A fluorescent sheet microscopy imaging device and method

Abstract

The application discloses a fluorescence light sheet microscopic imaging device and method, and belongs to the technical field of microscopic imaging. The device is composed of a low-coherence light source, a fiber coupler, a first collimator, a second collimator, a first moving platform, a second moving platform, a light sheet, a lens, a plane array camera and a computer. The method comprises a light sheet generation process and a light sheet imaging process. The light sheet imaging process is repeated to realize light sheet imaging of each section of a sample by moving different target imaging planes of the sample to an equal light path plane. The application realizes light sheet fluorescence imaging based on short-coherence light source interference. The thickness of the light sheet is reduced to the coherence length by taking advantage of the short coherence length of the short-coherence light, the longitudinal resolution is greatly improved, a small aperture is not needed, the excitation light intensity, bleaching and phototoxicity can be reduced, and in-vivo cell imaging can be realized without affecting cell activity. The plane array camera is used to collect the fluorescence signal of the focal plane of the sample, and the application has the characteristic of fast fluorescence signal collection speed.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microscopic imaging technology, and particularly relates to a fluorescent light sheet microscopic imaging device and method. BACKGROUND

[0002] The fluorescent microscopic imaging technology is a technology of irradiating excitation light onto a sample to make fluorescent substances in the sample generate fluorescent signals after being excited, collecting the fluorescent signals by using a camera, and imaging. The fluorescent microscopic imaging technology utilizes the characteristic that the intensity of the fluorescent signals is linearly related to the content of fluorescent substances in the sample within a certain range. The fluorescent microscopic imaging technology is widely applied in the fields of biomedicine and the like due to the characteristics of simple imaging process, low system cost, high time resolution, and extremely high sensitivity.

[0003] However, the traditional fluorescent microscopic imaging technology adopts a wide-field illumination mode, and the wide-field illumination mode does not have depth resolution capability, which leads to the inability to obtain three-dimensional information of the sample. In order to realize three-dimensional imaging of the sample, the sample needs to be fixed and sliced in advance, which is not only complicated to operate, but also cannot be used for live cell imaging.

[0004] In order to solve the defects of the traditional fluorescent microscopic imaging technology, the light sheet fluorescent microscopic technology is proposed, which adopts a unique light sheet illumination mode. The excitation light in the light sheet fluorescent microscopic technology is a thin light sheet parallel to the imaging plane of the sample. The light sheet is used to focus and illuminate the sample. Only the target plane and the sample near the target plane are excited to be imaged. Therefore, the light sheet illumination mode in the light sheet fluorescent microscopic imaging technology effectively improves the longitudinal resolution of the imaging system.

[0005] However, the longitudinal resolution of the light sheet fluorescent microscopic imaging technology is low due to the inability to eliminate the interference of the fluorescent signals near the target plane. In order to improve the longitudinal resolution, a pinhole is currently used to limit the out-of-focus position of the fluorescent light. However, the use of the pinhole will weaken the fluorescent signals entering the detector. In order to improve the intensity of the fluorescent signals, the intensity of the excitation light needs to be increased, which will increase the phototoxicity and affect the cell activity.

[0006] A Chinese patent application with publication number CN114878614A discloses an X-ray light sheet confocal three-dimensional fluorescent microscopic imaging system and method. The scheme places a micron-level X-ray slit on the side of the sample to shape the X-ray, so as to make the fluorescent signal layer excited in the sample thinner. Although the spatial resolution in the depth direction is improved, the improvement of the longitudinal resolution is very limited due to the diffraction effect of the slit. SUMMARY

[0007] To address the problems existing in the prior art, this invention provides a fluorescence light sheet microscopy imaging device and method. It achieves light sheet fluorescence imaging based on short-coherence light interference. Utilizing the short coherence length of short-coherence light, the light sheet thickness is reduced to the coherence length, significantly improving longitudinal resolution. Furthermore, it eliminates the need for a pinhole, reducing excitation light intensity, bleaching, and phototoxicity, enabling live cell imaging without affecting cell viability. This invention employs an area array camera to acquire fluorescence signals from the sample's focal plane, featuring rapid fluorescence signal acquisition.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a fluorescence light sheet microscopy imaging device, comprising a low-coherence light source, an optical fiber coupler, a first collimator, a second collimator, a first moving platform, a second moving platform, a beam splitter, a filter, a lens, an area array camera, and a computer; the low-coherence light source is optically connected to the optical fiber coupler via an optical fiber, and the excitation light output from the optical fiber coupler is split into two paths, namely a first excitation light and a second excitation light; the first excitation light output end of the optical fiber coupler is optically connected to the first collimator, and the second excitation light output end of the optical fiber coupler is optically connected to the second collimator. The first excitation light collimated by the first collimator and the second excitation light collimated by the second collimator form parallel light; the first collimator is mounted on the first moving platform; the second moving platform is used to place the sample; the first collimator directs the collimated first excitation light toward the sample; the second collimator directs the collimated second excitation light toward the sample through a beam splitter; the fluorescence excited by the sample is directed toward the lens through the beam splitter and filter, and then focused by the lens onto the area array camera; the area array camera is electrically connected to a computer, which acquires fluorescence images, filters out the excitation light through the filter, and processes the fluorescence images by the computer.

[0009] A fluorescence sheet microscopy imaging method, employing the aforementioned fluorescence sheet microscopy imaging device, includes the following steps:

[0010] Step 1: Light Sheet Generation Process

[0011] A low-coherence light source is activated, emitting excitation light. The excitation light enters the fiber coupler through an optical fiber, from which a first excitation light and a second excitation light are emitted. The first excitation light is collimated by a first collimator and then directed toward the sample, while the second excitation light is collimated by a second collimator and then directed toward the sample. At this point, the first and second excitation lights form parallel light beams. The first and second excitation lights will interfere at the equipathic surface inside the sample, causing the sample to fluoresce. The fluorescence emitted by the sample is then directed toward the area array camera via a beam splitter, a filter, and a lens.

[0012] Assuming the linewidth of low-coherence light is Δλ and the wavelength is λ, then L C =λ 2 / Δλ, where LC The coherence length is L; therefore, the first excitation light and the second excitation light are only coherent at a length L near the equioptic path plane 12. C Interference occurs within the range;

[0013] At coherence length L C Within the range, the expression for the interference intensity of the first excitation light and the second excitation light is:

[0014]

[0015] In the formula, I(x,y,z;t) is the interference intensity of the first excitation light and the second excitation light, I1(x,y,z;t) is the light intensity of the first excitation light, and I2(x,y,z;t) is the light intensity of the second excitation light. The phase difference between the first excitation light and the second excitation light;

[0016] At coherence length L C Within the specified range, the expression for the fluorescence intensity excited by the sample is:

[0017]

[0018] In the formula, F1(x,y,z;t) is the coherence length L. C The fluorescence intensity is within the range, where A is the fluorescence excitation efficiency, I1(x,y,z;t) is the intensity of the first excitation light, and I2(x,y,z;t) is the intensity of the second excitation light. The phase difference between the first excitation light and the second excitation light;

[0019] At coherence length L C Outside the range, the first excitation light and the second excitation light do not interfere with each other, and the expression for the fluorescence intensity excited by the sample is:

[0020] F2(x,y,z;t)=A[I1(x,y,z;t)+I2(x,y,z;t)]

[0021] In the formula, F2(x,y,z;t) is the coherence length L C The fluorescence intensity outside the range, A is the fluorescence excitation efficiency, I1(x,y,z;t) is the intensity of the first excitation light, and I2(x,y,z;t) is the intensity of the second excitation light;

[0022] Step 2: Light sheet imaging process

[0023] The second moving platform is activated to move the target imaging plane of the sample to the equioptic path surface. Then the first moving platform is activated to cause the first collimator to produce continuous small random shifts. Fluorescence images are continuously acquired by the area array camera and processed by the computer.

[0024] Assume that the number of fluorescence images acquired by the area array camera is N, and the light intensity of the fluorescence image is denoted as P(x,y;i), where i represents the light intensity of the fluorescence image. i The fluorescence images are acquired at various times, and each fluorescence image is equal to the superposition of the fluorescence intensity produced by the entire sample. Its expression is:

[0025] P(x,y;i)=∫(F1(x,y,z;t)+F2(x,y,z;t))dz

[0026] In the formula, P(x,y;i) ​​represents t i The light intensity of the fluorescence image at time t, F1(x,y,z;t) is the coherence length L C The fluorescence intensity within the range, F2(x,y,z;t) is the coherence length L C Fluorescent intensity outside the range;

[0027] Because the first collimator experiences continuous, minute, random shifts due to the first moving platform during fluorescence image acquisition, the phase difference between the first and second excitation beams is... The value is different at different times, that is, when t i ≠t j hour, F1(x,y,z;t) i )≠F1(x,y,z;t j ), while F2(x,y,z;t i ) = F2(x,y,z;t j Therefore, we have the following expression:

[0028] P(x,y;i)-P(x,y;i+1)≈F1(x,y,z;t i )-F1(x,y,z;t i+1 )

[0029] In the formula, P(x,y;i) ​​represents t i The fluorescence image intensity at time t, P(x,y;i+1) is the fluorescence intensity at time t. i+1 Fluorescence image intensity at time t, F1(x,y,z;t) i ) for t i Time coherence length L C Fluorescence intensity within the range, F1(x,y,z;t) i+1 ) for t i+1 Time coherence length L C Fluorescence intensity within the range;

[0030] Let the position of the equioptic path surface be z′, then the coherence length L C The expression for the fluorescence image of the sample tomography within the range is:

[0031]

[0032] In the formula, T(x,y;z′) is the coherence length L C The light intensity of the fluorescence image of the sample tomography within the range, P(x,y;i) is t i The fluorescence image intensity at time t, P(x,y;i+1) is the fluorescence intensity at time t. i+1 Fluorescence image intensity at any given time;

[0033] The position of the sample is adjusted by the second moving platform, and other target imaging planes of the sample are moved to the equioptic plane. The light sheet imaging process is repeated to finally achieve light sheet imaging of each section of the sample.

[0034] The beneficial effects of this invention are:

[0035] The fluorescence sheet microscopy imaging device and method of the present invention achieves fluorescence imaging of the sheet based on short coherence light interference. By utilizing the short coherence length of short coherence light, the thickness of the sheet is reduced to the coherence length, which greatly improves the longitudinal resolution. Furthermore, it eliminates the need for a pinhole, thereby reducing excitation light intensity, bleaching, and phototoxicity, and enabling live cell imaging without affecting cell viability. The present invention uses an area array camera to acquire fluorescence signals from the focal plane of the sample, which has the advantage of fast fluorescence signal acquisition speed. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structure of a fluorescent sheet microscopy imaging device according to the present invention;

[0037] Figure 2 Coherence length L C Diagram showing the relative position to the equioptic surface;

[0038] In the figure, 1—low coherence light source, 2—fiber optic coupler, 3—first collimator, 4—second collimator, 5—first moving platform, 6—second moving platform, 7—beam splitter, 8—lens, 9—area array camera, 10—computer, 11—sample, 12—equal optical path surface, 13—filter. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0040] like Figure 1As shown, a fluorescence light sheet microscopy imaging device includes a low-coherence light source 1, an optical fiber coupler 2, a first collimator 3, a second collimator 4, a first moving platform 5, a second moving platform 6, a beam splitter 7, a filter 13, a lens 8, an area array camera 9, and a computer 10. The low-coherence light source 1 is optically connected to the optical fiber coupler 2 via an optical fiber. The excitation light output from the optical fiber coupler 2 is split into two paths, namely a first excitation light and a second excitation light. The first excitation light output end of the optical fiber coupler 2 is optically connected to the first collimator 3, and the second excitation light output end of the optical fiber coupler 2 is optically connected to the second collimator 4. The first excitation light output end of the optical fiber coupler 2 is collimated by the first collimator 3. An excitation beam and a second excitation beam collimated by a second collimator 4 form parallel light; the first collimator 3 is mounted on a first moving platform 5; the second moving platform 6 is used to place the sample 11; the first collimator 3 directs the collimated first excitation beam toward the sample 11; the second collimator 4 directs the collimated second excitation beam toward the sample 11 through a beam splitter 7; the fluorescence excited by the sample 11 is directed toward a lens 8 through the beam splitter 7 and a filter 13, and then focused by the lens 8 onto a field array camera 9; the field array camera 9 is electrically connected to a computer 10, the field array camera 9 acquires fluorescence images, the filter 13 filters out the excitation beam, and the computer 9 processes the fluorescence images.

[0041] A fluorescence sheet microscopy imaging method, employing the aforementioned fluorescence sheet microscopy imaging device, includes the following steps:

[0042] Step 1: Light Sheet Generation Process

[0043] The low-coherence light source 1 is activated, and excitation light is emitted from it. The excitation light enters the fiber coupler 2 through the optical fiber, and the first excitation light and the second excitation light are emitted from the fiber coupler 2 respectively. The first excitation light is collimated by the first collimator 3 and then directed towards the sample 11. The second excitation light is collimated by the second collimator 4 and then directed towards the sample 11. At this time, the first excitation light and the second excitation light form parallel light. The first excitation light and the second excitation light will interfere at the equipathic surface 12 inside the sample 11, and cause the sample 11 to emit fluorescence. The fluorescence emitted by the sample 11 is directed towards the area array camera 9 through the beam splitter 7, the filter 13 and the lens 8 in sequence.

[0044] Assuming the linewidth of low-coherence light is Δλ and the wavelength is λ, then L C =λ 2 / Δλ, where L C The coherence length is L; therefore, the first excitation light and the second excitation light are only coherent at a length L near the equioptic path plane 12. C Interference occurs within the range, such as Figure 2 As shown;

[0045] At coherence length L C Within the range, the expression for the interference intensity of the first excitation light and the second excitation light is:

[0046]

[0047] In the formula, I(x,y,z;t) is the interference intensity of the first excitation light and the second excitation light, I1(x,y,z;t) is the light intensity of the first excitation light, and I2(x,y,z;t) is the light intensity of the second excitation light. The phase difference between the first excitation light and the second excitation light;

[0048] At coherence length L C Within the specified range, the expression for the fluorescence intensity excited by sample 11 is:

[0049]

[0050] In the formula, F1(x,y,z;t) is the coherence length L. C The fluorescence intensity is within the range, where A is the fluorescence excitation efficiency, I1(x,y,z;t) is the intensity of the first excitation light, and I2(x,y,z;t) is the intensity of the second excitation light. The phase difference between the first excitation light and the second excitation light;

[0051] At coherence length L C Outside the range, the first excitation light and the second excitation light do not interfere with each other, and the expression for the fluorescence intensity excited by sample 11 is:

[0052] F2(x,y,z;t)=A[I1(x,y,z;t)+I2(x,y,z;t)]

[0053] In the formula, F2(x,y,z;t) is the coherence length L C The fluorescence intensity outside the range, A is the fluorescence excitation efficiency, I1(x,y,z;t) is the intensity of the first excitation light, and I2(x,y,z;t) is the intensity of the second excitation light;

[0054] Step 2: Light sheet imaging process

[0055] The second moving platform 6 is activated to move the target imaging plane of sample 11 to the equi-optical path surface 12. Then the first moving platform 5 is activated to cause the first collimator 3 to produce continuous small random shifts. Fluorescence images are continuously acquired by the area array camera 9 and processed by the computer 9.

[0056] Assume that the number of fluorescence images acquired by the area array camera 9 is N, and the light intensity of the fluorescence image is denoted as P(x,y;i), where i represents the light intensity of the fluorescence image. i The fluorescence images are acquired at various times, and each fluorescence image is equal to the superposition of the fluorescence intensity produced by the entire sample 11. Its expression is:

[0057] P(x,y;i)=∫(F1(x,y,z;t)+F2(x,y,z;t))dz

[0058] In the formula, P(x,y;i) ​​represents t i The light intensity of the fluorescence image at time t, F1(x,y,z;t) is the coherence length L C The fluorescence intensity within the range, F2(x,y,z;t) is the coherence length L C Fluorescent intensity outside the range;

[0059] Because the first collimator 3 experiences continuous, minute, random shifts due to the first moving platform 5 during fluorescence image acquisition, the phase difference between the first excitation light and the second excitation light is... The value is different at different times, that is, when t i ≠t j hour, F1(x,y,z;t) i )≠F1(x,y,z;t j ), while F2(x,y,z;t i ) = F2(x,y,z;t j Therefore, we have the following expression:

[0060] P(x,y;i)-P(x,y;i+1)≈F1(x,y,z;t i )-F1(x,y,z;t i+1 )

[0061] In the formula, P(x,y;i) ​​represents t i The fluorescence image intensity at time t, P(x,y;i+1) is the fluorescence intensity at time t. i+1 Fluorescence image intensity at time t, F1(x,y,z;t) i ) for t i Time coherence length L C Fluorescence intensity within the range, F1(x,y,z;t) i+1 ) for t i+1 Time coherence length L C Fluorescence intensity within the range;

[0062] Let the position of the equipathic surface 12 be z′, then the coherence length L C The expression for the fluorescence image of sample 11 within the range is:

[0063]

[0064] In the formula, T(x,y;z′) is the coherence length L C The light intensity of the fluorescence image of sample 11 within the range, P(x,y;i) is t iThe fluorescence image intensity at time t, P(x,y;i+1) is the fluorescence intensity at time t. i+1 Fluorescence image intensity at any given time;

[0065] The position of sample 11 is further adjusted by the second moving platform 6, and other target imaging planes of sample 11 are moved to the equal optical path surface 12. The optical sheet imaging process is repeated, and finally optical sheet imaging of each section of sample 11 is achieved.

[0066] The solutions described in the embodiments are not intended to limit the scope of patent protection of this invention. All equivalent implementations or modifications that do not depart from the scope of this invention are included in the patent scope of this case.

Claims

1. A fluorescent light sheet microscopy apparatus, characterized by: The application relates to a low-coherence light sheet imaging device, which comprises a low-coherence light source, a fiber coupler, a first collimator, a second collimator, a first moving platform, a second moving platform, a light splitting sheet, a filter sheet, a lens, a plane array camera and a computer; the low-coherence light source is optically connected with the fiber coupler through an optical fiber; the excitation light output by the fiber coupler is divided into two paths, which are a first excitation light and a second excitation light; the first excitation light output end of the fiber coupler is optically connected with the first collimator, and the second excitation light output end of the fiber coupler is optically connected with the second collimator; the first excitation light collimated by the first collimator and the second excitation light collimated by the second collimator form parallel light; the first collimator is installed on the first moving platform; The second moving platform is used for placing a sample; the first collimator emits the collimated first excitation light to the sample; The second collimator emits the collimated second excitation light to the sample through the light splitting sheet; the fluorescent light excited by the sample is emitted to the lens through the light splitting sheet and the filter sheet, is focused by the lens and is emitted to the plane array camera; the plane array camera is electrically connected with the computer; the fluorescent image is collected by the plane array camera, the excitation light is filtered by the filter sheet, and the fluorescent image is processed by the computer.

2. A fluorescent light sheet microscopic imaging method using the fluorescent light sheet microscopic imaging apparatus according to claim 1, characterized by The application further relates to a low-coherence light sheet imaging method, which comprises the following steps: Step 1: light sheet generation process The low-coherence light source is started, excitation light is emitted from the low-coherence light source, the excitation light enters the fiber coupler through an optical fiber, the first excitation light and the second excitation light are emitted from the fiber coupler, the first excitation light is collimated by the first collimator and is emitted to the sample, the second excitation light is collimated by the second collimator and is emitted to the sample, the first excitation light and the second excitation light form parallel light at this time, the first excitation light and the second excitation light interfere at the equal optical path surface in the sample and excite the fluorescent light, and the fluorescent light excited by the sample is emitted to the plane array camera through the light splitting sheet, the filter sheet and the lens in sequence; Assuming that the linewidth of the low coherence light is Δλ and the wavelength is λ, L C = λ 2 / Δλ, where L C is the coherence length, therefore, the first excitation light and the second excitation light only produce interference within the coherence length L C range near the equal optical path surface (12); The coherent length L C The interference light intensity expression of the first excitation light and the second excitation light in the range of the coherent length L where I(x,y,z;t) is the intensity of the interference light of the first excitation light and the second excitation light, Ii(x,y,z;t) is the intensity of the first excitation light, I2(x,y,z;t) is the intensity of the second excitation light, is the phase difference of the first excitation light and the second excitation light; In the coherent length L C The expression of the fluorescence light intensity of the sample excitation is: where F1(x, y, z; t) is the coherent length L C the fluorescence intensity within the range A is the fluorescence excitation efficiency, I1(x, y, z; t) is the intensity of the first excitation light, I2(x, y, z; t) is the intensity of the second excitation light, is the phase difference between the first excitation light and the second excitation light; The coherent length L C Outside the range, the first excitation light and the second excitation light do not interfere, and the expression of the fluorescence light intensity of the sample excitation is: F2(x, y, z; t) = A [I1(x, y, z; t) + I2(x, y, z; t)] where F2(x, y, z; t) is the coherence length L C the fluorescence intensity outside the range, A is the fluorescence excitation efficiency, I1(x, y, z; t) is the light intensity of the first excitation light, and I2(x, y, z; t) is the light intensity of the second excitation light; Step 2: light sheet imaging process The target imaging plane of the sample is moved to the equal optical path surface by starting the second moving platform, then the first moving platform is started, the first collimator is continuously and slightly randomly offset by the first moving platform, the fluorescent image is continuously collected by the plane array camera, and the fluorescent image is processed by the computer; Assume that the number of fluorescence images collected by the area array camera is N, and the light intensity of the fluorescence image is P(x, y; i), wherein i represents the fluorescence image collected at t i The expression of each fluorescence image is equal to the superposition of the fluorescence light intensity generated by the whole sample, which is expressed as: P(x, y; i) = integral (F1(x, y, z; t) + F2(x, y, z; t)) dz In the formula, P(x,y;i) ​​represents t i The light intensity of the fluorescence image at time t, F1(x,y,z;t) is the coherence length L C The fluorescence intensity within the range, F2(x,y,z;t) is the coherence length L C Fluorescence intensity outside the range; Due to the continuous slight random offset of the first collimator caused by the first moving platform during the fluorescence image acquisition process, the phase difference between the first excitation light and the second excitation light The numerical values at different times are different, that is, when t i ≠t j , There are F1(x,y,z;t i )≠F1(x,y,z;t j ), and F2(x,y,z;t i )=F2(x,y,z;t j ), so there are the following expressions: P(x, y; i) - P(x, y; i + 1) = Fi(x, y, z; t i ) - Fi(x, y, z; t i+1 ) where P(x, y; i) is the fluorescence image intensity at time t i i+1, F1(x, y, z; t i+1 i+1) is the fluorescence image intensity at time t i i+1, F1(x, y, z; t i i+1) is the fluorescence image intensity at time t C i+1, F1(x, y, z; t i+1 i+1) is the fluorescence image intensity at time t i+1 i+1, F1(x, y, z; t C i+1) is the fluorescence image intensity at time t Let the position of the equal optical path plane be z', then the coherence length L C The expression of the fluorescence image of the sample tomography in the range is where T(x,y;z') is the coherence length L C the light intensity of the fluorescence image of the sample tomogram in the range P(x,y;i) is the light intensity of the fluorescence image at time t i the light intensity of the fluorescence image at time t i+1 the light intensity of the fluorescence image at time t The position of the sample is continuously adjusted by the second moving platform, the other target imaging planes of the sample are moved to the equal optical path surface, the light sheet imaging process is repeated, and finally the light sheet imaging of each section of the sample is realized.

Images