An image-guided microscopic illumination system with high resolution

Abstract

This disclosure provides microscope-based systems and methods that significantly improve image contrast and have better vertical / horizontal resolution. This disclosure uses confocal microscopy in replacement of conventional fluorescent microscopy, for example, adding a spinning disk or a confocal microscopy scanning unit on the prior invention. Using a spinning disk vastly improves the speed of image acquisition (allowing for imaging of fast dynamic processes and live specimens), and considerably reduces photo damage. Disclosed herein are some embodiments to demonstrate how to set up the spinning disk microscopy system for image-guided illumination and photolabeling.

Description

AN IMAGE-GUIDED MICROSCOPIC ILLUMINATION SYSTEM WITH HIGHRESOLUTIONCROSS REFERENCE OF RELATED APPLICATIONS

[0001] This non-provisional application claims priority to U.S. Provisional Patent Application No. 63 / 615,079, filed on December 27, 2023. This and all other extrinsic materials discussed herein are hereby incorporated by reference in their entirety.TECHNICAL FIELD

[0002] The present invention relates to a system and method for illuminating patterns on a sample, especially relating to a spinning disk microscopy system and method for illuminating varying patterns through a large number of fields of view consecutively at a high speed with confocal resolution.BACKGROUND

[0003] Spatial proteomics plays a pivotal role in advancing medical research by providing a comprehensive understanding of the spatial distribution of proteins within tissues. This technique enables researchers to study protein localization and interaction in their native environment, contributing significantly to the discovery of disease mechanisms and therapeutic targets. However, unlike transcriptomics where Polymerase chain reaction (PCR) is used to amplify the signals so that de novo transcriptomics like RNAseq are possible, no PCR-equivalent technology is yet available for proteomics.

[0004] To address protein amplification problems, U.S. Pat. No. 11,265,449 B2, has disclosed a microscope-based system designed for spatial proteomics, offering a foundation for studying protein spatial organization. With a unique integration of optical, photochemical, image processing, and mechatronic design, such systems and methods have abilities to process a high content of proteins, lipids, nucleic acids, or biochemical species for regulation,conversion, isolation, or identification in an area of interest based on user-defined microscopic image features, widely useful for cell or tissue sample experiments.

[0005] Despite the significant advancements in spatial proteomics tools, limitations remain in achieving the high resolution necessary for detailed protein localization studies. One critical challenge is the inherent thickness of tissue samples, which can scatter and absorb light during imaging, reducing spatial resolution and signal clarity. For effective spatial proteomics analysis, particularly in densely packed or heterogeneous tissues, achieving resolution comparable to confocal microscopy is essential.SUMMARY

[0006] To significantly improve the image contrast and have better vertical / horizontal resolution, the present invention intends to use confocal microscopy in replacement of conventional fluorescent microscopy, for example, adding a spinning disk or a confocal microscopy scanning unit on the prior invention. Using a spinning disk vastly improves the speed of image acquisition (allowing for imaging of fast dynamic processes and live specimens), and considerably reduces photo damage. Disclosed herein are some embodiments to demonstrate how to set up the spinning disk microscopy system for image- guided illumination and photolabeling.

[0007] To achieve the above objectives, the invention provides an image-guided microscopic illumination system, comprising a microscope, an imaging unit, an optical unit and an illuminating light source. The microscope comprises a stage, and the stage is configured to receive a biological sample. The imaging unit may comprise an imaging light source, a spinning disk assembly, and a camera, and the imaging light source is configured to generate an excitation light along an excitation light path through the spinning disk assembly onto the biological sample to induce an emission light by the biological along an emission light path that is imaged by the camera. The optical unit may comprise a beam expander anda pattern illumination device. The illuminating light source is optically coupled to the optical unit, the illuminating light source and the optical unit being configured to generate illumination light along an illumination light path to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

[0008] In a preferred embodiment, the microscope comprises a first dichroic mirror which is reflective in a first wavelength range includes the excitation light and the emission light and is transmissive in a second wavelength range that includes the illuminating light. The imaging unit further comprises a second dichroic mirror which is transmissive in a third wavelength range that includes the excitation light and is reflective in a fourth wavelength range that includes the emission light. Additionally, the second dichroic mirror is displaced within the spinning disk assembly. The imaging unit further comprises a filter which is transmissive in the fourth wavelength range that includes the emission light and blocks the second wavelength range associated with the illuminating light from reaching the camera. The spinning disk assembly is disposed between the first dichroic mirror and the filter along the emission light path. The imaging unit further comprises a second tube lens placed between the filter and the camera.

[0009] In one embodiment, the imaging unit is independently coupled to the microscope. In another embodiment, the imaging unit is incorporated into the optical unit.

[0010] In one embodiment, this system further comprises at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

[0011] To achieve the above objectives, the invention also provides another image-guided microscopic illumination system. The system comprises a microscope, an optical unit, and an illuminating light source. The microscope comprises a stage configured to receive a biological sample. The optical unit comprises a beam expander, a pattern illumination device, an imaging light source, a spinning disk assembly, and a camera. The imaging light source is configured to generate an excitation light along an excitation light path through the spinningdisk assembly onto the biological sample to induce light emission by the biological sample along an emission light path that is imaged by the camera. The illuminating light source is optically coupled to the optical system and configured to generate illumination light along an illumination light path to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

[0012] In a preferred embodiment, the optical unit further comprises a first dichroic mirror which is reflective in a first wavelength range includes the excitation light and is transmissive in a second wavelength range that includes the illuminating light. Additionally, the optical unit further comprises a second dichroic mirror which is transmissive in a third wavelength range that includes the excitation light and is reflective in a fourth wavelength range that includes the emission light. Also, the optical unit comprises a filter which is transmissive in the fourth wavelength range that includes the emission light and blocks the second wavelength range associated with the illuminating light from reaching the camera.

[0013] In one embodiment, the second dichroic mirror is further transmissive in the second wavelength range associated with the illuminating light.

[0014] In one embodiment, this system further comprising at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

[0015] To achieve the above objectives, the invention further provides still another image-guided microscopic illumination system. The system comprises a microscope, an imaging light source, a camera, and an illuminating light source. The microscope comprises a stage configured to receive a biological sample. The imaging light source is configured to generate an excitation light along an excitation light path that passes through a spinning disk assembly onto the biological sample to induce an emission light by the biological sample. The camera is configured to receive the emission light along an emission light path from the biological sample and form images of the biological sample. The illuminating light source is configured to generate illuminating light along an illumination light path that passes througha beam expander and a pattern illumination device to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

[0016] In a preferred embodiment, the system comprises a first dichroic mirror, and a second dichroic mirror. The first dichroic mirror is disposed in the excitation light path and the emission light path, the first dichroic mirror being reflective to the excitation light and the emission light. The second dichroic mirror is disposed in the excitation light path the emission light path, the second dichroic mirror being transmissive to the excitation light and reflective to the emission light. Additionally, the first dichroic mirror is also disposed in the illuminating light path and is transmissive to the illumination light.

[0017] In another preferred embodiment, the system comprises a first dichroic mirror, and a second dichroic mirror. The first dichroic mirror is disposed in the excitation light path, the first dichroic mirror being reflective to the excitation light. Additionally, the first dichroic mirror is also disposed in the illuminating light path and is transmissive to the illumination light. The second dichroic mirror disposed in the excitation light path and the emission light path, the second dichroic mirror being transmissive to the excitation light and reflective to the emission light. Also, the second dichroic mirror is disposed in the illuminating light path and is transmissive to the illumination light.

[0018] In another preferred embodiment, the system further comprises a filter. The filter is disposed in the emission light path and is transmissive the emission light but absorbs the illuminating light to block the illuminating light from reaching the camera.

[0019] In one embodiment, this system further comprising at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

[0020] To use the system of this disclosure, a biological sample, such as cell or tissue sample, can be prepared with photosensitizers or any photochemical probes suitable for performing photolabeling. The photosensitizers can be a photocatalyst conjugated with an antibody or a detectable tag (i.e. biotin). At one field of view, a microscopic image is taken.The system may further comprise one or more processors to process the captured image to determine where the biological sample would be illuminated (e.g. photo-activated or processed by photochemical reaction using a two-photon illumination light source). The processors then transfer the coordinated of points of interest to scanners for localized illumination. The stage of the microscope is then controlled to move to the next field of view again and again to repeat tis image-guided photoconversion process until enough samples or all the selected FOVs are processed. To more precisely select the desired target area, obtaining higher-resolution microscopic images is essential. Hence, the invention in this disclosure provides an imaged-guided illumination system incorporates a spinning disk assembly to achieve resolution improvement in comparison with those disclosed in U.S. Pat. No. 11,265,449 B2.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The embodiments will become more fully understood from the detailed description and accompanying drawings, which are given for illustration only, and thus are not limited to the present invention, and wherein:

[0022] FIG. 1 is a schematic diagram of a confocal microscopy system according to one embodiment of the present invention.

[0023] FIG.2A shows the spectrum of the transmission bands and reflective bands of the second dichroic mirror according to one embodiment of the present invention, which is compatible with the excitation wavelength (EX) and the emission wavelength (EM) of DAPI.

[0024] FIG.2B shows the spectrum of the transmission bands and reflective bands of the second dichroic mirror according to one embodiment of the present invention, which is compatible with the excitation wavelength (EX) and the emission wavelength (EM) of FITC.

[0025] FIG.2C shows the spectrum of the transmission bands and reflective bands of the second dichroic mirror according to one embodiment of the present invention, which iscompatible with the excitation wavelength (EX) and the emission wavelength (EM) of Alexa 568.

[0026] FIG.2D shows the spectrum of the transmission bands and reflective bands of the second dichroic mirror according to one embodiment of the present invention, which is compatible with the excitation wavelength (EX) and the emission wavelength (EM) of Cy5.

[0027] FIG. 3 is a schematic diagram of a confocal microscopy system according to another embodiment of the present invention.

[0028] FIG. 4 is a schematic diagram of a confocal microscopy system according to another embodiment of the present invention.

[0029] FIG. 5 is a schematic diagram of a confocal microscopy system according to still another embodiment of the present inventionDETAILED DESCRIPTION OF THE INVENTION

[0030] US Patent No. 11,265,449 B2, has disclosed a microscope-based system (referred to as “the prior invention” herein) comprising: a microscope, an imaging assembly comprising a camera and an imaging light source, an illuminating assembly comprising a pattern illumination device and an illuminating light source, and at least one processing module which is coupled to the microscope, the imaging assembly, and the illumination assembly. The processing module is configured to: (1) control the imaging assembly to acquire at least one image of a first field of view of the biological sample; (2) process the image automatically in real-time based on a predefined criterion, so as to determine coordinate information of an interested region; and (3) control the illuminating assembly to illuminate the first field of view with a light pattern corresponding to the interested region of the biological sample according to the coordinate information of the interested region. After the first of view has been imaged, processed, and illuminated, the processing module isconfigured to repeat the process for subsequent field of view. The prior invention is hereby incorporated by reference in their entirety.

[0031] A spinning disk in confocal microscopy refers to a specialized mechanism, typically a Nipkow disk or a disk with a series of microlenses, used to achieve rapid scanning and high-speed imaging. This method enables optical sectioning by selectively focusing light from the specimen through a patterned array of pinholes on the spinning disk. As the disk rotates, it scans multiple points simultaneously, allowing for faster acquisition of confocal images compared to single-point scanning systems. Spinning disk confocal microscopes are particularly useful for live-cell imaging, as they reduce photobleaching and phototoxicity while providing high temporal resolution. The technique is often used in biological and biomedical research to observe dynamic cellular processes in real-time.

[0032] A confocal microscopy systems provide by the various embodiments of this present disclosure may relate to for processing, for example but not limited to, a high content of proteins, lipids, nucleic acids, or biochemical species comprising an imaging light source, a photosensitizing light source, a pattern illumination device such as a set of dual-axis highspeed galvanometric scanning mirrors, a microscope body, a spinning disk to achieve confocal microscopy, a high-sensitivity camera, and a beam-expander device. To avoid any slowdown due to mechanical movement, the systems further incorporate a meticulously designed arrangement of multiple dichroic mirrors, which were used to allow multicolor imaging and femtosecond light illumination without movement of mechanical elements such as a turret or a shutter. It is therefore an object to process the proteins, lipids, nucleic acids, or biochemical species in an area of interest specified by fluorescent signals or structural signature of cell images. An additional object is to collect a large number of proteins, lipids, or nucleic acids through high content photolabeling and purification in order to identify biomolecules of interest in the area of interest by a mass spectrometer or a nucleic acid sequencer, followed by proteomic, metabolomic, or transcriptomic analyses.

[0033] Preferably, the systems of this disclosure may comprise at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length. The optical path used herein comprises the excitation light path, the emission light path, and the illuminating light path aforementioned. In one aspect, the at least one lens may be a tube lens, which is configured to affect the overall magnification and optical path length of the optical system. In another aspect, the at least one may be a scan lens, which is configured to ensure the laser beam remains focused on a flat plane during scanning, adapting to different scanning ranges or working distances. The skilled person in the art could add various lens, i.e. tube lens or scan lens, along the optical path with a flexibly adapt to different application needs, such as image magnification, scanning precision, or varying working distances.

[0034] Certain exemplary embodiments according to the present disclosure are described as below.

[0035] To clearly illustrate the arrangement of different optical paths, different line styles are used in FIGS. 1, 3-5 to represent the excitation light path (dashed lines), emission light path (bold solid lines), and illuminating light path (thin solid lines). More specifically, the excitation light travels along the excitation light path from the imaging light source, through the spinning disk assembly, into the objective. The emission light travels along the emission light path from the objective into the camera. The illumination light travels along the illumination light path from the illuminating light source, through the beam expander, through the pattern illumination device, through the tube lens, into the objective.

[0036] This disclosure provides an embodiment which is a confocal microscopy system for image-guided microscopic illumination. Please refer to FIG. 1. The confocal microscopy system 100 comprises a microscope 10, an imaging unit 20, an optical unit 30, and an illuminating light source 40. The microscope 10 comprises a stage 11 and an objective 12. The stage 11 is configured to be loaded with a sample S. The optical unit 30 and the imagingunit 20 are separately coupled to the different sites (i.e. side ports, or back port) of the microscope 10, and the illuminating light source 40 is coupled to the optical unit 30.

[0037] In this embodiment, the microscope may further comprise a first dichroic mirror 51 , which is highly reflective in a first wavelength range that includes the excitation light and the emission light and is transmissive in a second wavelength range of the illuminating light source. In one exemplary embodiment, the first dichroic mirror 51 is a single-edge dichroic beamsplitter, having an edge wavelength at 765 nm. Therefore, the first dichroic mirror could reflect various excitation light and emission light at the first wavelength range between 400 nm to 750 nm, and transmit illuminating light, i.e. at the second wavelength range between 700 nm to 1100 nm. Also, the microscope comprises a first tube lens 71, which is disposed along the illuminating light path.

[0038] In this embodiment, the imaging unit 20 may comprise an imaging light source 21, a spinning disk assembly 22 and a camera 23. The imaging light source 21 provides an excitation light at various wavelengths along with an imaging light path to induce light emission by the sample S. The imaging light source 21 can be a tungsten-halogen lamp, an arc lamp, a metal halide lamp, a LED light or a laser. The camera 23 can be various types of optical sensor with high quantum efficiency, so that as short exposure time is possible, such as photomultiplier (PMT), an EMCCD camera, a sCMOS camera, or photodiode array. In another embodiment, the imaging unit further comprises a second tube lens 72 placed along the imaging light path before the camera 23.

[0039] A spinning disk 22 is located in the intermediate image plane, intersecting the optical path. The scanning disk 22 can is rotatable and comprises an array of apertures, commonly referred to as pinholes. As such, the scanning disk may be referred to as a spinning pinhole disk. Preferably, the scanning disk 22 comprises a second rotatable disk coupled to the spinning pinhole disk for rotation therewith. Two disks are typically fixed to a common axle and rotate in unison with the shaft. The second disk comprises an array of lenses, ormicrolenses, and may be referred to as a spinning lens disk or collector disk. The two disks together form a spinning disk assembly, and the arrangement of the disks is such that the lens array and pinhole array are aligned with each other so that light incident on the lenses is focused on the pinholes. The lens disk may be omitted although this results in an inefficient use of the light and may adversely affect the signal to noise ratio of resulting images.

[0040] The preferred spinning disk 22 includes a second dichroic mirror 52, which is transmissive in a third wavelength range that includes multiple excitation light bands and is reflective in a fourth wavelength range that includes multiple emission light bands to the camera for imaging. For example, the second dichroic mirror 52 is a multiband dichroic having three or more bands in which light is transmitted or reflected. The user may choose ideal configurations according to the signal from the sample, e g. fluorescence imaging. Preferably, the second dichroic mirror 52 is highly transmissive (i.e., transmitting over 80%, or over 90% of incident light) of the excitation light projected from the imaging light source at various wavelength ranges. Also, the second dichroic mirror 52 is highly reflective (i.e., reflecting over 80%, or over 90% of incident light) of the emission light from the sample at various wavelength ranges. In one exemplary embodiment, the second dichroic mirror 52 has multiple transmission bands and reflection bands at various wavelength range as listed in Table 1.

[0041] Table 1

[0042] Based on this exemplary specification, the fluorescent dyes that users can utilize include DAPI (or EBFP) (see the spectrum as FIG. 2A) ' FITC (or Alexa 488, Atto 488, GFP) (see the spectrum as FIG. 2B) ' Alexa 568 (or Alexa 532, ATTO 532, ATTO 550, ATTO 565, Cy3, Cy3B, TRITC, RFP, mCherry, Texas Red )(see the spectrum as FIG. 2C) and Cy5 (or Alexa 647)(see the spectrum as FIG. 2D). In other embodiments, users may select an appropriate first dichroic mirror and second dichroic mirror according to the aforementioned principles, based on the spectrum of the dyes or fluorescent proteins intended to use.

[0043] In a preferred embodiment, the imaging unit further comprises a filter 53, which is transmissive in the fourth wavelength range that includes various emission light bands aforementioned and blocks the illuminating light from reaching the camera. In one exemplary embodiment, the filter 53 and the second dichroic mirror 52 share similar multiple transmission wavelength bands. However, the reflection wavelength band of the filter is altered to an absorbance band, allowing it to filter out noise that is not compatible with the fluorophore. Absorbance is typically expressed in terms of optical density (OD) values. Preferably, the OD value is at least larger than 4. In one exemplary embodiment, the filter 53 has multiple transmission bands and absorbance bands at various wavelength range as listed in Table 2.

[0044] Table 2ValueTransmission Band 1 Tavg > 90% 430 - 460 nm

[0045] In another exemplary embodiment, the filter 53 may be a lowpass dichroic mirror, allowing light with wavelengths below a certain cutoff to pass through, while reflecting or absorbing light with wavelengths above that cutoff wavelength. The cutoff wavelength can be determined according to the wavelength of the illuminating light used. In this embodiment, the cutoff wavelength is at least 700 nm. Preferably, the cutoff wavelength is 700nm, 750nm, 800nm, 850 nm or 900 nm.

[0046] In this embodiment, the optical unit 30 may comprise a beam expander 31, a pattern illumination device 32, and at least one reflective mirror 33. The reflective mirror 33 is a mirror configured to cause the illuminating light to be correctly aligned to the optical axis of the pattern illumination device 32. In this embodiment, the beam expander 31 is typically an optical device designed to adjust the diameter of a beam to modify its divergence angle or collimation. Advantageously, the beam expander is used to optimize optical performance and enhance system resolution. Optionally, the beam expander consists of two lenses as shown in FIG. 1. In a two-lens beam expander, the magnification is equal to the focal length of the second lens divided by that of the first lens. Preferably, the optical unit 30 further comprises a scan lens 6 provided between the pattern illumination device 32 and the objective 12.

[0047] Optionally, the pattern illumination device 32 may be galvanometric scanning mirrors to project specific light patterns onto the sample S, enabling precise spatial control of light distribution. Alternatively, DMD or SLM may be used for pattern illumination.

[0048] In this embodiment, the illuminating light source 40 provides an illuminating light through an illuminating light path to illuminate the sample S. In addition, the illuminating light source 40 is different from the imaging light source 21 for sample imaging. The illuminating light source here is only used to illuminate the regions of interest in the sample. In one preferred embodiment, the sample S may be incubated with a plurality of photosensitizers and fluorescent conjugated antibodies in the first step. After fluorescent imaging, the system may comprise one or more processors to perform real-time imaging processing to determine one or more regions of the interest in the image (or a field of view (FOV) of the sample S) and to generate a series of point coordinates to be scanned by the illuminating light source and the pattern illumination device. This illumination leads to free radical release of a photosensitizer, and thus results in biotinylation of amino acids in the scanned region. The cycle of acquiring microscopic images, imaging process, and illuminating the regions of interest is implemented in each selected FOV of the sample. The biotinylated proteins or biomolecules are then purified and performed subsequent experiments, such as mass spectrometry.

[0049] Preferably, the illuminating light source 40 can be a femtosecond laser may be used as the illumination light source to generate a two-photon effect for high axial illumination precision. The preferred wavelength range of the two-photon laser is from 700 nm to 1100 nm. This wavelength range is ideal for deep tissue imaging as it minimizes scattering and allows better penetration into biological samples.

[0050] Optionally, in this embodiment, additional tube lens may be disposed concurrently along the excitation light path and the emission light path between the spinning disk assemblyand the first dichroic mirror (not shown in FIG. 1). The additional tube lens can be disposed either in the imaging unit or in the microscope.

[0051] The microscope may have several side ports and one back port, and the imaging unit 20 and optical unit 30 may be connected to the microscope 10 with different ports. This disclosure provides a similar confocal microscopy system 100’ with previous system 100. However, in the system 100', the imaging unit and the optical unit are connected to ports different from those of the aforementioned system 100. Please refer to FIG. 3. In this embodiment, the imaging unit 20 is connected to the left port of the microscope 10 and the optical unit 30 is connected to the back port of the microscope 10. Optionally, a third tube lens 73 may be added along the illuminating light path. For example, the third tube lens 73 can be disposed in the microscope between the scan lens 6 and the first dichroic mirror 51.

[0052] In this embodiment, the specification of the first dichroic mirror 51 is exactly the opposite of that in system 100, which is highly transmissive in a first wavelength range that includes the excitation light and the emission light and is highly reflective in a second wavelength range of the illuminating light source. Therefore, the first dichroic mirror 51 may be a highpass dichroic mirror, and the cutoff wavelength can be determined according to the wavelength of the illuminating light used, such as 700 nm, 750 nm, 800 nm, 850 nm, or 900 nm.

[0053] This disclosure provides a third embodiment which is also a confocal microscopy system 200 for image-guided microscopic illumination. This system intergrades the imaging unit 20 into the optical unit 30 and will describe in detail. Because composition, variation or connection relationship to other elements of each detail elements of the confocal microscopy system 200 can refer to the previous embodiments, they have the save reference numbers in the drawings and are not repeated here.

[0054] As shown in FIG 4, the confocal microscopy system 200 for image-guided microscopic illumination comprises a microscope 10, an imaging unit 20, an optical unit 30,and an illuminating light source 40, wherein the imaging unit 20 is integrated into the optical unit 30, the illuminating light source 40 is coupled to the optical unit 30, and the optical unit 30 is coupled to the microscope 10.

[0055] The microscope 10 comprises a stage 11, an objective 12, and a first tube lens 71. The stage 11 is configured to be loaded with a sample S. The stage 11 of the microscope 10 can be a high-precision microscope stage.

[0056] In this embodiment, the optical unit 30 comprises an imaging unit 20, a beam expander 31, a pattern illumination device 32, at least one reflective mirror 33, a first dichroic mirror 51’ and a scan lens 6. Throughout the drawings, the same reference numerals are used to denote identical or equivalent elements, and their descriptions will not be repeated unless necessary.

[0057] The first dichroic mirror 51’ is placed between the pattern illumination device 32 and the scan lens 6, and here, the first dichroic mirror 51’ is provided with the same configuration as that of the first dichroic mirror 51 (FIG. 1) in previous embodiment.

[0058] In this embodiment, the imaging unit 20 may comprise an imaging light source21, a spinning disk assembly 22, a second dichroic mirror 52’, a filter 53’, a second tube lens 72, and a camera 23. The second dichroic mirror 52’ is placed in the spinning disk assembly22, and here, the second dichroic mirror 52’ is provided with the same configuration as that of the second dichroic mirror 52 (FIG. 1) in previous embodiment. Also, the filter 53’ is placed between the second dichroic mirror 52’ and the second tube lens 72, and here, the filter 53’ is provided with the same configuration as that of the filter 53 (FIG. 1) in previous embodiment.

[0059] Optionally, in this embodiment, additional tube lens may be disposed concurrently along the excitation light path and the emission light path between the spinning disk assembly and the first dichroic mirror (not shown in FIG. 4). The additional tube lens can be disposed either in the imaging unit or in the optical unit.

[0060] This disclosure provides a fourth embodiment which is a still another confocal microscopy system 300 for image-guided microscopic illumination. The major difference between the systems described in the previous embodiments and here is that the imaging light path and illuminating light path are integrated into the optical unit.

[0061] Hence, as shown in FIG. 5, the optical unit 30 here comprises a beam expander 31, a pattern illumination device 32, at least one reflective mirror 33, a first dichroic mirror 51”, and a scan lens 6. In this embodiment, the optical unit 30 further comprises an imaging light source 21, a spinning disk assembly 22, a second dichroic mirror 52”, a filter 53”, a second tube lens 72, and a camera 23. Because composition, variation or connection relationship to other elements of each detail elements of the confocal microscopy system 300 can refer to the previous embodiments, they have the save reference numbers in the drawings and are not repeated here.

[0062] In this embodiment, the microscope 10 comprises a stage 11, an objective 12, and a first tube lens 71. The stage 11 is configured to be loaded with a sample S. The stage 11 of the microscope 10 can be a high-precision microscope stage.

[0063] In this embodiment, the first dichroic mirror 51” is placed between the pattern illumination device 32 and the scan lens 6, and here, the first dichroic mirror 51” is provided with the same configuration as that of the first dichroic mirror 51 (FIG. 1) in previous embodiments.

[0064] In this embodiment, the second dichroic mirror 52” is placed along the common optical path of the excitation light path, emission light path, and illuminating light path. Also, the second dichroic mirror 52” is provided with the same configuration as that of the second dichroic mirror 52 (FIG. 1) in previous embodiments.

[0065] In this embodiment, the filter 53” is placed between the second dichroic mirror 52” and the second tube lens 72. Also, the filter 53” is provided with the same configuration as that of the filter 53” (FIG. 1) in previous embodiments.

[0066] The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present invention. It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described herein above. In addition, unless mention was made above to the contrary, it should be noted that all of the accompanying drawings are not to scale. A variety of modifications and variations are possible in light of the above teachings without departing from the scope and spirit of the invention, which is limited only by the following claims.

Claims

CLAIMSWhat is claimed is:

1. An image-guided microscopic illumination system, comprising: a microscope comprising a stage configured to receive a biological sample; an imaging unit, comprising an imaging light source, a spinning disk assembly, and a camera, the imaging light source being configured to generate an excitation light along an excitation light path through the spinning disk assembly onto the biological sample to induce an emission light by the biological along an emission light path that is imaged by the camera; an optical unit, comprising a beam expander and a pattern illumination device; and an illuminating light source, optically coupled to the optical unit, the illuminating light source and the optical unit being configured to generate illumination light along an illumination light path to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

2. The system according to claim 1, wherein the microscope further comprises a first dichroic mirror which is reflective in a first wavelength range includes the excitation light and the emission light and is transmissive in a second wavelength range that includes the illuminating light.

3. The system according to claim 1, wherein the imaging unit further comprises a second dichroic mirror which is transmissive in a third wavelength range that includes the excitation light and is reflective in a fourth wavelength range that includes the emission light.

4. The system according to claim 3, wherein the second dichroic mirror is displaced within the spinning disk assembly.

5. The system according to claim 1, wherein the imaging unit further comprises a filter which is transmissive in the fourth wavelength range that includes the emission light andblocks the second wavelength range associated with the illuminating light from reaching the camera.

6. The system according to claim 5, wherein the spinning disk assembly is disposed between the first dichroic mirror and the filter along the emission light path.

7. The system according to claim 1, wherein the imaging unit is incorporated into the optical unit.

8. The system according to claim 1, further comprising at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

9. The system according to claim 1, wherein the microscope further comprises a first dichroic mirror which is transmissive in a first wavelength range includes the excitation light and the emission light and is reflective in a second wavelength range that includes the illuminating light.

10. An image-guided microscopic illumination system, comprising: a microscope comprising a stage configured to receive a biological sample; an optical unit comprising a beam expander, a pattern illumination device, an imaging light source, a spinning disk assembly, and a camera, wherein the imaging light source is configured to generate an excitation light along an excitation light path through the spinning disk assembly onto the biological sample to induce light emission by the biological sample along an emission light path that is imaged by the camera; and an illuminating light source optically coupled to the optical system and configured to generate illumination light along an illumination light path to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

11. The system according to claim 10, wherein the optical unit further comprises a first dichroic mirror which is reflective in a first wavelength range includes the excitation light and is transmissive in a second wavelength range that includes the illuminating light.

12. The system according to claim 10, wherein the optical unit further comprises a second dichroic mirror which is transmissive in a third wavelength range that includes the excitation light and is reflective in a fourth wavelength range that includes the emission light.

13. The system according to claim 11, wherein the second dichroic mirror is further transmissive in the second wavelength range associated with the illuminating light.

14. The system according to claim 10, wherein the optical unit further comprises a filter which is transmissive in the fourth wavelength range that includes the emission light and blocks the second wavelength range associated with the illuminating light from reaching the camera.

15. The system according to claim 10, further comprising at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

16. An image-guided microscopic illumination system, comprising: a microscope comprising a stage configured to receive a biological sample; an imaging light source configured to generate an excitation light along an excitation light path that passes through a spinning disk assembly onto the biological sample to induce an emission light by the biological sample; a camera configured to receive the emission light along an emission light path from the biological sample and form images of the biological sample; and an illuminating light source configured to generate illuminating light along an illumination light path that passes through a beam expander and a pattern illumination device to illuminate the biological sample with a light pattern corresponding to one or more regions of interest in the biological sample.

17. The system according to claim 16, further comprising a first dichroic mirror disposed in the excitation light path and the emission light path, the first dichroic mirror being reflective to the excitation light and the emission light.

18. The system according to claim 17, wherein the first dichroic mirror is also disposed in the illuminating light path and is transmissive to the illumination light.

19. The system according to claim 16, further comprising a first dichroic mirror disposed in the excitation light path and the emission light path, the first dichroic mirror being transmissive to the excitation light and the emission light.

20. The system according to claim 19, wherein the first dichroic mirror is also disposed in the illuminating light path and is reflective to the illumination light.

21. The system according to claims 17 to 19, further comprising a second dichroic mirror disposed in the excitation light path the emission light path, the second dichroic mirror being transmissive to the excitation light and reflective to the emission light.

22. The system according to claim 16, further comprising a first dichroic mirror disposed in the excitation light path, the first dichroic mirror being reflective to the excitation light.

23. The system according to claim 19, wherein the first dichroic mirror is also disposed in the illuminating light path and is transmissive to the illumination light.

24. The system according to claim 19, further comprising a second dichroic mirror disposed in the excitation light path and the emission light path, the second dichroic mirror being transmissive to the excitation light and reflective to the emission light.

25. The system according to claim 21, wherein the second dichroic mirror is also disposed in the illuminating light path and is transmissive to the illumination light.

26. The system according to claim 16, further comprising at least one lens disposed along an optical path, wherein the lens is configured to adjust the focal length.

27. The system according to any one of the preceding claims, further comprising a filter, wherein the filter is transmissive to the emission light and blocks the illuminating light from reaching the camera.

28. The system according to any one of the preceding claims, wherein the excitation light travels along the excitation light path from the imaging light source, through the spinning disk assembly, into the objective.

29. The system according to any one of the preceding claims, wherein the emission light travels along the emission light path from the objective into the camera.

30. The system according to any one of the preceding claims, wherein the illumination light travels along the illumination light path from the illuminating light source, through the beam expander, through the pattern illumination device, into the objective.

31. The system according to any one of the preceding claims, wherein the pattern illumination device comprises a galvanometer scanning mirrors, a digital micromirror device, or a spatial light modulator.

32. The system according to any one of the preceding claims, wherein the camera comprises CCD, CMOS, photomultiplier tube, or photodiode.

33. The system according to any one of the preceding claims, wherein the second wavelength range that includes the illuminating light is from 700 nm to 1100 nm.

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