High resolution image guided micro-illumination system

Abstract

The present disclosure provides microscope-based systems and methods that can significantly improve image contrast and have better vertical / horizontal resolution. The present disclosure employs a confocal microscope to replace a conventional fluorescence microscope, such as adding a spinning disk or a confocal microscope scanning unit to the existing invention. The use of a spinning disk greatly improves the image acquisition speed (allowing imaging of fast dynamic processes and live samples), and greatly reduces light damage. The present disclosure provides some embodiments to demonstrate how to set up a spinning disk microscope system for image-guided illumination and light labeling.

Description

[0001] Priority Claim

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 615,097, filed December 27, 2023. The entire contents of the application, along with any external material discussed herein, are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to a system and method for illuminating patterns on a sample, and more particularly to a spinning disk microscope system and method for continuously illuminating different patterns in a large field of view at high speed with conjugate focal resolution. Background Technology

[0004] Spatial proteomics plays a crucial role in advancing medical research by providing a comprehensive understanding of the spatial distribution of proteins within tissues. This technology enables researchers to study the localization and interactions of proteins in their native environment, making significant contributions to the discovery of disease mechanisms and therapeutic targets. However, unlike transcriptomics, which uses polymerase chain reaction (PCR) to amplify signals and subsequently perform de novo transcriptomics such as RNAseq, proteomics currently lacks a technology equivalent to PCR.

[0005] To address the challenge of protein amplification, U.S. Patent No. 11,265,449 B2 discloses a microscope-based system designed for spatial proteomics, providing a foundation for studying the spatial organization of proteins. Through a unique integrated design of optics, photochemistry, image processing, and mechatronics, this system and method can handle high concentrations of proteins, lipids, nucleic acids, or biochemical species for modulation, transformation, separation, or identification within specific regions. It captures images of interest based on user-defined microscopic imaging features and has wide applications in cell or tissue sample experiments.

[0006] While significant advancements have been made in spatial proteomics tools, limitations remain in achieving the high resolution required for detailed protein localization studies. One key challenge is the inherent thickness of tissue samples, which scatters and absorbs light during imaging, thereby reducing spatial resolution and signal sharpness. Achieving resolution comparable to conjugate microscopy is crucial for effective spatial proteomics analysis, particularly in dense or heterogeneous tissues. Summary of the Invention

[0007] To significantly improve image contrast and achieve better vertical / horizontal resolution, this invention aims to replace traditional fluorescence microscopes with conjugate focal microscopes, for example, by adding a turntable or conjugate focal microscope scanning unit to existing inventions. Using a turntable greatly increases image acquisition speed (allowing imaging of fast-moving processes and live samples) and significantly reduces optical damage. This disclosure provides several embodiments demonstrating how to configure a turntable microscope system for image guidance illumination and light labeling.

[0008] To achieve the above objectives, the present invention provides an image-guided microscope illumination system, comprising a microscope, an imaging unit, an optical unit, and an illumination source. The microscope includes a stage configured to receive a biological sample. The imaging unit may include an imaging source, a rotating disk assembly, and a camera. The imaging source is configured to generate excitation light along an excitation optical path. The excitation light passes through the rotating disk assembly and illuminates the biological sample, inducing the biological sample to emit emitted light along an emission optical path, which is then imaged by the camera. The optical unit may include a beam expander and a pattern illumination device. The illumination source is optically coupled to the optical unit. The illumination source and the optical unit are configured to generate illumination light along the illumination optical path to illuminate the biological sample with light patterns corresponding to one or more regions of interest within the biological sample.

[0009] In a preferred embodiment, the microscope includes a first dichroic mirror that is reflective in a first wavelength range including excitation and emission light, and transmissive in a second wavelength range including illumination light. The imaging unit further includes a second dichroic mirror that is transmissive in a third wavelength range including excitation light and reflective in a fourth wavelength range including emission light. Furthermore, the second dichroic mirror is disposed within a rotating assembly. The imaging unit further includes a filter that is transmissive in the fourth wavelength range including emission light and blocks the second wavelength range associated with illumination light from reaching the camera. The rotating assembly is disposed between the first dichroic mirror and the filter along the emission light path. The imaging unit further includes a second lens barrel disposed between the filter and the camera.

[0010] In one embodiment, the imaging unit is independently coupled to the microscope. In another embodiment, the imaging unit is integrated into the optical unit.

[0011] In one embodiment, the system further includes at least one lens disposed along the optical path, wherein the lens is configured to adjust the focal length.

[0012] To achieve the above objectives, the present invention also provides another image-guided microscope illumination system. The system includes a microscope, an optical unit, and an illumination source. The microscope includes a stage configured to receive a biological sample. The optical unit includes a beam expander, a pattern illumination device, an imaging source, a turntable assembly, and a camera. The imaging source is configured to generate excitation light along an excitation optical path, which passes through the turntable assembly and illuminates the biological sample, thereby inducing the biological sample to emit emitted light along an emission optical path, which is then imaged by the camera. The illumination source is optically coupled to the optical unit and configured to generate illumination light along the illumination optical path to illuminate the biological sample with light patterns corresponding to one or more regions of interest within the biological sample.

[0013] In a preferred embodiment, the optical unit further includes a first dichroic mirror that is reflective in a first wavelength range including the excitation light and transmissive in a second wavelength range including the illumination light. Furthermore, the optical unit further includes a second dichroic mirror that is transmissive in a third wavelength range including the excitation light and reflective in a fourth wavelength range including the emitted light. Additionally, the optical unit includes a filter that is transmissive in the fourth wavelength range including the emitted light and blocks the second wavelength range associated with the illumination light from reaching the camera.

[0014] In one embodiment, the second dichroic mirror is further transmissive within a second wavelength range associated with the illumination light.

[0015] In one embodiment, the system further includes at least one lens disposed along the optical path, wherein the lens is configured to adjust the focal length.

[0016] To achieve the above objectives, the present invention also provides another image-guided microscope illumination system. The system includes a microscope, an imaging light source, a camera, and an illumination light source. The microscope includes a stage configured to receive a biological sample. The imaging light source is configured to generate excitation light along an excitation optical path, the excitation light passing through a rotating assembly to reach the biological sample, thereby inducing emission of light from the biological sample. The camera is configured to receive the emitted light from the biological sample along the emission optical path and form a plurality of images of the biological sample. The illumination light source is configured to generate illumination light along an illumination optical path passing through a beam expander and a pattern illumination device to illuminate the biological sample with light patterns corresponding to one or more regions of interest within the biological sample.

[0017] In a preferred embodiment, the system includes 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, and the first dichroic mirror is reflective of both the excitation light and the emission light. The second dichroic mirror is disposed in the excitation light path and the emission light path, and the second dichroic mirror is transmissive of the excitation light and reflective of the emission light. Furthermore, the first dichroic mirror is also disposed in the illumination light path and is transmissive of the illumination light.

[0018] In another preferred embodiment, the system includes a first dichroic mirror and a second dichroic mirror. The first dichroic mirror is disposed in the excitation light path and is reflective of the excitation light. Furthermore, the first dichroic mirror is also disposed in the illumination light path and is transmissive of the illumination light. The second dichroic mirror is disposed in both the excitation light path and the emission light path. The second dichroic mirror is transmissive of the excitation light and reflective of the emission light. Additionally, the second dichroic mirror is disposed in the illumination light path and is transmissive of the illumination light.

[0019] In another preferred embodiment, the system further includes a filter. The filter is disposed in the emitted light path, is transmissive to emitted light but absorbs incident light to block the incident light from reaching the camera.

[0020] In one embodiment, the system further includes at least one lens disposed along the optical path, wherein the lens is configured to adjust the focal length.

[0021] To use the system disclosed herein, biological samples, such as cell or tissue samples, can be prepared using photosensitizers or any suitable photochemical probes for photolabeling. The photosensitizer can be a photocatalyst bound to an antibody or a detectable tag (i.e., biotin). A microscopic image is captured in one field of view. The system may further include one or more processors to process the captured image to determine the location of the biological sample to be illuminated (e.g., photoactivation using a two-photon illumination source or treatment via a photochemical reaction). The processor then transmits the coordinates of the point of interest to a scanner for localized illumination. The microscope stage is then controlled to move repeatedly to the next field of view, repeating this image-guided light conversion process until sufficient sample has been processed or all selected fields of view have been completed. Obtaining higher resolution microscopic images is crucial for more precise selection of the desired target area. Therefore, the invention disclosed herein provides an image-guided illumination system that incorporates a turntable assembly, achieving improved resolution compared to the system disclosed in U.S. Patent No. 11,265,449. Attached Figure Description

[0022] The embodiments will be more fully understood through the following detailed description and accompanying drawings, which are for illustrative purposes only and therefore do not limit the invention, wherein:

[0023] [ Figure 1 [Illustration] is a schematic diagram of a conjugate focal microscope system according to an embodiment of the present invention.

[0024] [ Figure 2AThe spectrum of the transmission and reflection bands of a second dichroic mirror according to an embodiment of the present invention is illustrated, which is compatible with the excitation wavelength (EX) and emission wavelength (EM) of DAPI.

[0025] [ Figure 2B The spectrum of the transmission and reflection bands of a second dichroic mirror according to an embodiment of the present invention is illustrated, which is compatible with the excitation wavelength (EX) and emission wavelength (EM) of FITC.

[0026] [ Figure 2C The spectrum of the transmission and reflection bands of a second dichroic mirror according to an embodiment of the present invention is illustrated, which is compatible with the excitation wavelength (EX) and emission wavelength (EM) of Alexa 568.

[0027] [ Figure 2D The spectrum of the transmission and reflection bands of a second dichroic mirror according to an embodiment of the present invention is illustrated, which is compatible with the excitation wavelength (EX) and emission wavelength (EM) of Cy5.

[0028] [ Figure 3 [ ] is a schematic diagram of a conjugate focal microscope system according to another embodiment of the present invention.

[0029] [ Figure 4 [ ] is a schematic diagram of a conjugate focal microscope system according to another embodiment of the present invention.

[0030] [ Figure 5 [ ] is a schematic diagram of a conjugate focal microscope system according to another embodiment of the present invention. Detailed Implementation

[0031] US 11,265,449 B2 discloses a microscope-based system (referred to herein as the "Prior Invention") comprising: a microscope, an imaging assembly including a camera and an imaging light source, an illumination assembly including a patterned illumination device and an illumination light source, and at least one processing module 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 a biological sample; (2) automatically and in real-time process the image according to predefined criteria to determine coordinate information of a region of interest; and (3) control the illumination assembly to illuminate the first field of view with a light pattern corresponding to the region of interest of the biological sample, based on the coordinate information of the region of interest. After imaging, processing, and illuminating the first field of view, the processing module is configured to repeat this process for subsequent fields of view. The entire contents of the Prior Invention are incorporated herein by reference.

[0032] The rotating disk in conjugate 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 achieves optical sectioning by selectively focusing light from the sample onto a patterned array of pinholes on the disk. As the disk rotates, multiple points are scanned simultaneously, allowing for faster acquisition of confocal images compared to single-point scanning systems. Because rotating disk conjugate microscopy reduces photobleaching and phototoxicity while providing high temporal resolution, it is particularly useful for live-cell imaging. This technique is commonly used in biological and biomedical research to observe dynamic cellular processes in real time.

[0033] The conjugate focal microscopy systems disclosed in the embodiments herein can be used to process, for example, but not limited to, high concentrations of proteins, lipids, nucleic acids, or biochemical species. These systems include an imaging light source, a photosensitive light source, a patterning illumination device (e.g., a set of dual-axis high-speed galvanometer scanning mirrors), a microscope body, a turret for conjugate focal microscopy, a high-sensitivity camera, and a beam expander. To avoid speed reduction due to mechanical movement, the system further employs a carefully designed arrangement of multiple dichroic mirrors for multicolor imaging and femtosecond illumination without moving mechanical components such as turrets or shutters. Therefore, its purpose is to process proteins, lipids, nucleic acids, or biochemical species within regions of interest specified by fluorescent signals or structural features of cellular images. Another purpose is to collect large quantities of proteins, lipids, or nucleic acids through high-concentration photolabeling and purification, enabling the identification of biomolecules of interest within regions of interest using mass spectrometry or nucleic acid sequencers, followed by proteomics, metabolomics, or transcriptomics analysis.

[0034] Preferably, the system disclosed herein may include at least one lens disposed along the optical path, wherein the lens is configured to adjust the focal length. The optical path used in this disclosure includes the excitation optical path, the emission optical path, and the illumination optical path. In one embodiment, the at least one lens may be a barrel lens, and the barrel lens is configured to influence the overall magnification and optical path length of the optical system. In another embodiment, the at least one lens may be a scanning lens, and the scanning lens is configured to ensure that the laser beam remains focused on a plane during scanning to accommodate different scanning ranges or working distances. Those skilled in the art can add various lenses, such as barrel lenses or scanning lenses, along the optical path to flexibly adapt to different application requirements, such as image magnification, scanning accuracy, or varying working distances.

[0035] Some exemplary embodiments based on the content of this disclosure are described below.

[0036] To clarify, different optical paths and line types are shown in Figures 1 and 3 through 5 to represent the excitation optical path (dashed line), the emission optical path (thick solid line), and the illumination optical path (thin solid line). More specifically, the excitation light travels along the excitation optical path from the imaging source, passes through the turntable assembly, and enters the objective lens. The emission light travels along the emission optical path from the objective lens to the camera. The illumination light travels along the illumination optical path from the illumination source, passes through the beam expander, the pattern illumination device, and the lens barrel, and enters the objective lens.

[0037] This disclosure provides an embodiment of a conjugate focal microscope system with image-guided microscopy illumination. Please refer to Figure 1. The conjugate focal microscope system 100 includes a microscope 10, an imaging unit 20, an optical unit 30, and an illumination source 40. The microscope 10 includes a stage 11 and an objective lens 12. The stage 11 is configured to hold a sample S. The optical unit 30 and the imaging unit 20 are coupled to different locations (i.e., side ports or rear ports) of the microscope 10, respectively, and the illumination source 40 is coupled to the optical unit 30.

[0038] In this embodiment, the microscope may further include a first dichroic mirror 51, which has high reflectivity in a first wavelength range including excitation and emission light, and transmissivity in a second wavelength range of the illumination source. In an exemplary embodiment, the first dichroic mirror 51 is a single-edge dichroic mirror with an edge wavelength of 765 nm. Therefore, the first dichroic mirror can reflect various excitation and emission light within the first wavelength range (i.e., 400 nm to 750 nm) and transmit illumination light within the second wavelength range (i.e., 700 nm to 1100 nm). Furthermore, the microscope further includes a first tube lens 71, which is disposed along the illumination light path.

[0039] In this embodiment, the imaging unit 20 may include an imaging light source 21, a turntable assembly 22, and a camera 23. The imaging light source 21 provides excitation light of different wavelengths along the imaging optical path to induce the sample S to emit light. The imaging light source 21 may be a tungsten lamp, an arc lamp, a metal halide lamp, an LED light, or a laser. The camera 23 may be various types of optical sensors with high quantum efficiency, thereby enabling the shortest possible exposure time, such as a photomultiplier tube (PMT), an EMCCD camera, an sCMOS camera, or a photodiode array. In another embodiment, the imaging unit further includes a second lens barrel 72 positioned in front of the camera 23 along the imaging optical path.

[0040] Turntable 22 is located on the intermediate image plane and intersects the optical path. Turntable 22 is rotatable and includes an array of apertures, commonly referred to as pinholes. Therefore, the turntable can be called a rotating pinhole disk. Preferably, turntable 22 includes a second rotatable disk coupled to the rotating pinhole disk to rotate with it. The two turntables are typically fixed on a common axis and rotate synchronously with the axis. The second turntable includes an array of lenses or microlenses, which can be called a rotating lens disk or collecting disk. The two turntables together constitute a rotating turntable assembly, and the arrangement of the turntables aligns the lens array and the pinhole array with each other, so that light incident on the lenses is focused onto the pinholes. The lens disk can be omitted, although this would result in low light utilization efficiency and may adversely affect the signal-to-noise ratio of the resulting image.

[0041] The preferred turntable 22 includes a second dichroic mirror 52, which is transmissive in a third wavelength range including a plurality of excitation light bands and reflective in a fourth wavelength range including a plurality of emission light bands for imaging to a camera. For example, the second dichroic mirror 52 is a multi-band dichroic mirror with three or more bands of transmitted or reflected light. The user can select an ideal configuration based on the sample signal, such as for fluorescence imaging. Preferably, the second dichroic mirror 52 has high transmittance (i.e., transmits more than 80% or more than 90% of the incident light) for excitation light of different wavelength ranges projected from the imaging light source. Furthermore, the second dichroic mirror 52 has high reflectivity (i.e., reflects more than 80% or more than 90% of the incident light) for emission light from the sample in different wavelength ranges. In an exemplary embodiment, the second dichroic mirror 52 has a plurality of transmission and reflection bands in various wavelength ranges as listed in Table 1.

[0042] Table 1

[0043] Optical specifications numerical values Transmission band 1 Tavg > 90% 430 - 460 nm Transmission band 2 Tavg > 90% 500 - 550 nm Transmission band 3 Tavg >90% 580 - 620 nm Transmission band 4 Tavg >90% 660 - 900 nm Reflective band 1 Rabs >90% 370 - 410 nm Reflective band 2 Rabs >90% 470 - 490 nm Reflective band 3 Rabs >90% 559 - 570 nm Reflective band 4 Rabs >90% 630 - 650 nm

[0044] Based on the examples disclosed herein, fluorescent dyes that users can use include DAPI (or EBFP) (refer to the spectrum in Figure 2A), FITC (or Alexa 488, Atto 488, GFP) (refer to the spectrum in Figure 2B), Alexa 568 (or Alexa 532, ATTO 532, ATTO 550, ATTO 565, Cy3, Cy3B, TRITC, RFP, mCherry, Texas Red) (refer to the spectrum in Figure 2C), and Cy5 (or Alexa 647) (refer to the spectrum in Figure 2D). In other embodiments, users can select appropriate first and second dichroic mirrors according to the above principles based on the spectrum of the dye or fluorescent protein to be used.

[0045] In a preferred embodiment, the imaging unit further includes a filter 53, which is transmissive within a fourth wavelength range including the aforementioned emission bands and blocks illumination light from reaching the camera. In an exemplary embodiment, the filter 53 and the second dichroic mirror 52 share a similar plurality of transmissive wavelength bands. However, by changing the reflective wavelength band of the filter to an absorption band, the filter is allowed to filter out noise incompatible with the phosphor. Absorption brightness is typically expressed as an optical density (OD) value. Preferably, the OD value is at least greater than 4. In an exemplary embodiment, the filter 53 has a plurality of transmissive and absorption bands within each wavelength range as listed in Table 2.

[0046] Table 2

[0047] Optical specifications numerical values Transmission band 1 Tavg > 90% 430 - 460 nm Transmission band 2 Tavg > 90% 500 - 550 nm Transmission band 3 Tavg >90% 580 - 620 nm Transmission band 4 Tavg >90% 660 - 690 nm Absorption band 1 ODavg > 8, 370 – 410 nm Absorption band 2 ODavg > 8, 470 - 490 nm Absorption band 3 ODavg > 8, 559 - 570 nm Absorption band 4 ODavg > 8, 630 - 650 nm Absorption band 5 ODavg >4, 700 – 900 nm

[0048] In another exemplary embodiment, filter 53 may be a low-pass dichroic mirror to allow light with wavelengths below a certain cutoff wavelength to pass through, while reflecting or absorbing light with wavelengths above the cutoff wavelength. The cutoff wavelength can be determined based on the wavelength of the illumination light used. In this embodiment, the cutoff wavelength is at least 700 nm. Preferably, the cutoff wavelength is 700 nm, 750 nm, 800 nm, 850 nm, or 900 nm.

[0049] In this embodiment, the optical unit 30 may include a beam expander 31, a pattern illumination device 32, and at least one reflector 33. The reflector 33 is a mirror configured to properly align the illumination light with 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 beam diameter to modify its divergence angle or collimation. Advantageously, using a beam expander can optimize optical performance and improve system resolution. Optionally, the beam expander consists of two lenses, as shown in Figure 1. In a bilens beam expander, the magnification is equal to the focal length of the second lens divided by the focal length of the first lens. Preferably, the optical unit 30 further includes a scanning lens 6 disposed between the pattern illumination device 32 and the objective lens 12.

[0050] Optionally, the pattern illumination device 32 can be a galvanometer scanning mirror to project a specific light pattern onto the sample S, thereby achieving precise spatial control of the light distribution. Alternatively, a DMD or SLM can be used for pattern illumination.

[0051] In this embodiment, illumination source 40 provides illumination light to the sample S through an illumination optical path. Furthermore, illumination source 40 differs from imaging source 21 used for sample imaging. Here, the illumination source is used only to illuminate regions of interest within the sample. In a preferred embodiment, the sample S may be cultured with various photosensitizers and fluorescence-conjugated antibodies in a first step. After fluorescence imaging, the system may include one or more processors for performing real-time imaging processing to determine one or more regions of interest within the image (or a field of view, FOV, of the sample S) and generate a series of coordinates of points to be scanned by illumination sources and patterning illumination devices. Illumination causes the release of free radicals from the photosensitizers, leading to biotinylation of amino acids in the scanned regions. The cycle of acquiring microscopic images, image processing, and illuminating regions of interest is performed within each selected FOV of the sample. The biotinylated proteins or biomolecules are then purified and subjected to subsequent experiments, such as mass spectrometry analysis.

[0052] Preferably, the illumination source 40 can be a femtosecond laser, generating a two-photon effect with high axial illumination precision. The preferred wavelength range for the two-photon laser is between 700 nm and 1100 nm. This wavelength range is ideal for deep tissue imaging because it minimizes scattering and allows for better penetration into biological samples.

[0053] Optionally, in this embodiment, an additional lens barrel (not shown in Figure 1) can be simultaneously disposed between the turntable assembly and the first dichroic mirror along the excitation and emission light paths. The additional lens barrel can be disposed in the imaging unit or in the microscope.

[0054] The microscope may have multiple side ports and a rear port, and the imaging unit 20 and the optical unit 30 may be connected to the microscope 10 through different ports. This disclosure provides a conjugate focal microscope system 100' similar to the aforementioned system 100; however, in system 100', the ports to which the imaging unit and the optical unit are connected differ from those in the aforementioned system 100. Please refer to Figure 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 rear port of the microscope 10. Optionally, a third tube lens 73 may be additionally provided along the illumination path. For example, the third tube lens 73 may be disposed between the scanning lens 6 and the first dichroic mirror 51 in the microscope.

[0055] In this embodiment, the specifications of the first dichroic mirror 51 are exactly the opposite of those in system 100. The first dichroic mirror 51 has high transmittance in a first wavelength range including both excitation and emission light, and high reflectivity in a second wavelength range including both excitation and emission light. Therefore, the first dichroic mirror 51 can be a high-pass dichroic mirror, and the cutoff wavelength can be determined according to the wavelength of the illumination light used, for example, 700 nm, 750 nm, 800 nm, 850 nm, or 900 nm.

[0056] This disclosure provides a third embodiment, which is also a conjugate focal microscope system 200 configured with image-guided microscopic illumination. This system integrates the imaging unit 20 into the optical unit 30, as will be described in detail below. Since the composition, variations, or connections of the various detailed components of the conjugate focal microscope system 200 to other components can be referenced in the foregoing embodiments, reference numerals are retained in the figures and will not be repeated here.

[0057] As shown in Figure 4, the conjugate focal microscope system 200 configured with image-guided microscopy includes a microscope 10, an imaging unit 20, an optical unit 30, and a light source 40. The imaging unit 20 is integrated into the optical unit 30, the light source 40 is coupled to the optical unit 30, and the optical unit 30 is coupled to the microscope 10.

[0058] The microscope 10 includes a stage 11, an objective lens 12, and a first tube lens 71. The stage 11 is configured to hold a sample S. The stage 11 of the microscope 10 can be a high-precision microscope stage.

[0059] In this embodiment, the optical unit 30 includes an imaging unit 20, a beam expander 31, a pattern illumination device 32, at least one reflector 33, a first dichroic mirror 51', and a scanning lens 6. Throughout the figures, the same reference numerals are used to denote the same or equivalent components, and their descriptions will not be repeated unless necessary.

[0060] The first dichroic mirror 51' is located between the pattern illumination device 32 and the scanning lens 6. In this embodiment, the first dichroic mirror 51' has the same structure as the first dichroic mirror 51 (Figure 1) in the aforementioned embodiment.

[0061] In this embodiment, the imaging unit 20 may include an imaging light source 21, a turntable assembly 22, a second dichroic mirror 52', a filter 53', a second lens barrel 72, and a camera 23. The second dichroic mirror 52' ​​is disposed in the turntable assembly 22. In this embodiment, the second dichroic mirror 52' ​​has the same configuration as the second dichroic mirror 52 in the previous embodiment (Figure 1). Furthermore, the filter 53' is located between the second dichroic mirror 52' ​​and the second lens barrel 72. In this embodiment, the filter 53' has the same structure as the filter 53 in the previous embodiment (Figure 1).

[0062] Optionally, in this embodiment, an additional lens barrel (not shown in Figure 4) can be simultaneously disposed between the turntable assembly and the first dichroic mirror along the excitation and emission light paths. The additional lens barrel can be disposed in the imaging unit or in the optical unit.

[0063] This disclosure provides a fourth embodiment, which is another conjugate focal microscope system 300 configured with image-guided microscopic illumination. The main difference between the system described in the foregoing embodiments and that described in this embodiment is that the imaging optical path and the illumination optical path are integrated into an optical unit.

[0064] Therefore, as shown in Figure 5, the optical unit 30 of this embodiment includes a beam expander 31, a pattern illumination device 32, at least one reflector 33, a first dichroic mirror 51″, and a scanning lens 6. In this embodiment, the optical unit 30 further includes an imaging light source 21, a turntable assembly 22, a second dichroic mirror 52″, a filter 53″, a second lens barrel 72, and a camera 23. Since the composition, variations, or connection relationships of the various detailed components of the conjugate focal microscope system 300 with other components can be referred to in the foregoing embodiments, reference numerals are retained in the figures and will not be repeated here.

[0065] In this embodiment, the microscope 10 includes a stage 11, an objective lens 12, and a first tube lens 71. The stage 11 is configured to hold a sample S. The stage 11 of the microscope 10 can be a high-precision microscope stage.

[0066] In this embodiment, the first dichroic mirror 51″ is located between the pattern illumination device 32 and the scanning lens 6. In this embodiment, the first dichroic mirror 51″ has the same configuration as the first dichroic mirror 51 (Figure 1) in the previous embodiment.

[0067] In this embodiment, the second dichroic mirror 52″ is arranged along the common optical path of the excitation optical path, the emission optical path, and the illumination optical path. In addition, the second dichroic mirror 52″ has the same configuration as the second dichroic mirror 52 (Figure 1) in the aforementioned embodiment.

[0068] In this embodiment, filter 53″ is located between the second dichroic mirror 52″ and the second barrel lens 72. Furthermore, filter 53″ has the same configuration as filter 53 in the aforementioned embodiment (Figure 1).

[0069] This invention is not limited to the embodiments described herein, but can be modified or varied without departing from the scope of the invention. Those skilled in the art will understand that the invention is not limited to the specific descriptions and indications given above. Furthermore, unless otherwise stated above, it should be noted that all drawings are not to scale. Based on the foregoing teachings, various modifications and variations can be made without departing from the scope and spirit of the invention, which is limited only by the following claims.

Claims

1. An image-guided microscopic illumination system, comprising: A microscope includes a stage configured to receive a biological sample. An imaging unit includes an imaging light source, a turntable assembly, and a camera. The imaging light source is configured to generate an excitation light along an excitation light path. The excitation light passes through the turntable assembly and illuminates the biological sample to induce the biological sample to emit an emission light along an emission light path, which is then imaged by the camera. An optical unit, including a beam expander and a pattern illumination device; as well as An illumination source is optically coupled to the optical unit. The illumination source and the optical unit are configured to generate an illumination light along an illumination 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 of claim 1, wherein the microscope further comprises a first dichroic mirror, the first dichroic mirror being reflective in a first wavelength range including the excitation light and the emitted light, and transmissive in a second wavelength range including the illumination light.

3. The system of claim 1, wherein the imaging unit further includes a second dichroic mirror, the second dichroic mirror being transmissive in a third wavelength range including the excitation light and reflective in a fourth wavelength range including the emitted light.

4. The system as claimed in claim 3, wherein the second dichroic mirror is placed within the turntable assembly.

5. The system of claim 1, wherein the imaging unit further includes a filter that is transmissive in a fourth wavelength range including the emitted light and blocks a second wavelength range associated with the illumination light from reaching the camera.

6. The system of claim 5, wherein the turntable assembly is disposed between the first dichroic mirror and the filter along the emitted light path.

7. The system as claimed in claim 1, wherein the imaging unit is integrated into the optical unit.

8. The system of 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 of claim 1, wherein the microscope further comprises a first dichroic mirror, the first dichroic mirror being transmissive in a first wavelength range including the excitation light and the emitted light, and reflective in a second wavelength range including the illumination light.

10. An image-guided microscopic illumination system, comprising: A microscope includes a stage configured to receive a biological sample. An optical unit includes a beam expander, a pattern illumination device, an imaging light source, a turntable assembly, and a camera. The imaging light source is configured to generate an excitation light along an excitation light path. This excitation light passes through the turntable assembly and illuminates the biological sample, thereby inducing the biological sample to emit emitted light along an emission light path, which is then imaged by the camera. A light source, optically coupled to the optical system and configured to generate an illumination light along an illumination 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 of claim 10, wherein the optical unit further comprises a first dichroic mirror having reflectivity in a first wavelength range including the excitation light and transmissivity in a second wavelength range including the illumination light.

12. The system of claim 10, wherein the optical unit further comprises a second dichroic mirror, the second dichroic mirror being transmissive in a third wavelength range including the excitation light and reflective in a fourth wavelength range including the emitted light.

13. The system of claim 11, wherein the second dichroic mirror is further transmissive within the second wavelength range associated with the irradiated light.

14. The system of claim 10, wherein the optical unit further includes a filter that is transmissive in a fourth wavelength range including the emitted light and blocks the second wavelength range associated with the irradiated light from reaching the camera.

15. The system of 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, including a stage configured to receive a biological sample; An imaging light source is configured to generate an excitation light along an excitation optical path, the excitation light passing through a rotating disk assembly to reach the biological sample, thereby inducing an emission of light from the biological sample: A camera, configured to receive emitted light from the biological sample along an emitted light path and form a plurality of images of the biological sample: and A light source is configured to generate illumination light along a light path passing through a beam expander and a patterned 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 of claim 16 further includes a first dichroic mirror disposed in the excitation light path and the emission light path, the first dichroic mirror being reflective of the excitation light and the emission light.

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

19. The system of 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 of claim 19, wherein the first dichroic mirror is further disposed in the illumination path and reflects the illumination light.

21. The system of claims 17 to 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.

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

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

24. The system of 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 of claim 21, wherein the second dichroic mirror is further disposed in the illumination path and is transmissive to the illumination light.

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

27. The system as claimed in any of the preceding claims further includes a filter, wherein the filter is transmissive to the emitted light and blocks the illumination light from reaching the camera.

28. The system as described in any of the preceding claims, wherein the excitation light is transmitted from the imaging light source along the excitation light path, passes through the turntable assembly, and enters an objective lens.

29. The system as described in any of the preceding claims, wherein the emitted light is transmitted from the objective lens to the camera along the emitted light path.

30. The system as claimed in any of the preceding claims, wherein the illumination light is transmitted along the illumination path from the illumination source, through the beam expander, through the pattern illumination device, and into the objective lens.

31. The system as described in any of the preceding claims, wherein the pattern illumination device comprises a galvanometer scanning mirror, a digital micromirror device, or a spatial light modulator.

32. The system as described in any of the preceding claims, wherein the camera includes a CCD, CMOS, photomultiplier tube, or photodiode.

33. The system as described in any of the preceding claims, wherein the second wavelength range of the irradiating light is 700 nm to 1100 nm.

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