, and / or perform a method of capturing microscopic images of a sample 5 according to the present invention and the steps thereof.

[0142] A sample investigation system 1 according to the present invention is configured to initiate, control, perform and / or to be used a or in a method of capturing microscopic images of a sample 5 according to the present invention and the steps thereof.

[0143] An embodiment of the present invention’s system 1 may comprise:

[0144] - at least one microscope 101, 201 having an objective 102, at least one light source 105 or an optical coupling to an external light source 105, a primary specimen carrier 2 configured to receive, carry and or support a sample 5 or specimen 5,

[0145] - atleastone camera 112, 116, 120, 212, 216, and

[0146] - a control unit 300, 400, wherein:

[0147] - the control unit 300, 400 is formed according to the present invention, and

[0148] - the control unit 300, 400 is configured to control atleastone ofthe microscope 101, 201, the light source 105, the primary specimen carrier 2 and in particular a holder and / or stage therefor, and the camera with respect to their operation and cooperation.

[0149] Conventional methods of imaging a multi-color z-stack conduct a full z-stack scan to generate z - sections with one excitation followed by another z-stack scan at the next wavelength. Under this type of approach, the number of z-stack scans to be performed is proportional to the number of colors to be imaged in the stack.

[0150] Furthermore, single channel images of a z-stack slice are not collected concurrently or successively, such that they must be aligned subsequent to collection. This alignment is complicated by moving parts on the image collection chain, which increase the chance that successively collected single channel images of a z-stack slice may not be in alignment with one another, necessitating postcapture image alignment.

[0151] Also, after a full z-stack scan with one excitation followed by another z-stack scan at the next wavelength is performed, or even when a single full z-stack scan is performed, the remainder of the sample is often subjected to alternate imaging such as bright field imaging for subsequent z-stack site selection. However, performing such a site selection imaging process further extends the total time of a sample image capture process, and may also result in substantial sample photodamage.

[0152] Thus, multi-color imaging can become veiy time consuming, complicating high throughput image collection and limiting the types of samples that may be analyzed.

[0153] Disclosed herein are systems, methods and devices related to capturing a multicolor z-stack microscopic image set of a sample. Some such systems, methods and devices comprise one or more of focusing a microscope on a first z-stack slice, exposing the first z-stack slice to a first excitation wavelength, collecting a first emission image from emission resulting from the first excitation wavelength, exposing the first z-stack slice to a second excitation wavelength, collecting a second emission image from emission resulting from the second excitation wavelength, and then after collecting the first emission image and the second emission image, focusing the microscope on a second z-stack slice removed in a 'z' dimension from the first z-stack slice.

[0154] Such systems, methods and devices variously substantially reduce image collection time, simplify post-collection image assembly, and facilitate concurrent image site selection, resulting in a substantially faster, more efficient process.

[0155] Disclosed herein are systems and methods for the rapid, efficient capture of and assembly of multicolor z-stack microscopy image sets. Through practice of the disclosure herein, multicolor z- stack microscopy image sets are captured and assembled without requiring multiple collection passes through a sample or the concomitant loss of time or movement of imaging apparatus relative to the sample. Collection and assembly are in some cases accomplished in a time period comparable that to single color z-stack microscopy image set collection and assembly, and do not require image alignment

[0156] Central to many embodiments disclosed herein is the interleaving of multiple color z-stack slice image collection within a single slice capture event of a multiple color z-stack image collection process, rather than collecting single a complete set of single color z-stack slice image collection of a first color, followed by collecting a second complete set of single color z-stack slice image collection, and in some cases a third, fourth or even more complete single color z-stack image collection processes.

[0157] In z-stack image collection, the majority of the image collection time is often spent in moving or refocusing the microscope image collection column among the individual slabs of the z-stack. Furthermore, with each movement of the microscope image collection column, there is the possibility of slight repositioning of the sample relative to the microscope image collection column, such that image realignment must often be performed pursuant to z-stack image assembly

[0158] As disclosed herein, multicolor image collection is performed concurrently or successively on a single z-stack slice, ‘interleaved’ such that the microscope image collection column does not need to exit a z-stack slice until multiple color images have been collected. Similarly, the microscope image collection column does not need to perform more than a single pass through the sample at the image collection site.

[0159] Faster z-stack generation. Accordingly, through practice of the disclosure herein, the time spent on z- stack image collection for a multicolor image is reduced in some cases to only a fraction more than the time required for single color z-stack image capture. Accordingly, z-stack image collection for a multicolor image such as a two-color image is in some cases less than twofold the time for single color z-stack image collection, such as no more than 190%, 175%, 150%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, 101% or even 100% of the time of single color single color z-stack image collection. Similarly, z-stack image collection for a multicolor image such as a three color image is in some cases less than threefold the time for single color z-stack image collection, such as no more than 290%, 250%, 200, 150%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, 101% or even 100% of the time of single color single color z-stack image collection. Similarly, z-stack image collection for a multicolor image such as a four color image is in some cases less than threefold the time for single color z-stack image collection, such as no more than 390%, 350%, 300%, 250%, 200, 150%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, 101% or even 100% of the time of single color single color z-stack image collection. Similarly, z-stack image collection for a multicolor image such as a five color image is in some cases less than threefold the time for single color z-stack image collection, such as no more than 490%, 400%, 350%, 300%, 250%, 200, 150%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, 101% or even 100% of the time of single color single color z-stack image collection. Through practice of the disclosure herein, a decrease in the time required for multicolor image capture allows for increased throughput and content for a given image (e.g., an image of a sample). In some embodiments, by practice of the disclosure herein, a higher content image can be acquired in less time than other methods for multicolor image capture of a high content image (e.g., a multicolor image captured through mechanical manipulation of optical components). In some embodiments, by practice of the disclosure herein, a higher throughput of multicolor images can be acquired in less time than other methods for high throughput multicolor image capture (e.g., a multicolor image capture through mechanical manipulation of optical components).

[0160] Light capture timing. Interleaving, that is, concurrent or successive collection of multiple emission spectra at a single z-stack slice prior to moving the focus of the microscope image collection column to subsequent z-stack slice or z - section, is in various embodiments accomplished through a number of approaches consistent with the disclosure herein. Some such approaches are presented below in figure 2B, 3B, 4B, 5B, 6B, 7B, 8B, and 9B. The figures specify particular excitation wavelengths, but it is understood that a broad range of wavelengths are consistent with various embodiments of the disclosure herein, such as various wavelengths in the visible or otherwise excitation-consistent spectrum or that result in emission of light in the visible or otherwise detectible spectrum from a sample.

[0161] Approaches variously comprise concurrently or successively shining excitation energy of at least two wavelength onto a sample, and capturing resultant emission energy in either a single or more than one camera such as a CMOS camera.

[0162] For example, in some cases a single camera is used to capture emission spectra from a single sample arising from excitation energy that is projected onto a sample for nonoverlapping time spans, such that the emission spectra of the excitation energies do not temporally overlap, and such that the emission spectra may be captured by a single camera without overlapping. See, for example figure 2A and 2B.

[0163] Alternately, in some cases two cameras or more than two cameras are used to capture emission spectra from a single sample arising from excitation energy that is projected onto a sample for overlapping or nonoverlapping time spans. In these cases a dichroic filter may be used to separate emission spectra of different wavelengths, as shown in figures 3A and 3B.

[0164] These approaches are not in all cases mutually exclusive. Thus in some cases emission spectra from a single sample arising from excitation energy that is projected onto a sample for nonoverlapping time spans, such that the emission spectra of the excitation energies do not temporally overlap, but the emission spectra are nonetheless captured by distinct cameras. The cameras are configured to capture light only when their respective excitation energies are applied, as in figure 4A and figure 4B. Alternately, cameras may be configured to capture light continuously within a z-stack slice capture interval, as shown in figure 5A and figure 5B. In these cases, cameras may capture background z-slice images while their respective excitation energies are not applied to the sample.

[0165] Multiple color detection. The disclosure herein is consistent with multiple color z-stack image capture. Attention is paid above to two-color z-stack generation, but three, four or higher order color images or z-stack collection is also consistent with the disclosure herein. Furthermore, generally, the improvement in capture time is increased even more for higher order color combinations, as the conventional stack collection approaches scale with the number of colors collected while the approach of the disclosure herein does not

[0166] Multiple color detection is effected through use of one or more than one camera. In some cases three or more excitation wavelengths are projected onto a sample in succession such as nonoverlapping succession during a single z-stack slice image capture interval. As the corresponding emission spectra are temporally separated, they may be projected onto a single camera such as a CMOS camera at a single z-stack slice image capture interval. One such configuration is presented at Fig 6.

[0167] Alternately, multiple camera configurations such as two camera configurations are consistent with the disclosure herein. Some such approaches comprise a subset of the excitation spectra being projected onto a sample in succession such as nonoverlapping succession during a single z-stack slice image capture interval, to be captured by a single camera as above, while other emission spectrum or spectra are projected continuously onto the sample and collected either in a temporal pattern or continuously, as seen in figures 7, 8 or 9. In these cases, one light spectrum may in fact comprise background light intended to generate a conventional microscopy image such as a bright field image. Light may be exposed to the sample through the slide or from the opposite side of the slide, as seen in figure 8 or 9, respectively.

[0168] Electrically switching cameras and lasers conveys the benefit of retaining wavelength diversity without introducing the potential error that may accompany mechanical manipulation of an optical column or components during a z-scan or z - section image capture. Mechanical manipulation often involves rotating or translating structural components of an optical column, such as a camera or laser. Such movement may cause the optical column or sample to vibrate, causing misalignment or causing the sample to fall out of focus. Electrical switching is substantially gentler on the optical column, and minimizes inertial or mass movements such that vibration or loss of focus is minimized or eliminated

[0169] Wavelengths. The disclosure herein is consistent with a number of excitation and emission wavelengths, as well as white light or other light for bright field imaging to be performed concurrently with excitation an emission collection of a z-stack slice. Mention is made in the figures of 561 nm and 594 nm as preferred excitation energies. These wavelengths are in some cases preferred because they do not overlap with or result in emission spectra that overlap with some native mammalian cell auto fluorescence. However, as various wavelengths in the visible or otherwise excitation-consistent spectrum or that result in emission of light in the visible or otherwise detectible spectrum from a sample, a broad range of wavelengths are consistent with the disclosure, such as 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, or any wavelength encompassed by a range spanned by these numbers or adjacent to the range spanned by these numbers.

[0170] Alignment Z-stack image slices are aligned subsequent to capture so as to generate a collated 2-d or 3-d image. Collation requires that subsequent z-slice images be aligned precisely so as to compensate for off-set that may occur, for example through multiple passes through a sample pursuant to conventional z-stack image collection.

[0171] The disclosure herein substantially simplifies or in some cases alleviates the requirement for alignment of images of a single z-stack slice. This arises from the fact that multiple wavelength images of a z-stack slice are captured concurrently, interleaved in a single z-stack slice collection event, rather than occurring in subsequent passes through the sample to collect various colored z-stack images in series, in a process which introduces the risk of offset.

[0172] Accordingly, in some aspects of the disclosure herein, little or no alignment of multiple colored images of a particular z-series slice is required, and in some cases none is performed.

[0173] To further reduce the requirement for alignment or the risk of offset, in various embodiments of the disclosure herein the dichroic light splitter is fixed such that it does not revolve relative to the microscope image collection column. Such a configuration increases efficiency of movement of the microscope image collection column, reducing the risk of offset

[0174] An effect of this reduction in alignment burden is that the z-series collation time is reduced, to for example no more than 90%, 75%, 50%, 20%, 10% or less than 10% of the z-set collation time required when alignment of various color images of one or more or even each slice of the z-series is performed.

[0175] Additional innovations related to increases in z-stack capture. Efficient z-stack capture is predicated upon identification of sample sites from which the stack is to be taken. Often, z-stack sites are selected by first imaging a larger portion of the sample using, for example, bright field microscopy. This is often performed before an z-stack is captured, and may be performed again after a z-stack is collected to identify a second distinct site for z-stack capture.

[0176] Through some aspects of the disclosure herein, z-stack capture is accomplished concurrently with independent sample imaging such as that shown in figure 10 or 11. Through these approaches, z- stack capture and imaging for subsequent z-stack site selection are performed concurrently on a single sample or on a subsequent sample. In some cases the effect of this activity is to reduce the time necessary to generate a second or a plurality of z-stacks.

[0177] Z-stack capture often requires that the microscope remain in fluid contact with the transverse side of the slide upon which the sample is deposited. A challenge for z-stack capture is that some fluids do not maintain contact with the slide throughout a single or multiple z-stack capture events. Accordingly, in some cases a slide is configured to comprise a well or set of wells that may be partially or fully flooded with a contact liquid. In some cases the well may serve to maintain a reservoir of contact liquid, facilitating long term or iterative image capture without interruption.

[0178] Some slide configurations herein present a slide in proximity to a heating unit, such as that shown in figure 12. Such a configuration allows sample manipulation prior to concomitant with or between z- stack collection events. In some cases the well may serve to maintain a reservoir of contact liquid, facilitating long term or iterative image capture without interruption despite heating of the sample.

[0179] Samples. A broad range of samples are consistent with the disclosure herein. Samples suitable for analysis include live or preserved cells or tissues, such as FFPE cells, for example FFPE preserved cancer calls. Some samples are flash frozen or otherwise frozen samples, and are in some cases held at a freezing temperature by the slide configuration.

[0180] In some cases samples are treated to visualize specific or general nucleic acid distribution such as RNA or DNA distribution, or both RNA and DNA distribution. Alternately or in combination, samples are treated so as to visualize specific or general protein distribution, alone or in combination with other metabolites. Similarly, samples may be treated to visualize nuclear DNA position, chromatin structure, mitochondrial activity or distribution, or other organellar or metabolite localization or accumulation.

[0181] The methods and systems of the disclosure is further understood through closer investigation of the accompanying figures.

[0182] At figures 1A and IB, one sees a microscope configured for multi-color z-stack imaging. A high NA objective lens is positioned below a glass slide onto which a sample is deposited. A water immersion maintains contact between the objective and the bottom of the slide. The z-stack to be captured comprises three z-slices below the slide surface, a slice at the slide surface, and a plurality of slices through the sample deposited on the slide. Slices are separated from one another by a dz distance of about 35 Onm up to a total height of lOum above the surface of the slide.

[0183] At figure 2 A, one sees a microscope configured for dual wavelength interleaving of multi-color z-stack imaging with a single camera. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figures 1A and IB. Below the lens, one sees excitation light of 561 nm and 594 nm configured to be directed to a dichroic beam splitter. Light emitted from the sample passes through a dual-color filter and is captured by a CMOS camera, at bottom.

[0184] At figure 2B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging. From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0185] For each dz interval as indicated at bottom, two clock intervals are demarcated. The CMOS camera is configured to capture for both of these time intervals. The 561 nm laser and the 594 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0186] The activation of the lasers is in some cases electronic rather than mechanical. That is the lasers are alternately activated and deactivated rather than being mechanically directed toward and away from the sample or blocked from the sample by a structural gate. Accordingly, the substantial activity depicted in figure 2B is effected without mechanical motion, which may otherwise introduce vibration into the system, delaying imaging and in some cases necessitating refocusing. Similarly, the images may be captured without the sample or optics column being subjected to this mechanical motion, such that successive images used to generate a z-stack often do not need to be realigned. All of these features decrease overall image generation time.

[0187] At figure 3 A, one sees a microscope configured for dual wavelength interleaving of multi-color z-stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figures 1A or 2A. Below the lens, one sees excitation light of 561 nm and 594 nm configured to be directed to a first dichroic beam splitter. Light emitted from the sample is directed to a second dichroic beam splitter, such that emission light resulting from the two excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a single color filter to accommodate the wavelength of the emission light they are to collect.

[0188] At figure 3B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging. From top, one sees that the timing of 561 nm laser pulsing is continuous, the timing of 594 nm laser pulsing is continuous, the first CMOS camera is configured for light capture at intervals corresponding to the z-stack intervals, the second CMOS camera is configured for light capture at intervals corresponding to the z-stack intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0189] For each dz interval as indicated at bottom, one clock interval is demarcated. The CMOS cameras are configured to capture for the entirety of this interval. The 561 nm laser and the 594 nm laser are concurrently activated during the entirety of these intervals, such that both of the two lasers are exciting a given dz interval at a time, but such that for each dz interval, emission data is captured for each laser emission light at a separate CMOS camera. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0190] The activation of the cameras is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0191] At figure 4A, one sees a microscope configured for dual wavelength interleaving of multi-color z-stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figures 1A and 3A. Below the lens, one sees excitation light of 561 nm and 594 nm configured to be directed to a first dichroic beam splitter. Light emitted from the sample is directed to a second dichroic beam splitter, such that emission light resulting from the two excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a single color filter to accommodate the wavelength of the emission light they are to collect.

[0192] At figure 4B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging.

[0193] From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the first CMOS camera light capture intervals, the second CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0194] For each dz interval as indicated at bottom, two clock intervals are demarcated. The second CMOS camera is configured to capture emission light arising from excitation by the 561 nm laser. The first CMOS camera is configured to capture emission light arising from excitation by the 594 nm laser. The 561 nm laser and the 594 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0195] The activation of the lasers is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0196] At figure 5 A, one sees a microscope configured for dual wavelength interleaving of multi-color z-stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figures 1A, 3A, and 4A. Below the lens, one sees excitation light of 561 nm and 594 nm configured to be directed to a first dichroic beam splitter. Light emitted from the sample is directed to a second dichroic beam splitter, such that emission light resulting from the two excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a single color filter to accommodate the wavelength of the emission light they are to collect

[0197] At figure 5B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging.

[0198] From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the first CMOS camera light capture intervals, the second CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0199] For each dz interval as indicated at bottom, two clock intervals are demarcated. The second CMOS camera is configured to capture emission light passing though the filter targeting emission light from the 561 nm laser. The first CMOS camera is configured to capture emission light passing though the filter targeting emission light from the 594 nm laser. The 561 nm laser and the 594 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Camera 1 and camera 2, however, are configured to capture light throughout the dz interval, such that while their respective lasers are active, they are collecting emission light of their respective lasers, while during the remainder of the dz interval they are collecting background emission of the channel for which they are filtered. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0200] The activation of the lasers is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0201] At figure 6A, one sees a microscope configured for triple wavelength interleaving of multi-color z- stack imaging with a single camera. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figures 1A and IB. Below the lens, one sees excitation light of 561 nm, 594 nm and 405 nm configured to be directed to a dichroic beam splitter. Light emitted from the sample passes through a triple-color filter and is captured by a CMOS camera, at bottom.

[0202] At figure 6B, one sees a cartoon depiction of the timing regime to effect triple wavelength interleaving of multi-color z-stack imaging. From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the timing of 405 nm laser pulsing, the CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0203] For each dz interval as indicated at bottom, three clock intervals are demarcated. The CMOS camera is configured to capture for all three of these time intervals. The 561 nm laser, the 594 nm laser and the 405 nm are alternately activated in these intervals, such that only one of the lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to all three wavelengths, and emission data is captured for all three wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0204] The activation of the lasers is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0205] At figure 7A, one sees a microscope configured for triple wavelength interleaving of multi-color z- stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figure 2 A. Below the lens, one sees excitation light of 561 nm, 594 nm, and 405 nm configured to be directed to a first dichroic beam splitter. Light emitted from the sample is directed to a second dichroic beam splitter, such that emission light resulting from the three excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a color filter to accommodate the wavelength of the emission light they are to collect.

[0206] At figure 7B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging. From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the timing of the 405 nm light pulsing, the second CMOS camera light capture intervals, the first CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0207] For each dz interval as indicated at bottom, two clock intervals are demarcated. The second CMOS camera is configured to capture emission light arising from excitation by the 405 nm laser, which emits continuously but which is only captured at intervals of each dz time period. The first CMOS camera is configured to capture emission light arising from excitation by the 594 nm laser and the 561 nm laser. The 594 nm laser and the 561 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0208] The activation of the lasers and of the cameras is coordinated and in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0209] At figure 8A, one sees a microscope configured for triple wavelength interleaving of multi-color z- stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figure 2 A. Below the lens, one sees excitation light of 561 nm, and 594 nm configured to be directed to a first dichroic beam splitter from a laser source below the slide. Light a t a third wavelength is transmitted through the sample from above, and may be used to generate a transillumination brightfield image. Light emitted from the sample is directed to a second dichroic beam splitter, such that emission light resulting from the three excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a color filter to accommodate the wavelength of the emission light they are to collect

[0210] At figure 8B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging.

[0211] From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the timing of the 405 nm light pulsing, the second CMOS camera light capture intervals, the first CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm. For each dz interval as indicated at bottom, two clock intervals are demarcated. The second CMOS camera is configured to capture emission light arising from excitation or light arising from transillumination, which emits continuously and which is captured continuously in each dz time period. The first CMOS camera is configured to capture emission light arising from excitation by the 594 nm laser and the 561 nm laser. The 594 nm laser and the 561 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0212] The activation of the lasers and regulation of camera frame rate is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0213] At figure 9A, one sees a microscope configured for triple wavelength interleaving of multi-color z- stack imaging with dual camera imaging. At left, one sees the microscope configured for multi-color z-stack imaging similar to that of figure 2 A. Below the lens, one sees excitation light of 561 nm, and 594 nm configured to be directed to a first dichroic beam splitter from a laser source below the slide. Light at a third wavelength for back-illumination of the sample is transmitted through the sample from below, and may be used to generate a back-illumination brightfield image. Light emitted from the sample or arising from back-illumination of the sample is directed to a second dichroic beam splitter, such that emission light resulting from the three excitation wavelengths are directed separately to either a first or a second CMOS camera, each having a color filter to accommodate the wavelength of the emission light they are to collect

[0214] At figure 9B, one sees a cartoon depiction of the timing regime to effect dual wavelength interleaving of multi-color z-stack imaging.

[0215] From top, one sees the timing of 561 nm laser pulsing, the timing of 594 nm laser pulsing, the timing of the back illumination light, the second CMOS camera light capture intervals, the first CMOS camera light capture intervals, a clock timing of time intervals within a z-stack capture interval, and at bottom, time intervals during which the focal plane is advanced by the dz interval of 350 nm.

[0216] For each dz interval as indicated at bottom, two clock intervals are demarcated. The second CMOS camera is configured to capture emission light arising from excitation or light arising from back- illumination, which emits continuously and which is captured continuously in each dz time period. The first CMOS camera is configured to capture emission light arising from excitation by the 594 nm laser and the 561 nm laser. The 594 nm laser and the 561 nm laser are alternately activated in these intervals, such that only one of the two lasers is exciting a given dz interval at a time, but such that for each dz interval, the sample is exposed to both wavelengths, and emission data is captured for both wavelengths successively. Accordingly, images for each wavelength for each dz interval may be captured in a single sweep through the dz intervals of the sample.

[0217] The activation of the lasers and regulation of camera frame rate is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0218] At figure 10, one sees a first scheme for concurrent immersion liquid z-section capture and air spaced bright field imaging of a sample. Both microscope imaging devices are on the same side of the sample.

[0219] At figure 11, one sees a second scheme for concurrent z-section capture and bright field imaging of a sample. Both microscope imaging devices are on the same side of the sample. Five laser wavelengths are used to excite the sample to generate z - section images, and all five emission spectra, as well as transmission light, are captured by one of two cameras.

[0220] The activation of the lasers and regulation of the cameras is in some cases electronic rather than mechanical, conveying the stability and speed benefits discussed above and elsewhere herein.

[0221] At figure 12, one sees a slide configured with wells harboring immersion fluid opposite the sample. The slide comprises a series of slide frames which form wells capable of harboring immersion fluid (as shown) such as water. The slide comprises a flat slide surface for harboring a sample or samples (as shown) Such wells retain immersion fluid so as to maintain microscope contact with the slide surface over prolonged time or despite adverse conditions such as heating the sample.

[0222] The slide is configured to interface with a manipulation device capable of heating, cooling or otherwise manipulating the sample. Such as manipulation device comprises a frame and optionally a gasket layer to facilitate interface with the slide.

[0223] The slide is also configured to harbor an ITO layer that may be suitable for sample manipulation such as sample heating.

[0224] At figure 13, one sees a slide configuration lacking the ITO layer.

[0225] At figure 14, one sees a timeline for two channel and two camera image capture of a sample z-slice as part of an iterative sample z-slice capture workflow.

[0226] The figure depicts timing for Z movement, PC / Arduino Communication, a first wavelength activity, in this case 588 nm, a second wavelength activity, in this case 553 nm, first camera activity, a second camera activity, a first data transfer segment transferring data from the first camera, and a second data transfer segment transferring data from the second camera.

[0227] Along the y-axis one sees exemplary timescales for the components mentioned above. In particular, in this case movement of the optics column along the z axis (S) takes 29 ms, from tO to tl, and is followed by a 20 ms segment, from tl to t2, of communication (D) between the PC and Arduino Z stage or optical column controller, and relates to column stabilization and focusing.

[0228] Next one sees concurrent 100 ms activation, from t2 to t3, of the first laser excitation beam, in this case 588 nm, and camera 1. Activation of these imaging components is electrically directed and does not comprise mechanical movement, which eliminates an otherwise negative impact on focusing and timing when cameras, lasers or cameras and lasers are mechanically moved so as to be positioned for sample excitation and image capture.

[0229] Next one sees deactivation of the first laser excitation beam and camera 1 at t3, and transfer of data collected by camera 1 in the t2-t3 interval from sample excitation by the 588 nm laser.

[0230] Simultaneously, one sees concurrent 100 ms activation of the second laser activation beam, in this case at 553 nm, and camera 2, Activation of these imaging components is electrically directed and does not comprise mechanical movement, which eliminates an otherwise negative impact on focusing and timing when cameras, lasers or cameras and lasers are mechanically moved so as to be positioned for sample excitation and image capture.

[0231] In particular, camera 2 data collection occurs concurrently with camera 1 data transmission, such that camera 1 data transmission does not contribute to overall duration of the z-slice image capture.

[0232] Next one sees deactivation of the second laser excitation beam and camera 2 at t4, and transfer of data collected by camera 2 in the t3-t4 interval from sample excitation by the 553 nm laser.

[0233] Simultaneously, one sees movement of the optics column along the z axis (S) to a new z slice, which takes 29 ms, and which is followed by a 20 ms segment, of communication (D) between the PC and Arduino Z stage or optical column controller, and relates to column stabilization and focusing. In particular, S and D events concurrently with camera 2 data transmission, such that camera 2 data transmission does not contribute to overall duration of the z-slice image capture.

[0234] The total duration for a z-slice focusing and image capture and spans from tO to t4, and spans 249ms in this example.

[0235] A few features of this workflow warrant particular emphasis. Firstly, the transition from laser 1 and camera 1 to laser 2 and camera 2 is mediated electronically rather than mechanically, such that there is no movement delay S or PC / Arduino D delay intervening in the transition from laser 1 and camera 1 to laser 2 and camera 2. Secondly, camera 1 is not involved in collection of laser 2 emission, such that it may transfer laser 1 collection data concurrently with laser 2 and camera 2 activity. As a result, camera 1 data transfer does not contribute to the overall time of imaging of the z section. Similarly, camera 2 is not involved in movement S or PC / Arduino D, or in collection of laser 1 emission, such that it may transfer laser 2 collection data concurrently with optics column movement and refocusing, and with laser 1 and camera 1 activity. Thus, data transfer does not contribute to z-slice collection time, and data collection for a single z slice is not interrupted by an intervening refocusing as would be necessitated by mechanical transitioning from a first to a second camera.

[0236] In this example, the z-slice collection workflow is performed a total of 32 times pursuant to z-slice collection for the sample.

[0237] At figure 15, one sees a timeline for three channel and three camera image capture of a sample z-slice as part of an iterative sample z-slice capture workflow.

[0238] The figure depicts timing for Z movement, PC / Arduino Communication, a first wavelength activity, in this case 588 nm, a second wavelength activity, in this case 553 nm, a third wavelength activity, in this case 405 nm, a first camera activity, a second camera activity, and a third camera activity. Not shown but occurring in the z-slice capture are a first data transfer segment transferring data from the first camera, a second data transfer segment transferring data from the second camera, and a third data transfer segment transferring data from the third camera.

[0239] Along the y-axis one sees exemplary timescales for the components mentioned above. In particular, in this case movement of the optics column along the z axis (S) takes 29 ms, from tO to tl, and is followed by a 20 ms segment, from tl to t2, of communication (D) between the PC and Arduino Z stage or optical column controller, and relates to column stabilization and focusing. Next one sees concurrent 100 ms activation, from t2 to t3, of the first laser excitation beam, in this case 588 nm, and camera 1. Activation of these imaging components is electrically directed and does not comprise mechanical movement, which eliminates an otherwise negative impact on focusing and timing when cameras, lasers or cameras and lasers are mechanically moved so as to be positioned for sample excitation and image capture.

[0240] Next one sees deactivation of the first laser excitation beam and camera 1 at t3. Not shown but initiated at this point is transfer of data collected by camera 1 in the t2-t3 interval from sample excitation by the 588 nm laser.

[0241] Simultaneously, one sees concurrent 100 ms activation of the second laser activation beam, in this case at 553 nm, and camera 2, Activation of these imaging components is electrically directed and does not comprise mechanical movement, which eliminates an otherwise negative impact on focusing and timing when cameras, lasers or cameras and lasers are mechanically moved so as to be positioned for sample excitation and image capture.

[0242] In particular, camera 2 data collection occurs concurrently with camera 1 data transmission, such that camera 1 data transmission does not contribute to overall duration of the z-slice image capture. Next one sees deactivation of the second laser excitation beam and camera 2 at t4.

[0243] Not shown but initiated at this point is transfer of data collected by camera 2 in the t4-t5 interval from sample excitation by the 553 nm laser.

[0244] Simultaneously, one sees concurrent 100 ms activation of the third laser activation beam, in this case at 405 nm, and camera 3, Activation of these imaging components is electrically directed and does not comprise mechanical movement, which eliminates an otherwise negative impact on focusing and timing when cameras, lasers or cameras and lasers are mechanically moved so as to be positioned for sample excitation and image capture.

[0245] In particular, camera 3 data collection occurs concurrently with camera 2 data transmission, such that camera 2 data transmission does not contribute to overall duration of the z-slice image capture. Next one sees deactivation of the third laser excitation beam and camera 3 at t5.

[0246] Not shown but initiated at this point is transfer of data collected by camera 2 at t5 from sample excitation by the 405 nm laser. Simultaneously, one sees movement of the optics column along the z axis (S) to a new z slice, which takes 29 ms, and which is followed by a 20 ms segment, of communication (D) between the PC and Arduino Z stage or optical column controller, and relates to column stabilization and focusing.

[0247] In particular, S and D events concurrently with camera 3 data transmission, such that camera 3 data transmission does not contribute to overall duration of the z-slice image capture.

[0248] The total duration for a z-slice focusing and image capture and spans from tO to t5, and spans 349ms in this example.

[0249] A few features of this workflow warrant particular emphasis. Firstly, the transitions from laser 1 and camera 1 to laser 2 and camera 2, and from laser 2 and camera 2 to laser 3 and camera 3, are mediated electronically rather than mechanically, such that there is no movement delay S or PC / Arduino D delay intervening in the transition from laser 1 and camera 1 to laser 2 and camera 2 or from laser 2 and camera 2 to laser 3 and camera 3. Secondly, camera 1 is not involved in collection of laser 2 emission, such that it may transfer laser 1 collection data concurrently with laser 2 and camera 2 activity. Similarly, camera 2 is not involved in collection of laser 3 emission, such that it may transfer laser 2 collection data concurrently with laser 3 and camera 3 activity.

[0250] As a result, camera 1 data transfer and camera 2 data transfer does not contribute to the over all time of imaging of the z section. Similarly, camera 3 is not involved in movement S or PC / Arduino D, or in collection of laser 1 emission, such that it may transfer laser 3 collection data concurrently with optics column movement and refocusing, and with laser 1 and camera 1 activity. Thus, data transfer does not contribute to z-slice collection time, and data collection for a single z slice is not interrupted by an intervening refocusing as would be necessitated by mechanical transitioning from a first to a second camera.

[0251] In this example, the z-slice collection workflow is performed a total of 32 times pursuant to z-slice collection for the sample.

[0252] At figure 16, one sees a schematical block diagram elucidating an embodiment for a control unit according to the present invention.

[0253] At figure 17 one sees a schematical block diagram elucidating an embodiment for a control unit according to the present invention. At figure 18, one sees an optics column configured to capture sections of a sample to form a z-stack consistent with the disclosure herein. The optics column comprises two light sources lambda 1’ and lambda 2’, a first of which emits at either 588 or 650 nm, while the second emits at 553 nm. The light sources direct light to a first beamsplitter / dichronic-1, labeled 106 in the figure, so that they may be redirected to the sample at a selected z-stack plane, while their resultant emission spectra pass through the beamsplitter Ito a beamsplitter / dichronic 2, which directs the emission resulting from laser 1 excitation through a single color filter 114 to camera 2 at 116, while directing the emission resulting from laser 2 excitation through single color filter 110 to camera 1.

[0254] The excitation and emission spectra passage through a high NA objective lens 102, through a water immersion contact 104 and a glass slide on 170 um in this case, through a sample on a 1 mm glass slide, contained in a chamber and immersed in a fluid. Alternate embodiments image the sample from below the glass slide onto which the sample is attached.

[0255] A few features of this optics column warrant highlighting. Firstly, as two lasers are used, the light wavelength directed to the sample is modulated by a simple electrical on / off gating rather than by changing the wavelength generated by a single laser or by changing a filter or beamsplitter in front of a multiwavelength laser. Thus, the excitation frequency to which the sample is exposed is changed without mechanical adjustment of any features of the optics column.

[0256] Similarly, a fixed beamsplitter directs the emission spectra from laser 1 and laser 2 to distinct cameras 2 and 1, such that direction of the emission spectra to cameras is effected without mechanical adjustment of any features of the optics column.

[0257] Rather, coordination in the optics column is electrical, in that cameras are activated in synchrony with laser activation and deactivation, purely through electric control. In alternate embodiments cameras 2 and 1 are left on continually. In either case, two channel (or in alternates such as three laser embodiments or larger laser embodiments) excitation wavelength switching and concurrent emission capture switching for a given z- section is effected without mechanical manipulation of the optics column. Multiple wavelengths are captured during a single positioning of the optics column at a given z - section, such that time spent adjusting the optics column to a new z - section is minimized or reduced, and mechanical manipulation of the optics column while it is directed at a particular z - section is minimized or reduced.

[0258] Thus, the optics column presented herein substantially reduces the negative impact that mechanical manipulation of the often relatively massive optics column, such as impacts on sample focusing and image orientation. Time does not need to be spent on allowing the column to settle or cease vibrating after mechanical manipulation pursuant to laser switching for a given z - section, and time does not need to be spent re-orienting images captured at different excitation wavelengths for a given z - section. Consequently, z - section capture is accomplished rapidly and efficiently without having to accommodate refocusing pursuant to ameliorating the effects of mechanical manipulation during a z - section capture, and image assembly from z - sections is accomplished without having to realign various wavelength images if a given z -section.

[0259] At figure 19, one sees an optics column configured to capture sections of a sample to form a z-stack consistent with the disclosure herein. The optics column of figure 19 differs from that of figure 18 in that beamsplitter / dichronic 1 (106) and beamsplitter / dichronic 2 (108) are separated by a multiband emission filter (122) that does not exclude the emission spectrum emitted by the sample following excitation by laser 1 (588nm) or laser 2 (553nm), but filters light of at least one other wavelength.

[0260] Consistent with the features of the optics column of figure 18, above, the emission filter of figure 19 effects emission light filtration without mechanical motion within the optics column. Thus, like the optics column of figure 18, the optics column presented herein substantially reduces the negative impact that mechanical manipulation of the often relatively massive optics column leads to, such as impacts on sample focusing and image orientation. Time does not need to be spent on allowing the column to settle or cease vibrating after mechanical manipulation pursuant to laser switching for a given z - section, and time does not need to be spent re-orienting images captured at different excitation wavelengths for a given z -section. Consequently, z - section capture is accomplished rapidly and efficiently without having to accommodate refocusing pursuant to ameliorating the effects of mechanical manipulation during a z - section capture, and image assembly from z - sections is accomplished without having to realign various wavelength images if a given z -section.

[0261] At figure 20, one sees an optics column configured to capture sections of a sample to form a z-stack consistent with the disclosure herein. The optics column of figure 20 differs from that of figure 19 in that the optics column comprises a third laser , in this case projecting an excitation beam lambda 0 at a wavelength of 405 nm. This excitation beam yields an emission spectrum that is captured by camera 2 (116), which also captures the emission spectrum arising from the 588 nm excitation energy laser.

[0262] Consistent with the features of the optics column of figures 18 and 19, above, the optics column of figure 20 effects emission light capture from three excitation wavelengths without mechanical motion within the optics column. Data from camera 2 may be distinguished as to whether it arises from lambda 0 (405nm) or lambda 1 (588 nm) either by the wavelength of the emission spectrum or in some embodiments by timing of the light transmitted to camera 2. That is, excitation by laser lambda 0 and laser lambda 1 may be distinguished in that the lasers are not simultaneously active, such that timing of receipt of excitation light by camera 2 allows one to distinguish the laser from which the emission light arose. This timing is effected in many cases electrically rather than mechanically, such that transition between the lasers does not require mechanical manipulation of the optics column.

[0263] Thus, like the optics column of figures 18 and 19, and elsewhere herein, the optics column presented herein substantially reduces the negative impact that mechanical manipulation of the often relatively massive optics column leads to, such as impacts on sample focusing and image orientation. Time does not need to be spent on allowing the column to settle or cease vibrating after mechanical manipulation pursuant to laser switching for a given z - section, and time does not need to be spent re-orienting images captured at different excitation wavelengths for a given z -section. Consequently, z - section capture is accomplished rapidly and efficiently without having to accommodate refocusing pursuant to ameliorating the effects of mechanical manipulation during a z - section capture, and image assembly from z - sections is accomplished without having to realign various wavelength images if a given z -section.

[0264] These and further aspects of the present invention will also be described in detail in the following passages:

[0265] Returning to the disclosure more generally, one of the main motivations underlying the present invention is to enable the acquisition of wide-field fluorescence-based images from different wavelengths or excitation wavelengths of involved light or general electromagnetic radiation during a single z-stack scan of a biological specimen or sample 5 immobilized on a substrate such as microscope slide and serving as a primary specimen or sample carrier.

[0266] A conventional method of imaging a multi-color z-stack is to conduct a full z-stack scan with a single kind excitation, e.g. single color or wavelength of involved light or general electromagnetic radiation followed by another z-stack scan at another wavelength. This makes the whole process quite slow, in particular in cases where large areas of the sample 5 need to be scanned, this translates into many tens of hours of image acquisition.

[0267] According to one key idea the present invention’s concept, interleaving the various color imaging by temporally switching an underlying light source on and off at a rate that is synced with the rate at which the CMOS camera 112, 116, 120, 212, 216 or a general camera 112, 116, 120, 212, 216 is capturing images.

[0268] In one embodiment of the present invention, a wide field multi-channel fluorescence microscope or general microscope 101 with through focus scanning configuration is provided to collect multiple single z plane images from a sample 5.

[0269] Two or more excitation channels that interleave or that are interleaved in time - in its operation - in order to illuminate the sample 5 and / or to excite different fluorophores within the sample 5.

[0270] The detection of photons emitted from the sample 5 in general and / or from different fluorophores may be achieved by a single camera or by plural or multiple (at least two) cameras, each having one or more corresponding wavelength bands, in particular matching to one or plural of the emission wavelengths of the sample 5 and in particular of one or plural of the fluorophores provided in the sample 5.

[0271] The detection camera(s) 112, 116, 120, 212, 216 may have a matching frame rate and an exposure time fitting to the timing of the interleaved excitation light sources.

[0272] In a configuration 1, a single camera 112 shall have a transmission spectral band to detect photons from all fluorophores. In one embodiment, odd frames collect photons from a fluorophore 1, illuminated and / or excited e.g. by an illumination and / or excitation channel with a wavelength of e.g. 561 nm for the illumination or excitation radiation or light, and even frames collect photons from a fluorophore 2, excited by excitation channel with a wavelength of e.g. 594 nm for the illumination or excitation radiation or light.

[0273] In a configuration 2, a first camera 112 and second camera 116 have transmission spectral bands to detect photons from a fluorophore 1 and a fluorophore 2, respectively.

[0274] In an alternative configuration 2b, the exposure times of two camera 112, 116 are interleaved together with the timing of the illumination and / or excitation channels: E.g. 2ndcamera 116 is on to collect from fluorophore 1 as illumination and / or excitation at 561 nm is on, but off when illumination and / or excitation at 561 nm is off, and 1stcamera 112 is on to collect from fluorophore 2 as illumination and / or excitation at 594 nm excitation is on, but off when illumination and / or excitation at 594 nm is off.

[0275] Before and after, any concretely mentioned distinct wavelength - such as 561 nm or 594 nm or the like - is mentioned as an example only and any other wavelength, combination of wavelengths, spectra, each being discrete and / or continuous, disjunct (non-overlapping) or overlapping may be used for illumination, excitation and / or detection for realizing the present invention’s concepts.

[0276] In a further alternative configuration 2c, two cameras are always turned on at a frame rate matching a combined clock rate of two illumination and / or excitation channels, e.g. 2ndcamera 116 is on to collect from fluorophore 1 as illumination and / or excitation at 561 nm excitation is on (referred to as a 1stframe), and also on (referred to as a 2nd frame) when illumination and / or excitation at 561 nm is off. 1stcamera 112 is on to collect from fluorophore 2 as illumination and / or excitation at 594 nm excitation is on (2ndframe), and also on (1stframe) when illumination and / or excitation at 594 nm is off.

[0277] One comparable simple implementation of the present invention’s gist is realized by employing a single camera 112 in order to image from the various color channels. For example, when using two illumination and / or excitation lasers, one can use a clock signal to synchronize the z-stack movement, e.g. the relative movement between the sample 5 and / or specimen 5 and the focus and / or focal plane of the underlying microscope 110, the camera acquisition and the timing of operating the two illumination and / or excitation light sources, e.g. turning them on and off.

[0278] Before and after, a slice sj or z-stack slice - with j = 0, 1, 2, ..., N and N being an integer describing a maximum number of slices or layers - may refer to an ideal slice or plane in the underlying sample 5 or specimen 5, to which the focus or focal plane may be set on in order to capture an image from said slice or plane of the sample 5 or specimen 5. As shown in figure IB, the slices or planes sj may be parallel to the XY plane of an underlying frame of reference XYZ. The index j distinguishes slices sj located at different positions in the height direction Z (Z direction) and thereby refer to respective coordinate values zj in the height or Z direction.

[0279] For the sake of simplicity, it may be assumed that the time allotted for each slice sj may be 100 ms (100 • IO3s). The first clock pulse triggers the first laser to turn ON for a duration that must not exceed 50 ms. At the same time and synchronized with the turning ON of the first laser, the CMOS camera 112 is triggered to collect light emitted as a result of reflection and / or fluorescence in the sample 5 due the illumination and / or excitation by the first light source or laser. In this case the exposure of the camera must not exceed the duration of the pulse of the laser. The next clock pulse triggers the second laser in the next 50 ms timeslot. This next pulse triggers the camera 112 again to start another round of acquisition which now captures the reflection and / or fluorescence due to the second light or laser pulse. At the end of this period the z-position zj for slice sj of the sample 5 is moved to zk such that it allows the imaging of the next slice sk of the sample and from here the cycle continues.

[0280] There may be used arbitrary value for j and k. But it is advantageous to have k = j + 1, to start from an initial or primary height zO in the sample and to set zj = zO + j • dz, dz being e height increment and the index j counting the number of increments.

[0281] In the example just presented, a 32 slice z-stack tile (i.e. a tile that has 32 slices sj and 32 different Z positions / height zj) will take at 3200 ms. In order to reduce the total image time of such a single tile to be below a second, the clock will have to operate at about 67 Hz. Being able to image an entire z- stack of a single tile below one second has direct impact on the ability of the system to image more area per unit time.

[0282] Certain embodiments of the present invention might involve one or plural polarizing beam-splitters 106, 108. This is advantageous for separating the illumination and / or excitation light or radiation from the received radiation or light from reflection or fluorescence. The reason for this is that the laser or general light source input is polarized and hence a polarizing beam-splitter (PBS) 106, 108 can prevent the excitation light from travelling into the camera path directly. The refection and / or fluorescence light, radiation or signal does not or not necessarily carry or maintain a particular polarization and thus a good or sufficient portion of this radiation or signal can travel through the PBS 106, 108 in order to be imaged.

[0283] The optional use of one or plural dual bandpass filters 110, 114, 118, 122 whose bandpass wavelengths are adjusted to the optimum fluorescence spectra will reduce the opportunity of crosstalk and further reduce the chances of leakage reflection and / or excitation radiation or light making its way to the imager.

[0284] An alternative implementation extends the basic concept by adding a second camera 116 in addition to a first camera 112. This is inter alia shown in connection with the embodiments of figures 2A, 3A, 4A, 5 A, 7A, 8A, 9A, 10, and 11. Here an advantage is based on the fact that both light sources or lasers can be operated continuously. The received radiation of reflection (e.g. refection and / or fluorescence) is separated with the assistance of a dichroic mirror or beam splitter 108 that is placed after the first PBS / dichroic 106. The two cameras 112, 116 get triggered simultaneously and the exposure of the cameras 112, 116 can be maintained for the entire duration of the imaging of a current slice sj.

[0285] The exposure time of each camera 112, 116 can be adjusted within the time window of the slice sj in order to suit the amount of signal impinging on the respective camera 112, 116.

[0286] The cameras 112, 116 in this case may be operated at potential lower framerate.

[0287] Additional optional bandpass filters 110, 114 before each camera 112, 116 may ensure that any chance of cross talk is further reduced.

[0288] Further concepts 3 and 4 may extend the key ideas presented in concepts 1 and 2 as described above in order to accommodate a third channel of illumination and / or detection:

[0289] In some embodiments, the biological sample 5 is imaged using a third illumination and / or excitation such as the one shown in the figures above. A potential light source or laser can be a "purple” laser acting at a wavelength of e.g. 405 nm. With the judicious use of a triple color or triple bandpass filter 118 one can envisage the use of a single camera 112 with all three lasers being pulsed and synchronized with the frame rate of the camera 112. This is demonstrated in figures 6A and 6B.

[0290] Adding in this case an additional second camera 116 can allow one of the lasers to be operated continuously. Such a configuration is demonstrated in figures 7A, 8A, and 9A.

[0291] In the embodiment elucidated with figure 14 and in view of having two cameras 112, 116 and two illumination and reception / detection channels, capturing a complete tile or z-stack of 32 slices sj regarding an underlying specimen 5 or sample 5 may require 32 • 249 ms for illumination, detection, and communication, which includes 29 ms for movement in the Z direction and 20 ms for confirmation that the z-stage is in a stable position (e.g., PC / Arduino Comm) for each slice sj, resulting in 8.090 seconds per 32-slices tile.

[0292] Accordingly, various embodiments herein allow for a total time for two channel imaging of a z-stack of 32 slices sj of a sample of no more than 40 seconds, 20 seconds, 15 seconds, 12 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds or less than 2 seconds. For example, some embodiments described herein allow for a total time for two channel imaging of a z-stackof 32 slices sj of a sample of no more than 4 seconds. In another example, some embodiments described herein allow for a total time for two channel imaging of a z-stack of 32 slices sj of a sample of between 1-7 seconds, 1-6 seconds, 1-5 seconds, or 1-4 seconds.

[0293] In the embodiment elucidated with figure 15 and in view of having two cameras 112, 116 and three illumination and reception / detection channels, capturing a complete tile or z-stack of 32 slices sj regarding an underlying specimen 5 or sample 5 may require 32 • 349 ms for illumination, detection, and communication resulting in 11.168 seconds capture time per 32-slices tile resulting in a total time (including extra times for XY direction movement) of 11.610 seconds.

[0294] Accordingly, various embodiments herein allow for a total time for three or more than three channel imaging of a z-stack of 32 slices sj of a sample of no more than 40 seconds, 20 seconds, 15 seconds, 12 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds or less than 2 seconds.

[0295] Figure 16 is a schematical block diagram elucidating an embodiment for a control unit 300 according to the present invention. Its components an interconnected via control and / or acquisition lines 350, that may be formed as a bus.

[0296] Its components may inter alia include:

[0297] - a PC or general computer 301, possibly but not necessarily run under Linux OS,

[0298] - a 1stcamera (mono)Cl, 307 (mono), possibly but not necessarily run under USB 3.0,

[0299] - a 2ndcamera (mono) C2, 306, possibly but not necessarily run under USB 3.0,

[0300] - a 1stautofocus unit 308, possibly but not necessarily run under ethernet,

[0301] - a Z-stage unitZl, 309, possibly but not necessarily run under CAN-to-USB,

[0302] - a Tip-Tilt stage TpTltl, 310, possibly but not necessarily run under CAN-to-USB,

[0303] - an XY stage unitXY, 311, possibly but not necessarily run under CAN-to-USB,

[0304] - a temperature controller TCI, 312, possibly but not necessarily run under USB,

[0305] - (optionally) a brightfield LED BFLED1, connected to a microcontroller unitMCUl,

[0306] - a (first) laser unit Laserl, 305, e.g. a 5 or five color laser engine, connected to both the PC, 301 and a 2ndmicrocontroller unit MCU2, 302,

[0307] - a 2ndmicrocontroller unit MCU2, 302 for operating, controlling and / or pulsing the laser, possibly but not necessarily run under USB, - a 1stsyringe pump SP1, 313, connected to a 3rdmicrocontroller unitMCU3, 303,

[0308] - a 2ndsyringe pump SP2, 314, connected to the 3rdmicrocontroller unitMCU3, 303,

[0309] - a 1strotary valve RV1, 315, connected to the 3rdmicrocontroller unitMCU3, 303,

[0310] - a 2ndrotary valve RV2, 316, connected to the 3rdmicrocontroller unit MCU3, 303,

[0311] - (optionally) a 3rdrotary valve RV3, connected to the 3rdmicrocontroller unit MCU3, 303,

[0312] - a 1stdegasser DG1, 317, connected to the 3rdmicrocontroller unitMCU3, 303,

[0313] - a 1ststage XYZPrSiprl to move a / the needle, possibly but not necessarily run under USB,

[0314] - a 1stflow sensor FS1, 319, connected to a 4thmicrocontroller unitMCU4,

[0315] - a 1stpressure sensor PSI, 320, connected to a 4thmicrocontroller unit MCU4,

[0316] - a 1sttemperature sensor TS1, 321, connected to a 4thmicrocontroller unit MCU4,

[0317] - a 3rdmicrocontroller unit MCU3, 303, possibly but not necessarily run under USB, and

[0318] - a 4thmicrocontroller unit MCU4, 304, possibly but not necessarily run under USB.

[0319] Figure 17 is a schematical block diagram elucidating an embodiment for a control unit 400 according to the present invention. Its components an interconnected via control and / or acquisition lines 450, that may be formed as a bus.

[0320] Its components may inter alia include a PC or general computer PC, 401, to which a 1stmicrocontroller unit MCU1, 402, a 2ndmicrocontroller unit MCU2, 403, a 3rdmicrocontroller unit MCU3, 404, and one or plural camera units 406 are connected to.

[0321] The 1stmicrocontroller unit MCU1, 402 may have connected thereto one or plural light source units and / or lasers 405, one or plural brightfield light source or LED units 407, one or plural temperature sensor units 408, one or plural autofocus units 409, one or plural stage units 410. The 1stmicrocontroller unit MCU1, 402 may may trigger the one or plural camera units 406.

[0322] The 2ndmicrocontroller unit MCU2, 403 may have connected thereto one or plural immersion delivery units 411, one or plural probe sampling units 412, and one or plural buffer sampling units 413.

[0323] The 3rdmicrocontroller unit MCU3, 404 may have connected thereto one or plural sensors and / or sensor fusion units 414.

[0324] In view of the configuration and operation of the control unit 400, data from the camera(s) 406 may directly go to the PC, however the cameras may be triggered from one of the MCUs 403, 403, 404 which also may synchronize the pulsing of the laser engine 406 and the stages or z-stage 410, the autofocus 409 and xy-stage 410.

[0325] The (two) brightfield LEDs 407 and the temperature controllers 408 may be for each of the two flowcell holders, the latter serving as primary sample carriers or for holding a primary sample carrier.

[0326] The immersion delivery unit 411 pumps an immersion liquid or fluid, e.g. water, using a single syringe pump to a flow-cell 9.

[0327] The probe sampling unit 412 may comprise of an xyz- stage that may move a needle that extracts samples 5 or specimens 5 out of a 96- well plate. There may a rotary valve and a syringe pump be involved and / or shared with the buffer sampling unit 413.

[0328] The buffer sampling unit 413 may comprise a syringe pump - possibly shared with the probe sampling unit 412, and two rotary valves. Also a degasser may be in line with buffer sources / bottles.

[0329] The sensor fusion unit 414 of the system may comprise of all the sensors that are built into the system and that monitor the health of the instrument and sensors that aid in the successful operation of the instrument For example, slide proximity sensors, vibrations sensors, flow-cell holder clamp sensors and the like may be involved.

[0330] The configurations of control units 300 and 400 as shown in figures 16 and 17 may partially or completely be combined with each other.

[0331] Figures 18, 19, and 20 schematically show an upright configuration in which the imaging is to be performed from above an underlying flow-cell or sample or specimen carrier, namely in the height direction Z.

[0332] As shown in figures 18 and 19, two light sources or excitation lasers are used.

[0333] In the configuration of figure 18, individual filters 110, 114 are used in front of the cameras 112, 116.

[0334] Figure 19 demonstrates to usage of a multiband filter 122 before the entry to the 2nd dichroic beamsplitter 108 or prism 108.

[0335] In the configuration of figure 20, an approach with three light sources or lasers is demonstrated. The dichroic beamsplitters 106, 108 and multiband emission filter 122 are engineered and configured to accommodate the delivery of three emission and three detection or fluorescence channels.

[0336] One underlying and embracing key idea of the present invention and its embodiments is multicolor illumination and / or excitation imaging with scanning through colors / frequencies per slice - e.g. per focus in the Z or height direction of the underlying sample 5 - instead of scanning through all positions or heights zj in the Z or height direction of the focus per color / frequency.

[0337] In some cases this scanning is effected without mechanical manipulation of an optics column component during a particular z - section scanning event, such that the optics column does not need to be refocused pursuant to multichannel or multiwavelength image collection for a particular z - section. Similarly, in some cases images for a particular z - section may be assembled without realignment or alignment confirmation, for example because the optics column does not move and is not subject to potential movement arising from mechanical adjustment or adjustment of moving parts pursuant to laser switching, light splitting, camera switching or image capture.

[0338] According to this aspect of the present invention, only a single pass and thus only a single movement of the optics / focus in z direction is required per z - section image capture. In some cases no mechanical manipulation is required for, for example, laser light switching, camera switching, beam splitting, or image capture. Consequently, in some cases images for a particular z - section may be assembled without realignment or alignment confirmation, for example because the optics column does not move and is not subject to potential movement arising from mechanical adjustment or adjustment of moving parts pursuant to laser switching, light splitting, camera switching or image capture.

[0339] Consistent with the elimination of mechanical motion in the optics column pursuant to individual z - section imaging, the disclosure herein often comprises enabling multichannel - e.g. multicolor - imaging by electronic switching, rather than mechanical switching, between the light sources or lasers, cameras, and the Z direction motion or movement of the sample carrying stage of the underlying microscope 101, 202 and the coordinated timing thereof.

[0340] Electrical switching may have a faster timing than mechanical switching between light sources (wavelengths) and / or other system components for imaging, e.g. mechanical wheel filters for multiwavelength imaging.

[0341] Furthermore, electrical switching does not impact optics alignment, such that refocusing during image capture and image alignment subsequent to image capture is minimized or not required. Consequently, image capture and image assembly may both be completed substantially more quickly than in systems in the art

[0342] A possible timing for each component in an individual multi-channel image capture may be designed as given in the following:

[0343] - The light source or laser is switched ON and OFF with a time span below 1 ms and / or each light source channel or laser channel might need to be pulsed for as low as 25 ms. In alternate embodiments a laser is switched on or off in no more than 10ms, 5ms, 2ms, 1ms, 0.5 ms, 0.2 ms, 0.1ms, 0.05 ms, 0.02 ms, 0.01 ms or less. Similarly in some cases a light source channel may be pulsed for no more than 500 ms, 200 ms, 100 ms, 50 ms, 20 ms, 10 ms, 5 ms, 2 ms, 1 ms or less than 1 ms.

[0344] - The camera timing is preferably synchronized to the timing of the light sources or lasers. The exposure time can be equal or shorter than the pulse duration of the light source or laser, for example no more than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than 5%the pulse duration.

[0345] - The current data transfer from a camera to a memory may be 67 ms in full-frame, or in some cases no more than 500 ms, 200 ms, 100ms, 67 ms, 50 ms, 20 ms, 10 ms or less. Data transfer is in some cases effected concurrently with image capture, such as image capture for a camera other than the camera from which data transfer is effected. Consequently, in some cases image data transfer does not impact overall z - section capture time. A timeline for embodiments where data transfer overlaps data capture and thus does not impact overall z - section imaging time is shown in figure 14.

[0346] In addition to the advantages mentioned above, a faster tile capture resulting from decreased timing from each component is achieved, wherein a tile comprises a multiple number of (e.g. 32) images from different z-stack slices sj of the sample 5 at different heights zj.

[0347] In some cases a single z - section of a tile imaged using two lasers is captured in no more than 2,000 ms, 1,500 ms, 1,000 ms, 900 ms, 800 ms, 700 ms, 600 ms, 500 ms, 400 ms, 350 ms, 300 ms, 250 ms, 200 ms, 150 ms, 100 ms or less than 100 ms, In some cases a single z - section of a tile imaged using three lasers is captured in no more than 2,000 ms, 1,500 ms, 1,000 ms, 900 ms, 800 ms, 700 ms, 600 ms, 500 ms, 400 ms, 350 ms, 300 ms, 250 ms, 200 ms, 150 ms, 100 ms or less than 100 ms, Similarly, a tile of 30 z - sections is in some cases collected in no more than 15 seconds, 10 seconds, 9 seconds, 8 seconds, 7.5 seconds, 7 seconds, 6 seconds, 5 seconds, 4 s, 3 s or less than 3 seconds,

[0348] Thus the present disclosure is less time consuming, bears less repositioning errors, and improves the quality of alignment and thus compatibility of the captured images. These benefits arises from one or more of a number of aspects of the present disclosure, such as not employing mechanical manipulation of an optical column pursuant to or interrupting an individual z - section imaging, relying upon electrical manipulation of laser activity and camera activity, not using mechanical manipulation of optical column components such as filters or beamsplitters, transferring data concurrently or in parallel with camera data collection rather than in series, and reducing or eliminating the need to orient images of a particular z - section relative to one another.

[0349] Similarly, some embodiments of the present disclosure exhibit reduced photodamage in image collection. This arises from the sample material being subjected to less light / laser exposure since the light / laser is only on during the image capture. Accordingly, samples variously exhibit no more than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less of the photobleaching exhibited in samples conventionally imaged, such as imaged using an approach that comprises mechanical manipulation of an optics column constituent pursuant to, during, or interrupting a particular z - section capture event.

[0350] Besides the configurations, in which the sample carrier / slide 2 carries the specimen 5 on its upper surface and in which the specimen 5 is imaged from below the lower surface 2b of the sample slide / carrier 2 with the immersion liquid 104 therebetween, alternatively imaging from above the sample 5 is possible, too.

[0351] Figures 18 to 20 demonstrate such alternative optical arrangements with the objective lens 102 imaging the sample 5 from the top of the sample 5 with the immersion liquid 104 in between the lens 102 and a cover glass 6 of an underlying flow cell carrying and comprising the sample 5.

[0352] Both optical configurations (imaging from below or from above the sample 5) can be used with the present invention’s multi-channel capture principles and systems as described herein.

[0353] In all the embodiments the temporal requirements for data transfer and / or control operations shall preferably be taken into account In this regard the time slot D for "PC / Arduino Comm" may be used to consider the communication between the PC and the Z-stage and / or its controller. The time slot D is the PC communication with the Z-stage to enable the Z-stage to be in a stable position prior to image capture. In the sense of the present invention, a so called single tile is a 3D z-stack comprising 32 individual images of the sample at various z-positions zj focusing on respective slices sj or z-stack slices sj of the sample 5. A final 3D image may then be constructed from each of the tiles.

[0354] The above described and further aspects of the present invention inter alia regarding configuration, function, technical application and / or technical effect may also be based on the following numbered embodiments. 1. A method of capturing microscopic images of a sample 5, comprising: A focusing a microscope 101 on the sample 5 at a first position zj in a direction Z of a height of the sample 5 as a first z-stack slice sj, B exposing the sample 5 and at least the first z-stack slice sj to electromagnetic radiation and in particular to light of a first illumination and / or excitation wavelength XI, C collecting a first emission image from emission of electromagnetic radiation or light by the sample 5 and in particular by the first z-stack slice sj resulting from exposing the sample 5 and at least the first z- stack slice sj to the electromagnetic radiation or light of the first illumination and / or excitation wavelength XI, D exposing the sample 5 and at least the first z-stack slice sj to electromagnetic radiation and in particular to light of a second illumination and / or excitation wavelength X2, E collecting a second emission image from emission of electromagnetic radiation or light by the sample 5 and in particular by the first z-stack slice sj resulting from exposing the sample 5 and at least the first z-stack slice sj to the electromagnetic radiation or light of the second illumination and / or excitation wavelength X2, and F after collecting the first emission image and the second emission image, focusing the microscope 101 on the sample 5 at a second position zk in the direction Z of height of the sample 5 as a second z-stack slice sk removed in the direction Z of height from the first z-stack slice sj. 2. The method according to any previous embodiment, such as embodiment 1, wherein a multicolor z-stack microscopic image set of the sample 5 is captured. 3. The method according to any previous embodiment, wherein steps A to F are partially or completely repeated: - by starting with an initial position zO in the height direction Z of the sample 5 referring to an initial z-stack slice sO of the sample 5, - by ending with a final position zN in the height direction Z of the sample 5 referring to an final z-stack slice sN of the sample 5, and - by successively incrementing with an increment dz a position of a focus or a focal plane of an underlying illuminating and / or imaging device in the height direction Z of the sample 5 starting initial position zO and ending at the final position zN. 4. The method according to any previous embodiment, such as embodiment 3, the following relations I and II are fulfilled in the repeated process zj = zO + j • dz, with j = 0, ..., N, and I zN = zO + N • dz, II with zO denoting the initial position zO in the height direction Z of the sample 5 referring to an initial z-stack slice sO of the sample 5, with zN denoting the final position zN in the height direction Z of the sample 5 referring to an final z-stack slice sN, dz begin the increment in the height direction Z of the sample 5, and with j is an integer counting index ranging from 0 to N, N being a natural number larger than 0. 5. The method according to any previous embodiment, wherein collecting with respect to a the first z-stack slice sj of the sample 5 a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 are performed concurrently prior to focusing the microscope 101 on a second z-stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj. 6. The method according to any previous embodiment, wherein collecting with respect to a the first z-stack slice sj of the sample 5 a first emission image from emission resulting from the first illumination and / or excitation wavelength XI and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength X2 are performed sequentially prior to focusing the microscope 101 on a second z-stack slice sk removed in the a z dimension or direction Z of height from the first z-stack slice sj. 7. The method according to any previous embodiment, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope 101 on the first z-stack slice sj more or one than once. 8. The method according to any previous embodiment, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope 101 on the first z-stack slice sj again after collecting the first emission image and the second emission image. 9. The method according to any previous embodiment, wherein capturing the multicolor z-stack microscopic image set comprises at least one of: - collecting a brightfield image of the first z-stack slice sj, - collecting a transmission illuminated image of the first z-stack slice sj, - collecting a back illuminated image of the first z-stack slice sj, and - collecting an unilluminated image of the first z-stack slice sj. 10. The method according to any previous embodiment, wherein capturing the multicolor z-stack microscopic image set is accomplished in at least one of - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample 5, - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set of the sample 5, and - a time that is less than twice as long as it takes to collect a monocolor or monochromic z-stack microscopic image set on the microscope 101. 11. The method according to any previous embodiment, wherein capturing the multicolor z-stack microscopic image set is accomplished in at least one of a time that is less than 110% as long as it takes - to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample 5, - to collect a monocolor or monochromic z- stack microscopic image set of the sample 5, and - to collect a monocolor or monochromic z-stack microscopic image set on the microscope 101. 12. The method according to any previous embodiment, wherein the multicolor z-stack microscopic image set comprises or is based on at least two colors, three colors, four colors, five colors of electromagnetic radiation and in particular of light for exposing the sample 5 and at least a respective z-stack slice sj thereof, wherein a color is defined by a wavelength, a weighted discrete and / or continuous plurality of wavelengths, and / or a spectrum of wavelengths. 13. The method according to any previous embodiment, wherein a first emission image and a second emission image of a z-stack slice sj of the sample and in particular of a first z- stack slice sj of the sample 5 are at least one of: - aligned upon image capture, - not subjected to post capture image alignment relative to one another, and - not offset relative to one another upon image capture. 14. The method according to any previous embodiment, wherein the first illumination and / or excitation wavelength XI and the second illumination and / or excitation wavelength X2 pass through a common beam splitter 106 that is in particular fixed relative to the microscope 101. 15. The method according to any previous embodiment, wherein no mechanical image capture part of the microscope 101 moves or is moved relative to the sample 5 pursuant to capturing an image of the first z-stack slice sj. 16. The method according to any previous embodiment, wherein the first illumination and / or excitation wavelength XI and the second illumination and / or excitation wavelength X2 through a common beam splitter 106 that is attached to a turret that moves or is configures to move relatively to the microscope 101. 17. The method according to any previous embodiment, wherein each first image of a z-stack slice sj of the sample 5 is captured though a first camera 112, in particular with respect to a first color and / or wavelength XI. 18. The method according to any one of any previous embodiment, such as embodiments 1 to 16, wherein at least one first image of a z-stack slice sj of the sample 5 image is captured though a first camera 112, in particular with respect to a first color and / or wavelength XI, and at least another one first image of the z-stack slice sj of the sample 5 is captured though a second camera 116, in particular with respect to a second color and / or wavelength X2. 19. The method according to any previous embodiment, wherein the sample 5 is or comprises a fluorophore labeled nucleic acid and in particular at least one of an RNA molecule and a DNA molecule. 20. The method according to any previous embodiment, wherein the sample 5 is or comprises at least one of a fluorophore labeled protein, a flash frozen sample, a fresh frozen sample, an FFPE preserved sample, wherein in particular the FFPE preserved sample is subjected to at least one of a nucleic acid assay and a protein assay. 21. The method according to any previous embodiment, comprising collecting a bright field image of a distinct region of the sample 5 using a second microscope 201. 22. The method according to any previous embodiment, such as embodiment 21, wherein the bright field image of the distinct region of the sample 5 is collected concurrently. 23. The method according to any previous embodiment, such as embodiment 21 or 22, wherein the second microscope 201 is positioned at least one of: - adjacent to a microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5, - on an opposite sample side relative to the microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5, and - on an opposite slide side relative to the microscope 101 that captures the multicolor z-stack microscopic image set of the sample 5. 24. Control unit 300, 400 for controlling a sample and / or sample investigation system 1 and configured to initiate, control, and / or perform a method according to any previous embodiment, such as embodiments and the steps thereof. 25. Sample investigation system 1 and configured to initiate, control, perform and / or to be used a or in a method according to any one of any previous embodiments, such as embodiments 1 to 23 and the steps thereof. 26. System 1 according to any previous embodiment, such as embodiment 25, comprising: - at least one microscope 101, 201 having an objective 102, - at least one light source or an optical coupling to an external light source, - a primary specimen carrier 2 configured to receive, carry and or support a sample 5 or specimen 5, - at least one camera 112, 116, 120, 212, 216, and - a control unit 300, 400, wherein: - the control unit 300, 400 is formed according to any previous embodiment, such as embodiment 24, and - the control unit 300, 400 is configured to control at least one of the microscope 101, 201, the light source, the primary specimen carrier 2 and in particular a holder and / or stage therefor, and the camera with respect to their operation and cooperation. 27. A method of capturing a multicolor z-stack microscopic image set of a sample, comprising focusing a microscope on a first z-stack slice by performing a mechanical manipulation, exposing the first z- stack slice to a first excitation wavelength, collecting a first emission image from emission resulting from the first excitation wavelength, exposing the first z-stack slice to a second excitation wavelength, collecting a second emission image from emission resulting from the second excitation wavelength, without subjecting the microscope to mechanical manipulation pursuantto exposing the first z-stack slice to the second excitation wavelength or collecting the second emission image, and then after collecting the first emission image and the second emission image, focusing the microscope on a second z-stack slice removed in a ‘z’ dimension from the first z-stack slice by performing a mechanical manipulation on the microscope. 28. The method of any previous embodiment, such as embodiment 27, wherein collecting a first emission image from emission resulting from the first excitation wavelength and collecting a second emission image from emission resulting from the second excitation wavelength are performed concurrently prior to focusing the microscope on a second z- stack slice removed in a ‘z’ dimension from the first z-stack slice. 29. The method of any previous embodiment, such as embodiment 27, wherein collecting a first emission image from emission resulting from the first excitation wavelength and collecting a second emission image from emission resulting from the second excitation wavelength are performed sequentially prior to focusing the microscope on a second z-stack slice removed in a ‘z’ dimension from the first z-stack slice. 30. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z- stack microscopic image set is accomplished without focusing the microscope on the first z-stack slice one than once. 31. The method of any previous embodiment, such as embodiment27, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope on the first z-stack slice again after collecting the first emission image and the second emission image. 32. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a brightfield image of the firstz-stack slice. 33. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a transmission illuminated image of the first z-stack slice. 34. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a back illuminated image of the first z-stack slice. 35. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting an unilluminated image of the first z-stack slice. 36. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image set of a comparable sample. 37. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image set of the sample. 38. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image set on the microscope. 39. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z-stack microscopic image set of a comparable sample. 40. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z-stack microscopic image set of the sample. 41. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z-stack microscopic image set on the microscope. 42. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 1 second. 43. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 350 ms. 44. The method of any previous embodiment, such as embodiment 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 250 ms. 45. The method any previous embodiment, such as embodiments 27-42, wherein the multicolor z-stack microscopic image set comprises two colors. 46. The method any previous embodiment, such as embodiments 27- 42, wherein the multicolor z-stack microscopic image set comprises three colors. 47. The method any previous embodiment, such as embodiments 27- 42, wherein the multicolor z- stack microscopic image set comprises four colors. 48. The method any previous embodiment, such as embodiments 27- 42, wherein the multicolor z-stack microscopic image set comprises five colors. 49. The method of any previous embodiment, such as embodiment 27, wherein the first z-stack slice first emission image and the first z-stack slice second emission image are aligned upon image capture. 50. The method of any previous embodiment, such as embodiment 27, wherein the first z- stack slice first emission image and the first z-stack slice second emission image are not subjected to post capture image alignment relative to one another. 51. The method of any previous embodiment, such as embodiment 27, wherein the first z-stack slice first emission image and the first z-stack slice second emission image are not offset relative to one another upon image capture. 52. The method of any previous embodiment, such as embodiment 0, wherein the first excitation wavelength and the second excitation wavelength pass through a common beam splitter that is fixed relative to the microscope. 53. The method of any previous embodiment, such as embodiment 52, wherein no mechanical microscope image capture part moves relative to the sample pursuant to capturing an image of the first z-stack slice. 54. The method of any previous embodiment, such as embodiment 27, wherein the first excitation wavelength and the second excitation wavelength pass through a common beam splitter that is attached to a turret that moves relative to the microscope. 55. The method any previous embodiment, such as embodiments 27 - 54, wherein each first z-stack slice image is captured though a first camera. 56. The method any previous embodiment, such as embodiments 27 - 54, wherein at least one first z-stack slice image is captured though a first camera and at least one first z-stack slice image is captured though a second camera. 57. The method of any previous embodiment, such as embodiment 27, wherein the sample comprises a fluorophore labeled nucleic acid. 58. The method of any previous embodiment, such as embodiment 57, wherein the nucleic acid is an RNA molecule. 59. The method of any previous embodiment, such as embodiment 57, wherein the nucleic acid is a DNA molecule. 60. The method of any previous embodiment, such as embodiment 27, wherein the sample comprises a fluorophore labeled protein. 61. The method of any previous embodiment, such as embodiment 27, wherein the sample is a flash frozen sample. 62. The method of any previous embodiment, such as embodiment 27, wherein the sample is an FFPE preserved sample. 63. The method of any previous embodiment, such as embodiment 62, wherein the FFPE preserved sample is subjected to at least one nucleic acid assay and at least one protein assay. 64. The method any previous embodiment, such as embodiments 27 - 63, comprising collecting a bright field image of a distinct region of the sample using a second microscope. 65. The method of any previous embodiment, such as embodiment 64, wherein the bright field image of the distinct region of the sample is collected concurrently. 66. The method of any previous embodiment, such as embodiment 64, wherein the second microscope is positioned adjacent to the microscope that captures the multicolor z-stack microscopic image set 67. The method of any previous embodiment, such as embodiment 64, wherein the second microscope is positioned on an opposite sample side relative to the microscope that captures the multicolor z-stack microscopic image set 68. The method of any previous embodiment, such as embodiment 64, wherein the second microscope is positioned on an opposite slide side relative to the microscope that captures the multicolor z-stack microscopic image set. 69. The method of any previous embodiment, such as embodiment 27, comprising transmitting the first image concurrently with collecting the second emission image on a z - section of a sample. 70. A sample image assembled from at least 10 z - sections collected by a microscope optics column, wherein the sample image comprises data from at least a first excitation wavelength and a second excitation wavelength, wherein no z - section is subjected to more than 1 focusing event by the microscope optics column, and wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength does not comprise mechanical manipulation of the microscope optics column. 71. The sample image of any previous embodiment, such as embodiment 70, wherein the sample image comprises at least 30 z - sections. 72. The sample image of any previous embodiment, such as embodiment 70, wherein the microscope optics column comprises at least 2 lasers. 73. The sample image of any previous embodiment, such as embodiment 72, wherein access of the lasers to the sample is controlled using electrical manipulation. 74. The sample image of any previous embodiment, such as embodiment 70, wherein the microscope optics column comprises at least 2 cameras. 75. The sample image of any previous embodiment, such as embodiment 72, wherein access of the cameras to emission radiation from the sample is controlled using electrical manipulation. 76. The sample image of any previous embodiment, such as embodiment 70, wherein the data from at least a first excitation wavelength and a second excitation wavelength arising from a z - section are not subjected to post capture image orientation adjustment. 77. The sample image of any previous embodiment, such as embodiment 70, wherein the microscope optics column does not return to a z - section after imaging that z - section. 78. The sample image of any previous embodiment, such as embodiment 70, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength comprises electrical manipulation of the microscope optics column. 79. The sample image of any previous embodiment, such as embodiment 78, wherein the electrical manipulation of the microscope optics column comprises deactivation of a first laser generating the first excitation wavelength. 80. The sample image of any previous embodiment, such as embodiment 78, wherein the electrical manipulation of the microscope optics column comprises activation of a second laser generating the second excitation wavelength. 81. The sample image of any previous embodiment, such as embodiment 70, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength does not comprise mechanical manipulation of a first camera. 82. The sample image of any previous embodiment, such as embodiment 81, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength comprises electrical deactivation of a first camera. 83. The sample image of any previous embodiment, such as embodiment 82, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength comprises electrical activation of a second camera. 84. The sample image of any previous embodiment, such as embodiment 83, wherein electrical deactivation of the first camera and electrical activation of the second camera are coordinated. 85. The sample image of any previous embodiment, such as embodiment 83, wherein electrical deactivation of the first camera and electrical activation of the second camera occur no more than 2 ms from one another. 86. The sample image of any previous embodiment, such as embodiment 70, wherein the sample image is collected in a single pass through the z - sections of the sample. 87. The sample image of any previous embodiment, such as embodiment 70, wherein a z -section image is collected in no more than 500 ms. 88. The sample image of any previous embodiment, such as embodiment 70, wherein a z -section image is collected in no more than 350 ms. 89. The sample image of any previous embodiment, such as embodiment 70, wherein a z -section image is collected in no more than 250 ms. 90. The sample image of any previous embodiment, such as embodiment 70, wherein the sample image comprises data from a third excitation wavelength. 91. The sample image of any previous embodiment, such as embodiment 70, wherein the sample image comprises transmission light data. 92. A method of reducing z - section image collection time for a three dimensional sample image of a sample, comprising focusing on a first z - section, and capturing a first z - section image, wherein capturing the first z - section image comprises subjecting the sample to a first excitation wavelength from a first laser, collecting first emission wavelength light from the sample in a first camera, subjecting the sample to a second excitation wavelength from a second laser, and collecting second emission wavelength light from the sample in a second camera, wherein none of the first laser, second laser, first camera and second camera are manipulated mechanically independently during the capturing. 93. The method of any previous embodiment, such as embodiment 92, wherein the first laser, second laser, first camera and second camera are manipulated electronically by activation and deactivation rather than mechanically by independent repositioning. 94. The method of any previous embodiment, such as embodiment 92, wherein multiple images are captured at the first z-section prior to changing microscope focus from the first z-section to the second z-section, thereby generating a z-section dataset for the sample wherein the focusing the microscope on a z-section occurs no more time than the number of z-sections. 95. The method of any previous embodiment, such as embodiment 92, wherein the method does not comprise refocusing the microscope on the sample at the first z-section. 96. A microscopy optics column comprising a first excitation laser, a second excitation laser, a first camera, a second camera, an objective lens, and a beam splitter, wherein access of a first beam of the first excitation laser to a sample is gated electronically, wherein access of a second beam of the second excitation laser to the sample is gated electronically, wherein access of the first camera to a first emission spectrum is gated electronically, wherein access of the second camera to a second emission spectrum is gated electronically, wherein first camera data transmission is concurrent with second camera image collection activity. As used herein, the term "mechanical” refers to the physical adjustment of position of an object, often through activation of a motor, gears or other generation or conveyance of force. Mechanical manipulation of, for example a component of an optics column, changes the position of that component relative to the remainder of the optics column. Mechanical manipulation of the optics column as a whole, may occur pursuant to focusing or changing the position of the optics column relative to the sample or changing a property of any component of the optics column (e.g., changing a emission or excitation wavelength of a component of the optics column).

[0355] As used herein, the term "electrical” refers to the activation or inactivation of a component through use of an electrical signal alone rather than through or in combination with the physical adjustment of position of an object, such as occurs using a motor.

[0356] As used herein, the term "about” when used in the context of a scalar value refers to a range spanning 10% less than to 10% more than that that value, while in the context of a range the term refers to a larger range spanning from 10% below the stated lower limit to 10% above the greater limit

[0357] List of Reference Signs

[0358] 1 sample / specimen investigation arrangement / assembly / system

[0359] 2 (primary) specimen carrier, glass slide

[0360] 2a first and / or upper surface of (primary) specimen carrier or glass slide 2

[0361] 2b second and / or lower surface of (primary) specimen carrier or glass slide 2

[0362] 2r rim, rim portion of (primary) specimen carrier or glass slide 2

[0363] 5 (biological) sample, (biological) specimen, (biological) section

[0364] 6 (cover) glass slide

[0365] 6a first and / or upper surface of (cover) glass slide 6

[0366] 6b second and / or lower surface of (cover) glass slide 6

[0367] 6r rim, rim portion of (cover) glass slide 6

[0368] 7 frame, flow cell frame

[0369] 7c channel, flow cell channel

[0370] 7e (ITO, indium tin oxide) electrode

[0371] 7g gasket, flow cell gasket

[0372] 8 (investigation) medium / reagent / solution

[0373] 9 flow cell, fluid cell, investigation compartment

[0374] 10 sample / specimen holder and / or setting arrangement / assembly

[0375] 100 (immersion) microscope, microscope arrangement / assembly

[0376] 101 (first / lst) microscope

[0377] 102 (optical) objective

[0378] 103 (imaging) slide frame

[0379] 104 immersion liquid, immersion fluid, immersion water

[0380] 105 light source unit, light source(s), Laser unit, laser(s)

[0381] 106 1st(dichroic) beamsplitter

[0382] 108 2nd(dichroic) beamsplitter

[0383] 110 1st(single) color filter

[0384] 112 1st(CMOS) camera

[0385] 114 2nd(single) color filter

[0386] 116 2nd(CMOS) camera

[0387] 118 (triple) color filter

[0388] 120 (CMOS) camera

[0389] 122 (multiband) emission filter

[0390] 201 (second / 2nd) microscope

[0391] 202 (optical) objective 204 (air) space / gap

[0392] 206 3rd(dichroic) beamsplitter

[0393] 208 4th(dichroic) beamsplitter

[0394] 210 3rd(single) color filter

[0395] 212 3rd(CMOS) camera

[0396] 214 4th(single) color filter

[0397] 216 4th(CMOS) camera

[0398] 300 control system, control unit

[0399] 301 (general) computer, PC

[0400] 400 control system, control unit

[0401] 401 (general) computer, PC

[0402] C clock signal

[0403] D (control signal for) communication with superordinate control unit

[0404] DT (control signal) for data transfer dz increment in z position,

[0405] IJj control signal for imaging or capturing images by a camera, frate rate signal; j = 1, ..., 4

[0406] L, Lj control signal for illumination by a light source / laser

[0407] N (maximum) Number of (imaging) slices / z-positions (in z stack)

[0408] S control signal for stage movement, stage movement control signal sj (imaging) slice or z-stack slice with number j / at jthz-position (in z stack) - corresponding to spatial position zj, 1stz-stack slice sk (imaging) slice or z-stack slice with number k / at kthz-position (in z stack) - corresponding to spatial position zk, 2ndz-stack slice t time tj (specific) instance of time; j = 0, 1, 2, ... tj (specific) clock time; j = 0, 1, 2, ... x spatial direction, length direction

[0409] X direction of longitudinal extension y spatial direction, width direction

[0410] ¥ direction of transversal or width extension z spatial direction, height direction zj spatial position in z direction, sample / specimen slice / layer position zk spatial position in z direction, sample / specimen slice / layer position

[0411] Z direction of height or height extension

[0412] Xj (jth) reception and / or transmission and / or detection wavelength / color of number j; with j = 0, 1, 2, ... (jth) illumination and / or excitation wavelength / color of number j; with j = 0, 1, 2, ...

[0413] 58

Claims

Claims1. A method of capturing microscopic images of a sample (5), comprising:(A) focusing a microscope (101) on the sample (5) at a first position (zj ) in a direction (Z) of a height of the sample (5) as a first z-stack slice (sj),(B) exposing the sample (5) and at least the first z-stack slice (sj) to electromagnetic radiation and in particular to light of a first illumination and / or excitation wavelength (XI),(C) collecting a first emission image from emission of electromagnetic radiation or light by the sample (5) and in particular by the first z-stack slice (sj) resulting from exposing the sample (5) and at least the first z-stack slice (sj) to the electromagnetic radiation or light of the first illumination and / or excitation wavelength (XI),(D) exposing the sample (5) and at least the first z-stack slice (sj) to electromagnetic radiation and in particular to light of a second illumination and / or excitation wavelength (X2),(E) collecting a second emission image from emission of electromagnetic radiation or light by the sample (5) and in particular by the first z-stack slice (sj) resulting from exposing the sample (5) and at least the first z-stack slice (sj) to the electromagnetic radiation or light of the second illumination and / or excitation wavelength (X2), and(F) after collecting the first emission image and the second emission image, focusing the microscope (101) on the sample (5) at a second position (zk) in the direction (Z) of height of the sample (5) as a second z-stack slice (sk) removed in the direction (Z) of height from the first z-stack slice (sj).

2. The method according to claim 1, wherein a multicolor z-stack microscopic image set of the sample (5) is captured.

3. The method according to any one of the preceding claims, wherein steps (A) to (F) are partially or completely repeated:- by starting with an initial position (zO) in the height direction (Z) of the sample (5) referring to an initial z-stack slice (sO) of the sample (5),- by ending with a final position (zN) in the height direction (Z) of the sample (5) referring to a final z-stack slice (sN) of the sample (5), and- by successively incrementing with an increment (dz) a position of a focus or a focal plane of an underlying illuminating and / or imaging device in the height direction (Z) of the sample (5) starting initial position (zO) and ending at the final position (zN).

4. The method according to claim 3, the following relations (I) and (II) are fulfilled in the repeated process zj = zO + j • dz, with j = 0, N, and (I) zN = zO + N • dz, (II) with zO denoting the initial position (zO) in the height direction (Z) of the sample (5) referring to an initial z-stack slice (sO) of the sample (5), with zN denoting the final position (zN) in the height direction (Z) of the sample (5) referring to an final z-stack slice (sN), dz begin the increment in the height direction (Z) of the sample (5), and with j is an integer counting index ranging from 0 to N, N being a natural number larger than 0.

5. The method according to any one of the preceding claims, wherein collecting with respect to a the first z-stack slice (sj) of the sample (5) a first emission image from emission resulting from the first illumination and / or excitation wavelength (XI) and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength (X2) are performed concurrently prior to focusing the microscope (101) on a second z-stack slice (sk) removed in the a z dimension or direction (Z) of height from the first z-stack slice (sj).

6. The method according to any one of the preceding claims, wherein collecting with respect to a the first z-stack slice (sj) of the sample (5) a first emission image from emission resulting from the first illumination and / or excitation wavelength (XI) and collecting a second emission image from emission resulting from the second illumination and / or excitation wavelength (X2) are performed sequentially prior to focusing the microscope (101) on a second z-stack slice (sk) removed in the a z dimension or direction (Z) of height from the first z-stack slice (sj).

7. The method according to any one of the preceding claims, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope (101) on the first z-stack slice (sj) more or one than once.

8. The method according to any one of the preceding claims, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope (101) on the first z-stack slice (sj) again after collecting the first emission image and the second emissionimage.

9. The method according to any one of the preceding claims, wherein capturing the multicolor z-stack microscopic image set comprises at least one of:- collecting a brightfield image of the first z-stack slice (sj ),- collecting a transmission illuminated image of the first z-stack slice (sj),- collecting a back illuminated image of the first z-stack slice (sj), and- collecting an unilluminated image of the first z-stack slice (sj).

10. The method according to any one of the preceding claims, wherein capturing the multicolor z-stack microscopic image set is accomplished in at least one of- a time that is less than twice as long as it takes to collect a monocolor or monochromic z- stack microscopic image set of a comparable sample (5),- a time that is less than twice as long as it takes to collect a monocolor or monochromic z- stack microscopic image set of the sample (5), and- a time that is less than twice as long as it takes to collect a monocolor or monochromic z- stack microscopic image set on the microscope (101).

11. The method according to any one of the preceding claims, wherein capturing the multicolor z-stack microscopic image set is accomplished in at least one of a time that is less than 110% as long as it takes- to collect a monocolor or monochromic z-stack microscopic image set of a comparable sample (5),- to collect a monocolor or monochromic z-stack microscopic image set of the sample (5), and- to collect a monocolor or monochromic z-stack microscopic image set on the microscope (101).

12. The method according to any one of the preceding claims, wherein the multicolor z-stack microscopic image set comprises or is based on at least two colors, three colors, four colors, five colors of electromagnetic radiation and in particular of light for exposing the sample (5) and at least a respective z-stack slice (sj) thereof, wherein a color is defined by a wavelength, a weighted discrete and / or continuous plurality of wavelengths, and / or a spectrum of wavelengths.

13. The method according to any one of the preceding claims, wherein a first emission image anda second emission image of a z-stack slice (sj ) of the sample and in particular of a first z-stack slice (sj) of the sample (5) are at least one of:- aligned upon image capture,- not subjected to post capture image alignment relative to one another, and- not offset relative to one another upon image capture.

14. The method according to any one of the preceding claims, wherein light of a first illumination and / or excitation wavelength (XI) and light of a second illumination and / or excitation wavelength (X2) pass through a common beam splitter (106) that is in particular fixed relative to the microscope (101).

15. The method according to any one of the preceding claims, wherein no mechanical image capture part of the microscope (101) moves or is moved relative to the sample (5) pursuant to capturing an image of the first z-stack slice (sj).

16. The method according to any one of the preceding claims, wherein light of a first illumination and / or excitation wavelength (XI) and light of a second illumination and / or excitation wavelength (X2) pass through a common beam splitter (106) that is attached to a turret that moves or is configures to move relatively to the microscope (101).

17. The method according to any one of the preceding claims, wherein each first image of a z- stack slice (sj) of a sample (5) is captured though a first camera (112), in particular with respect to a first color and / or wavelength (XI).

18. The method according to any one of claims 1 to 16, wherein at least one first image of a z- stack slice (sj) of the sample (5) image is captured though a first camera (112), in particular with respect to a first color and / or wavelength (XI), and at least another one first image of the z-stack slice (sj) of the sample (5) is captured though a second camera (116), in particular with respect to a second color and / or wavelength (X2) .

19. The method according to any one of the preceding claims, wherein the sample (5) is or comprises a fluorophore labeled nucleic acid and in particular at least one of an RNA molecule and a DNA molecule.

20. The method according to any one of the preceding claims, wherein the sample (5) is or comprises at least one of a fluorophore labeled protein, a flash frozen sample, a fresh frozensample, an FFPE preserved sample, wherein in particular the FFPE preserved sample is subjected to at least one of a nucleic acid assay and a protein assay.

21. The method according to any one of the preceding claims, comprising collecting a bright field image of a distinct region of the sample (5) using a second microscope (201).

22. The method according to claim 21, wherein the bright field image of the distinct region of the sample (5) is collected concurrently.

23. The method according to claim 21 or 22, wherein the second microscope (201) is positioned at least one of:- adjacentto a microscope (101) that captures the multicolor z-stack microscopic image set of the sample (5),- on an opposite sample side relative to the microscope (101) that captures the multicolor z-stack microscopic image set of the sample (5), and- on an opposite slide side relative to the microscope (101) that captures the multicolor z- stack microscopic image set of the sample (5).

24. Control unit (300, 400) for controlling a sample and / or sample investigation system (1), which is configured to initiate, control, and / or perform a method according to any one of the preceding claims and the steps thereof.

25. Sample investigation system (1), which configured to initiate, control, perform and / or to be used a or in a method according to any one of claims 1 to 23 and the steps thereof.

26. System (1) according to claim 25, comprising:- at least one microscope (101, 201) having an objective (102),- at least one light source (105) or an optical coupling to an external light source (105),- a primary specimen carrier (2) configured to receive, carry and or support a sample (5) or specimen (5),- atleastone camera (112, 116, 120, 212, 216), and- a control unit (300, 400), wherein:- the control unit (300, 400) is formed according to claim 24, and- the control unit (300, 400) is configured to control at least one of the microscope (101,201), the light source (105), the primary specimen carrier (2) and in particular a holder and / or stage therefor, and the camera with respect to their operation and cooperation.

27. A method of capturing a multicolor z-stack microscopic image set of a sample, comprising focusing a microscope on a first z-stack slice by performing a mechanical manipulation, exposing the first z-stack slice to a first excitation wavelength, collecting a first emission image from emission resulting from the first excitation wavelength, exposing the first z-stack slice to a second excitation wavelength, collecting a second emission image from emission resulting from the second excitation wavelength, without subjecting the microscope to mechanical manipulation pursuant to exposing the first z-stack slice to the second excitation wavelength or collecting the second emission image, and then after collecting the first emission image and the second emission image, focusing the microscope on a second z-stack slice removed in a ‘z’ dimension from the first z-stack slice by performing a mechanical manipulation on the microscope.

28. The method of claim 27, wherein collecting a first emission image from emission resulting from the first excitation wavelength and collecting a second emission image from emission resulting from the second excitation wavelength are performed concurrently prior to focusing the microscope on a second z-stack slice removed in a ‘z’ dimension from the first z- stack slice.

29. The method of claim 27, wherein collecting a first emission image from emission resulting from the first excitation wavelength and collecting a second emission image from emission resulting from the second excitation wavelength are performed sequentially prior to focusing the microscope on a second z-stack slice removed in a ‘z’ dimension from the first z-stack slice.

30. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope on the first z-stack slice more than once.

31. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished without focusing the microscope on the first z-stack slice again after collecting the first emission image and the second emission image.

32. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a brightfield image of the first z-stack slice.

33. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a transmission illuminated image of the first z-stack slice.

34. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting a back illuminated image of the first z-stack slice.

35. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set comprises collecting an unilluminated image of the first z-stack slice.

36. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image set of a comparable sample.

37. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image set of the sample.

38. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than twice as long as it takes to collect a monocolor z-stack microscopic image seton the microscope.

39. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z- stack microscopic image set of a comparable sample.

40. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z- stack microscopic image set of the sample.

41. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in a time that is less than 110% as long as it takes to collect a monocolor z- stack microscopic image seton the microscope.

42. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 1 second.

43. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 350 ms.

44. The method of claim 27, wherein capturing the multicolor z-stack microscopic image set is accomplished in no more than 250 ms.

45. The method of any one of claims 27 to 42, wherein the multicolor z-stack microscopic image set comprises two colors.

46. The method of any one of claims 27 to 42, wherein the multicolor z-stack microscopic image set comprises three colors.

47. The method of any one of claims 27 to 42, wherein the multicolor z-stack microscopic image set comprises four colors.

48. The method of any one of claims 27 to 42, wherein the multicolor z-stack microscopic image set comprises five colors.

49. The method of claim 27, wherein the first z-stack slice first emission image and the first z- stack slice second emission image are aligned upon image capture.

50. The method of claim 27, wherein the first z-stack slice first emission image and the first z- stack slice second emission image are not subjected to post capture image alignment relative to one another.

51. The method of claim 27, wherein the first z-stack slice first emission image and the first z- stack slice second emission image are not offset relative to one another upon image capture.

52. The method of claim 0, wherein the first excitation wavelength and the second excitation wavelength pass through a common beam splitter that is fixed relative to the microscope.

53. The method of claim 52, wherein no mechanical microscope image capture part moves relative to the sample pursuant to capturing an image of the first z-stack slice.

54. The method of claim 27, wherein the first excitation wavelength and the second excitationwavelength pass through a common beam splitter that is attached to a turret that moves relative to the microscope.

55. The method of any one of claims 27 to 54, wherein each first z-stack slice image is captured though a first camera.

56. The method of any one of claims 27 to 54, wherein at least one first z-stack slice image is captured though a first camera and at least one first z-stack slice image is captured though a second camera.

57. The method of claim 27, wherein the sample comprises a fluorophore labeled nucleic acid.

58. The method of claim 57, wherein the nucleic acid is an RNA molecule.

59. The method of claim 57, wherein the nucleic acid is a DNA molecule.

60. The method of claim 27, wherein the sample comprises a fluorophore labeled protein.

61. The method of claim 27, wherein the sample is a flash frozen sample.

62. The method of claim 27, wherein the sample is an FFPE preserved sample.

63. The method of claim 62, wherein the FFPE preserved sample is subjected to at least one nucleic acid assay and at least one protein assay.

64. The method of any one of claims 27 to 63, comprising collecting a bright field image of a distinct region of the sample using a second microscope.

65. The method of claim 64, wherein the bright field image of the distinct region of the sample is collected concurrently.

66. The method of claim 64, wherein the second microscope is positioned adjacent to the microscope that captures the multicolor z-stack microscopic image set67. The method of claim 64, wherein the second microscope is positioned on an opposite sample side relative to the microscope that captures the multicolor z-stack microscopic image set68. The method of claim 64, wherein the second microscope is positioned on an opposite slide side relative to the microscope that captures the multicolor z-stack microscopic image set69. The method of claim 27, comprising transmitting the first image concurrently with collecting the second emission image on a z - section of a sample.

70. A sample image assembled from at least 10 z - sections collected by a microscope optics column, wherein the sample image comprises data from at least a first excitation wavelength and a second excitation wavelength, wherein no z - section is subjected to more than 1 focusing event by the microscope optics column, and wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength does not comprise mechanical manipulation of the microscope optics column.

71. The sample image of claim 70, wherein the sample image comprises at least 30 z - sections.

72. The sample image of claim 70, wherein the microscope optics column comprises at least 2 lasers.

73. The sample image of claim 72, wherein access of the lasers to the sample is controlled using electrical manipulation.

74. The sample image of claim 70, wherein the microscope optics column comprises at least 2 cameras.

75. The sample image of claim 72, wherein access of the cameras to emission radiation from the sample is controlled using electrical manipulation.

76. The sample image of claim 70, wherein the data from at least a first excitation wavelength and a second excitation wavelength arising from a z - section are not subjected to post capture image orientation adjustment.

77. The sample image of claim 70, wherein the microscope optics column does not return to a z - section after imaging that z - section.

78. The sample image of claim 70, wherein switching from collecting data generated by the firstexcitation wavelength to data generated by the second excitation wavelength comprises electrical manipulation of the microscope optics column.

79. The sample image of claim 78, wherein the electrical manipulation of the microscope optics column comprises deactivation of a first laser generating the first excitation wavelength.

80. The sample image of claim 78, wherein the electrical manipulation of the microscope optics column comprises activation of a second laser generating the second excitation wavelength.

81. The sample image of claim 70, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength does not comprise mechanical manipulation of a first camera.

82. The sample image of claim 81, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength comprises electrical deactivation of a first camera.

83. The sample image of claim 82, wherein switching from collecting data generated by the first excitation wavelength to data generated by the second excitation wavelength comprises electrical activation of a second camera.

84. The sample image of claim 83, wherein electrical deactivation of the first camera and electrical activation of the second camera are coordinated.

85. The sample image of claim 83, wherein electrical deactivation of the first camera and electrical activation of the second camera occur no more than 2 ms from one another.

86. The sample image of claim 70, wherein the sample image is collected in a single pass through the z - sections of the sample.

87. The sample image of claim 70, wherein a z -section image is collected in no more than 500 ms.

88. The sample image of claim 70, wherein a z -section image is collected in no more than 350 ms.

89. The sample image of claim 70, wherein a z -section image is collected in no more than 250 ms.

90. The sample image of claim 70, wherein the sample image comprises data from a third excitation wavelength.

91. The sample image of claim 70, wherein the sample image comprises transmission light data.

92. A method of reducing z - section image collection time for a three dimensional sample image of a sample, comprising focusing on a first z - section, and capturing a first z - section image, wherein capturing the first z - section image comprises subjecting the sample to a first excitation wavelength from a first laser, collecting first emission wavelength light from the sample in a first camera, subjecting the sample to a second excitation wavelength from a second laser, and collecting second emission wavelength light from the sample in a second camera, wherein none of the first laser, second laser, first camera and second camera are manipulated mechanically independently during the capturing.

93. The method of claim 92, wherein the first laser, second laser, first camera and second camera are manipulated electronically by activation and deactivation rather than mechanically by independent repositioning.

94. The method of claim 92, wherein multiple images are captured at the first z-section prior to changing microscope focus from the first z-section to the second z-section, thereby generating a z-section dataset for the sample wherein the focusing the microscope on a z- section occurs no more time than the number of z-sections.

95. The method of claim 92, wherein the method does not comprise refocusing the microscope on the sample at the first z-section.

96. A microscopy optics column comprising a first excitation laser, a second excitation laser, a first camera, a second camera, an objective lens, and a beam splitter, wherein access of a first beam of the first excitation laser to a sample is gated electronically, wherein access of a second beam of the second excitation laser to the sample is gated electronically, wherein access of the first camera to a first emission spectrum is gated electronically, wherein access of the second camera to a second emission spectrum is gated electronically, wherein first camera data transmission is concurrent with second camera image collection activity.

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