CN115113384B - Flat light sheet microscope and imaging method of sample - Google Patents
Flat light sheet microscope and imaging method of sample Download PDFInfo
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Abstract
The present disclosure relates to a tiled light sheet microscope and a method of imaging a sample. The imaging method is as follows. Generating a first laser beam of a first wavelength range and generating a second laser beam of a second wavelength range; the first laser beam is first optically modulated and the second laser beam is second optically modulated. The first optically modulated first laser beam and the second optically modulated second laser beam are combined. The combined laser beams are directed onto an illumination path and first and second excitation light sheets corresponding to the first and second wavelength ranges are generated by scanning the laser beams. The two excitation light sheets are tiled along the propagation direction of the excitation light to illuminate the sample. Collecting fluorescence emitted by the sample. In this way, simultaneous at least two-color imaging can be performed on various sample tissues without affecting spatial resolution and without increasing image acquisition time.
Description
Technical Field
The present disclosure relates to precision optical instruments and methods of use thereof, and more particularly to tiled light sheet microscopes and methods of imaging samples associated therewith.
Background
Due to the opacity of biological tissue, 3D fluorescence imaging of transparent tissue is accomplished primarily by imaging tissue sections of physical sections. Tissue transparentization techniques enable light sheet microscopy to be used for high-speed, high spatial resolution three-dimensional imaging of biological tissue structures by transparentizing the biological tissue. More importantly, the optical slicing of the illumination sheet replaces physical tissue slicing, so that related sample preparation, image acquisition and image analysis become more efficient and practical. Thus, the combination of tissue transparentization using light sheet microscopy and 3D fluorescence imaging is rapidly becoming an important method for observing the cellular and subcellular structures of large multicellular organisms such as organoids, embryos, organs and even whole model animals.
Multicolor 3D fluorescence imaging (especially bicolor imaging) is commonly used to confirm the spatial relationship of different organelles in biological samples. A common method of performing bi-color 3D imaging under an optical sheet microscope is to turn the excitation laser on and off sequentially at each image plane, illuminating the sample at different excitation wavelengths. The emitted fluorescence of different colors is filtered through a dual wavelength channel filter and then focused on the same detection camera. Despite its simple structure, this approach has fluorescent crosstalk between the two color channels due to the use of a dual wavelength channel filter. By replacing the dual channel filter with a filter wheel or dichroic mirror comprised of two single wavelength channel filters, the emitted fluorescence of different wavelengths can be separated and directed to different detection cameras or different sensor areas of the same detection camera to reduce fluorescence crosstalk. However, since it is difficult to simultaneously hold the excitation light sheets of two wavelengths in focus and avoid fluorescence interference, two-color imaging is still performed sequentially with two configurations, which doubles the image acquisition time, and the images seen by the two color ranges are not images at the same instant. Sequential two-color imaging can cause more serious problems for high resolution 3D imaging of large transparent tissues, since the process can last from hours to days or even longer for single-color high resolution three-dimensional imaging of large volumes of biological tissue, which can double the imaging time.
Tiled light microscopy (TLS-SPIM) has been successfully applied to high resolution 3D imaging of transparent tissue. TLS-SPIM is superior to conventional optical sheet microscopes in achieving higher spatial resolution and better optical sectioning capability by translating a thin tunable excitation light sheet on the image plane along the propagation direction of the excitation light and acquiring additional images. By using thin tiled sheets of light, using the latest tissue transparency and tissue expansion techniques in combination, biological tissue can be imaged with isotropic spatial resolution from a few micrometers to tens of nanometers. However, the tiling of the light sheet reduces the imaging throughput of the tiled light sheet microscope due to the need to collect additional images. Sequential bi-color imaging is therefore a greater problem for TLS-SPIM, as it doubles the already extended image acquisition time even further. The present disclosure is provided to address the above-mentioned deficiencies in the background art.
Disclosure of Invention
It is intended to provide a tiled light sheet microscope and an imaging method of a sample capable of performing simultaneous at least two-color imaging of various sample tissues without affecting spatial resolution and without increasing image acquisition time.
In a first aspect, embodiments of the present disclosure provide a tiled light sheet microscope. The tiled light sheet microscope includes a first laser configured to generate a first laser beam of a first wavelength range. The tiled light sheet microscope also includes a first Spatial Light Modulator (SLM) assembly configured to modulate the first laser beam. The tiled light sheet microscope also includes a second laser configured to generate a second laser beam of a second wavelength range. The tiled light sheet microscope also includes a second Spatial Light Modulator (SLM) assembly configured to modulate the second laser beam. The tiled light sheet microscope also includes combining optics configured to combine the first laser beam modulated with the first SLM assembly and the second laser beam modulated with the second SLM. The tiled light sheet microscope further includes a galvanometer configured to direct the combined laser beam onto an illumination path by deflecting the galvanometer angle and to generate two excitation light sheets corresponding to the first wavelength range and the second wavelength range by scanning the laser beam, the galvanometer further arranged to be conjugate to respective optical modulation planes of the first SLM assembly and the second SLM assembly. The tiled light sheet microscope further includes an excitation objective lens disposed at an end of the illumination path to illuminate a sample to be detected, and having an entrance pupil conjugated to respective optical modulation planes of the first SLM assembly and the second SLM assembly. The tiled light sheet microscope also includes a detection objective configured to collect fluorescence emitted by the sample to be detected.
In a second aspect, embodiments of the present disclosure provide a method of imaging a sample. The imaging method includes generating a first laser beam of a first wavelength range and generating a second laser beam of a second wavelength range. The imaging method includes first optically modulating the first laser beam and second optically modulating the second laser beam. The imaging method includes combining a first optically modulated first laser beam and a second optically modulated second laser beam. The imaging method includes directing the combined laser beams onto an illumination path and generating first and second excitation light sheets corresponding to the first and second wavelength ranges by scanning the laser beams. The imaging method comprises independently tiling the two excitation light sheets along the propagation direction of the excitation light to illuminate the sample. The imaging method includes collecting fluorescence emitted by the sample.
With the tiled light sheet microscope and sample imaging method according to various embodiments of the present disclosure, it is possible to perform simultaneous at least two-color imaging of various sample tissues, maintaining the same spatial resolution and without increasing the image acquisition time.
Drawings
In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. The same reference numerals with letter suffixes or different letter suffixes may represent different instances of similar components. The accompanying drawings illustrate various embodiments by way of example in general and not by way of limitation, and together with the description and claims serve to explain the disclosed embodiments. Such embodiments are illustrative and not intended to be exhaustive or exclusive of the present apparatus or method.
FIG. 1 illustrates a flow chart of a method of imaging a sample using a tiled light sheet microscope in accordance with an embodiment of the present disclosure;
fig. 2 (a) shows an example of the principle of simultaneous two-color 3D imaging of an imaging method of a sample according to an embodiment of the present disclosure;
fig. 2 (b) shows another example of the principle of simultaneous two-color 3D imaging of an imaging method of a sample according to an embodiment of the present disclosure;
FIG. 3 shows a schematic block diagram of a tiled light sheet microscope in accordance with an embodiment of the present disclosure;
FIGS. 4 (a) -4 (h) are schematic diagrams illustrating calibration of a tiled light sheet microscope using dye solution for co-color bi-color imaging, according to embodiments of the present disclosure; and
fig. 5 (a) -5 (f) show detailed results of using the simultaneous two-color imaging method according to an embodiment of the present disclosure for the vortex worm Schmidtea mediterranea.
Detailed Description
In order to better understand the technical solutions of the present disclosure, the following detailed description of the present disclosure is provided with reference to the accompanying drawings and the specific embodiments. Embodiments of the present disclosure will be described in further detail below with reference to the drawings and specific embodiments, but not by way of limitation of the present disclosure.
The terms "first," "second," and the like, as used in this disclosure, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises" and the like means that elements preceding the word encompass the elements recited after the word, and not exclude the possibility of also encompassing other elements.
Fig. 1 illustrates a flow chart of a method of imaging a sample using a tiled light sheet microscope, in accordance with an embodiment of the present disclosure. Fig. 1 illustrates two-color 3D imaging as an example, a scheme of laser beams in two wavelength ranges may be used for two-color 3D imaging (each wavelength range corresponds to each color channel), but the disclosure is not limited thereto, and a scheme of laser beams in multiple wavelength ranges may be extended to implement multi-color 3D imaging, which is not repeated herein. The imaging method of the present disclosure is particularly applicable to large-sized samples of transparentized tissue, but is not limited thereto, and may be applicable to other types and sizes of tissue samples, and is not described herein. The transparentized tissue includes biological tissue subjected to transparentization treatment, or biological tissue that has been transparent in a natural state.
As shown in fig. 1, the imaging method includes generating a first laser beam of a first wavelength range (step 101) and generating a second laser beam of a second wavelength range (step 102), respectively. Thus, the first laser beam and the second laser beam can flexibly control the respective start timing and optical parameters according to specific requirements. In some embodiments, the first and second laser beams may be simultaneously activated and continuously generated, providing a basis for simultaneous illumination (e.g., simultaneous illumination) of the same image plane by subsequent excitation light sheets of different color channels and maintaining focus thereon.
The first laser beam may be first optically modulated (step 103) and the second laser beam may be second optically modulated (step 104), respectively. Thereby, the first optical modulation and the second optical modulation can be performed independently of each other. For example, in the case of a synchronous generation of the first laser beam and the second laser beam, the first optical modulation and the second optical modulation can accordingly be carried out synchronously, whereby a synchronous illumination of the same image plane by the excitation light sheet of the subsequent different color channels and a focusing thereof are achieved. By means of independent generation of laser beams in different wavelength ranges and independent optical modulation thereof, compared with sequential bicolor imaging, the imaging method can remarkably reduce image acquisition time and improve imaging flux under the condition that spatial resolution is not affected. In particular, separate optical paths may be provided for laser beams (illumination light) of different wavelength ranges, so that individual controls on each optical path may be utilized to flexibly obtain an optical grid of an excitation light sheet or a group of discrete optical grids according to specific needs (e.g., without limitation, spatial separation). In some embodiments, separate SLM assemblies may be utilized to obtain a desired optical grid or a set of discrete optical grids via loading of different phase maps, which may be scanned by a galvanometer to obtain corresponding excitation light sheets, e.g., excitation light sheets of different wavelength ranges (e.g., different colors) (see fig. 2 (a)) and a set of discrete excitation light sheets of different wavelength ranges (e.g., different colors) (see fig. 2 (b)). As an example, in fig. 2 (a), two-color imaging (color distinguishing the logo in the figure by texture) may be performed simultaneously using two spatially separated tiled light sheets with different excitation wavelengths tiled at different positions. As an example, in fig. 2 (b), two spatially separated sets of discontinuous tiles may be used for simultaneous bi-color imaging, as compared to tiling one excitation light sheet for each color at a time in fig. 2 (a), and tiling one set of discontinuous excitation light sheets for each color at a time in fig. 2 (b), thereby further improving imaging throughput. In this disclosure, the expression "two excitation light sheets" is intended to encompass two excitation light sheets corresponding to two wavelength ranges (e.g., corresponding to two color ranges) as shown in fig. 2 (a), as well as two sets of discontinuous tiled excitation light sheets corresponding to two wavelength ranges (e.g., corresponding to two color ranges) as shown in fig. 2 (b).
In TLS-SPIM, illumination light may be phase modulated via an SLM assembly to generate an excitation light sheet, adjusting the light sheet intensity distribution, and tiling the light sheet to image the entire field of view (FOV). The misalignment of the corresponding excitation light sheet can be corrected and kept in focus under different imaging conditions, e.g. in each tile, via independent phase modulation of illumination light of different wavelength ranges by independent SIM components, thus achieving a satisfactory spatial resolution.
The first optically modulated first laser beam and the second optically modulated second laser beam may be combined (step 105), and the combined laser beams may then be directed onto an illumination path and first and second excitation light sheets corresponding to the first and second wavelength ranges may be generated by scanning the laser beams (step 106). In view of the independent generation of laser beams of different wavelength ranges and their independent optical modulation, the generated first and second excitation light sheets can be independently and flexibly controlled and adjusted, e.g. to illuminate the same image plane and to remain focused at all times, e.g. to be spatially separated (as shown in fig. 2 (a) and 2 (b)), and fluorescent cross-talk between different color channels can be reduced. In some embodiments, combining the first optically modulated first laser beam and the second optically modulated second laser beam may be achieved by a semi-transparent semi-reflective treatment and/or a polarization beam splitting treatment.
In step 107, the two excitation light sheets may be tiled along the propagation direction of the excitation light to illuminate the sample and collect fluorescence emitted by the sample (in response to illumination of the tiled excitation light sheets) (step 108). Thus, the first excitation light sheet and the second excitation light sheet may be tiled independently. In some embodiments, tiling of the excitation light sheets may be achieved by loading the respective SLM assemblies with sets of phase diagrams, which may benefit from independent operation of the corresponding SIM assemblies of the excitation light sheets of different wavelength ranges, such that the individual excitation light sheets after tiling, which correspond to different wavelength ranges, may also remain spatially separated from each other, thereby further reducing fluorescence cross-talk. In some embodiments, the fluorescence emitted by the sample comprises fluorescence of a first wavelength range that is excited by an excitation light sheet generated by a laser beam of the first wavelength range and fluorescence of a second wavelength range that is excited by an excitation light sheet generated by a laser beam of the second wavelength range. The fluorescence thus collected is mixed (mixture of different colors), and subsequently, the collected mixed fluorescence can be separated into fluorescence of two color ranges and imaged accordingly. In some embodiments, separate imaging components may be employed to achieve fluorescence imaging of different color ranges, e.g., fluorescence imaging of different color ranges may be performed simultaneously, such that the superimposed composite image may present less distorted and more detailed information.
Fig. 3 shows a schematic block diagram of a tiled light sheet microscope in accordance with an embodiment of the present disclosure. As shown in fig. 3, the tiled light sheet microscope can include a first laser and a second laser (not shown). The first laser may be configured to generate a first laser beam of a first wavelength range (illustrated as 561nm for excitation of red fluorescence) and the second laser may be configured to generate a second laser beam of a second wavelength range (illustrated as 488nm for excitation of green fluorescence). In some embodiments, the first laser and the second laser may be turned on and continuously operated at the same time, and in combination with the synchronous operation of the relevant optical components, synchronous (simultaneous) imaging of fluorescence of different colors may be achieved. In some embodiments, the laser beam of the first wavelength range is used to excite fluorescence of a first wavelength range (i.e., a first color) and the laser beam of the second wavelength range is used to excite fluorescence of a second wavelength range (i.e., a second color).
The tiled light sheet microscope is used for dividing the excited fluorescence of different colors into independent light paths before beam combination. As shown in fig. 3, the tiled light sheet microscope further comprises a first SLM assembly 301 configured to modulate the first laser beam and a second SLM assembly 302 configured to modulate the second laser beam. In some embodiments, the first SLM assembly 301 and the second SLM assembly 302 may be configured to operate independently to generate two independently controlled and spatially separable excitation light sheets. This is by way of example only, the first SLM assembly 301 and the second SLM assembly 302, which operate independently, may flexibly perform optical modulation operations for laser beams of different wavelength ranges as required in accordance with various embodiments of the present disclosure, including, but not limited to, at least one or several of light sheet intensity distribution, misalignment correction, tiling of laser light sheets, and focus preservation at image planes under different imaging conditions, and the like. In particular, the first SLM assembly 301 and the second SLM assembly 302 may be configured to enable independent tiling of two excitation light sheets corresponding to the first wavelength range and the second wavelength range by loading phase diagrams separately. In some embodiments, the independent tiling of the two excitation light sheets may enable the individual excitation light sheets corresponding to different wavelength ranges after tiling to remain spatially separated from each other, thereby further reducing fluorescence crosstalk.
The tiled light microscope also includes combining optics 303 configured to combine, i.e., combine, the first laser beam modulated with the first SLM assembly 301 and the second laser beam modulated with the second SLM assembly 302. A half mirror may be used as the combining optics 303 as shown in fig. 3 to allow the first laser beam modulated by the first SLM assembly 301 to pass through and the second laser beam modulated by the second SLM assembly 302 to reflect and match in the optical path to reduce the divergence of the combined beam. In some embodiments, a polarizing beamsplitter may also be used as the combining optics to achieve similar optical processing.
The combined beam may be directed onto the illumination path by deflecting the angle of a galvanometer 304, said galvanometer 304 being further arranged to be conjugate to the respective optical modulation planes of said first SLM assembly 301 and said second SLM assembly 302, and to generate two excitation light sheets corresponding to said first wavelength range and said second wavelength range by scanning the laser beam. By way of example, two illumination paths are shown in fig. 3, which are separated on either side of imaging chamber 305, and excitation objectives 306a and 306b, which are disposed at the ends of each illumination path for illuminating the sample to be tested, so that, regardless of where the sample is in imaging chamber 305, the excitation objective on one side is brought into proximity, the sample to be tested can be illuminated with the excitation objective on the nearer side at a closer transmission distance. But this is merely an example and it is also possible to provide only one illumination path on one side of the imaging chamber 305.
The detection objective 307 may be used to collect the mixed-color fluorescence emitted by the excitation of the sample to be detected. Next, the mixed-color fluorescence collected by the detection objective 307 may be separated into fluorescence of two color ranges, for example, red fluorescence and green fluorescence, using a spectroscopic optical member 308. In some embodiments, the beam splitting optical component 308 may be a half mirror, but it may also be other optical components/components, so long as the fluorescence of each color can be separated. As shown in fig. 3, the separated fluorescence of the first color may be further imaged to the first detection camera 309b using the first barrel lens 309a after being filtered by the first filter (not shown); the separated fluorescence of the second color may be further imaged to the second detection camera 310b using a second barrel lens 310a after being filtered by the second filter (not shown), and the first barrel lens 309a and the second barrel lens 310a may have the same focal length.
In some embodiments, the first SLM assembly 301, the second SLM assembly 302, the first detection camera 309b and the second detection camera 310b may operate in a synchronized manner, such that fluorescence imaging of different colors may be performed in synchronization, such that the superimposed composite image may present less distorted and more detailed information.
The flow of imaging is described in detail below using another detailed example of the construction of a tiled light sheet microscope as shown in fig. 3. The sample to be tested may be immersed in an imaging buffer in imaging chamber 305 and then mounted on a sample holder driven by a 3D translation stage for 3D imaging.
The excitation laser beams of 488nm and 561nm wavelengths may be expanded to a beam diameter of about 8mm (l1=30 mm, l2=250 mm) and sent to two identical first and second SLM assemblies 301 and 302 for phase modulation, respectively. Each of first SLM assembly 301 and second SLM assembly 302 may be a binary SLM assembly, and may be comprised of a polarizing beam splitting prism, a half wave plate and a 1280 x 1024 binary SLM (as shown in fig. 3).
In some embodiments, each modulated laser beam may be focused on a corresponding optical slit 311a (or 311 b) to block unwanted diffraction orders and beam combining to form a combined beam using, for example, half mirror as combining optics 303. Two binary SLMs may be conjugated with the galvanometer 304 by a relay lens (l3=300 mm, l4=175 mm). Galvanometer 304 may direct two illumination beams to a designated one of the two symmetrical illumination paths by deflecting its initial angle and generate two excitation light sheets corresponding to different colors by scanning the laser beam. The modulated laser beam may be further conjugated to the entrance pupil of two excitation objectives 306a and 306b (e.g., mitutoyo MY 5X-802) through two pairs of relay lenses (l5=l6=150mm), respectively, to illuminate the sample from two opposite directions.
Fluorescence emitted from the sample may be collected using, for example, mitutoyo MY10X-804 detection objectives 309b and 310b for tissue imaging at micron-scale spatial resolution. Detection objectives 309b and 310b with higher Numerical Apertures (NA) may be used for tissue imaging with sub-micron spatial resolution. The collected fluorescence can be split by using a half mirror for long-range imaging as a spectroscopic optical member 308, and after passing through two single-band bandpass filters, further imaged onto two detection cameras using two cylindrical mirrors 309a and 310a having the same focal length (l=150 mm) to capture tissue images.
In some embodiments, the tiled light sheet microscope can be calibrated and then used for simultaneous bi-color imaging. The imaging chamber 305 may be filled with a mixture of Alexa Fluor 488 and Alexa Fluor 561 dye solution for calibration. Since the laser light having a wavelength of 488nm can excite the two dyes, respectively, the excited laser beams can be seen on the two detection cameras 309b and 310 b. The detection paths of the two color channels are calibrated with respect to the 488nm excitation beam so that the two detection cameras 309b and 310b are focused on the same image plane. Meanwhile, the FOV and magnification of both the detection cameras 309b and 310b are the same. Next, phase maps for modulating the two laser beams may be generated, respectively. Two sets of calibrated phase maps can be loaded sequentially into the two SLM assemblies 301 and 302, tiling excitation light sheets of two color ranges at different locations during tiling to image the entire FOV while minimizing fluorescence cross-talk. During imaging, both SLM assemblies 301 and 302 and both detection cameras 309b and 310b are synchronized with the same trigger signal.
Fig. 4 (a) -4 (h) show schematic diagrams of calibration of tiled light sheet microscopes using dye solutions for co-color bi-color imaging, according to embodiments of the present disclosure.
As shown in fig. 4 (a), 488nm excitation light sheet was tiled at 6 tiling positions (naod=0.05, naid=0.015) by loading a set of phase maps, thereby generating a 488nm tiling light sheet as shown in fig. 4 (b). As shown in fig. 4 (c), 561nm excitation light sheet (naod=0.05, naid=0.015) was tiled at 6 tile positions spatially separated from 488nm excitation light sheet by loading a set of phase maps, thereby generating 561nm tiling light sheet shown in fig. 4 (d).
Fig. 4 (e) -4 (h) show schematic diagrams of groups of discrete excitation light sheets by tiling. As shown in fig. 4 (e), a set of phase maps was loaded to tile 488nm discrete excitation light sheet sets (naod=0.07, naid=0.02) to produce 488nm discrete excitation light sheet sets as shown in fig. 4 (f). As shown in fig. 4 (g), by registering a set of phase diagrams to tile 561nm discrete excitation light sheet sets, the waists of each excitation light sheet are separated from the waists of 488nm discrete excitation light sheets (naod=0.07, naid=0.02), resulting in 561nm discrete excitation light sheets shown in fig. 4 (h).
To evaluate the ability of the imaging methods according to various embodiments of the present disclosure to be applied to simultaneous bicolor imaging, a labeling neuron pool (fluorescein) and an axon projection (rhodomine B) of about 5-fold expanded vortex worm Schmidtea Mediterraterranea was imaged using a calibration process of a tiled light sheet microscope for bicolor imaging using dye solutions according to embodiments of the present disclosure shown in fig. 4 (a) -4 (d).
The excitation light sheets of 488nm and 561nm were tiled at six positions in the image plane as shown in FIGS. 4 (a) -4 (d), at about 1.5X1.5X4. Mu.m 3 The spatial resolution of (2) imaging the extended vortex insects, the resolution corresponding to 0.3X0.3X0.8 μm taking into account the 5-fold expansion ratio of the vortex insects 3 . By aligning 10 to 1.5X1.5X1.2mm in one hour 3 Is imaged, and the vortex is imaged in two color ranges. Tiling 488nm excitation light sheets from tiling position 1 to 6 on each image plane to image the distribution of neurons; in the imaging process, 561nm excitation light sheets are tiled from tiling positions 4 to 6, and then 561nm excitation light sheets are tiled from tiling positions 1 to 3 to image the projection of the neuron axons, and the maximum distance between the two light sheets is always kept. Thereby reducing the imaging time by half and increasing the imaging flux by a factor of two compared with sequentially performing two-color imaging. This advantage will be more pronounced as the sample size increases.
The results of evaluation imaging are shown in fig. 5 (a) -5 (f). Fig. 5 (a) and 5 (b) show the transverse and axial Maximum Intensity Projections (MIPs) of a planar neuron pool. Fig. 5 (c) and 5 (d) show the lateral and axial MIPs of planar neuronal axon projections. Fig. 5 (e) and 5 (f) show lateral and axial MIPs of the vortex shedding worm, showing both the pool of neurons and the neuronal axon projections. The relationship between planar neurons and the neuron axon projections can be clearly seen from the imaging results.
The above description is intended to be illustrative and not restrictive. For example, the above-described examples (or one or more aspects thereof) may be used in combination with each other. For example, other embodiments may be used by those of ordinary skill in the art upon reading the above description. In addition, in the above detailed description, various features may be grouped together to streamline the disclosure. This is not to be interpreted as an intention that the disclosed features not being claimed are essential to any claim. Rather, the disclosed subject matter may include less than all of the features of a particular disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description as examples or embodiments, with each claim standing on its own as a separate embodiment, and it is contemplated that these embodiments may be combined with one another in various combinations or permutations. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.
The above embodiments are only exemplary embodiments of the present disclosure, and are not intended to limit the present invention, the scope of which is defined by the claims. Various modifications and equivalent arrangements of parts may be made by those skilled in the art, which modifications and equivalents are intended to be within the spirit and scope of the present disclosure.
Claims (20)
1. A tiled light sheet microscope, the tiled light sheet microscope comprising:
a first laser configured to generate a first laser beam of a first wavelength range;
a first spatial light modulator assembly configured to modulate the first laser beam;
a second laser configured to generate a second laser beam of a second wavelength range;
a second spatial light modulator assembly configured to modulate the second laser beam;
combining optics configured to combine the first laser beam modulated with the first spatial light modulator assembly and the second laser beam modulated with the second spatial light modulator;
a galvanometer configured to direct the combined laser beam onto an illumination path by deflecting the galvanometer angle and to generate two excitation light sheets corresponding to the first wavelength range and the second wavelength range by scanning the laser beam, the galvanometer further arranged to be conjugate to respective optical modulation planes of the first spatial light modulator assembly and the second spatial light modulator assembly, wherein the first spatial light modulator assembly and the second spatial light modulator assembly are configured to operate independently to generate two independently controlled and spatially separable excitation light sheets;
an excitation objective lens disposed at an end of the illumination path to illuminate a sample to be detected, and having an entrance pupil conjugated with respective optical modulation planes of the first and second spatial light modulator assemblies; and
and a detection objective configured to collect fluorescence emitted by the sample to be detected.
2. The tiled light sheet microscope of claim 1, wherein the first laser and the second laser are on and continuously operating simultaneously.
3. The tiled light sheet microscope of claim 1, wherein the first spatial light modulator assembly and the second spatial light modulator assembly are configured to enable independent tiling of two excitation light sheets corresponding to the first wavelength range and the second wavelength range by loading phase maps, respectively.
4. A tiled light sheet microscope according to claim 3 wherein the independent tiling of the two excitation light sheets is such that individual excitation light sheets corresponding to different wavelength ranges after tiling remain spatially separated from each other.
5. The tiled light sheet microscope of claim 1, wherein the combining optics comprises a half mirror and/or a polarizing beamsplitter.
6. The tiled light sheet microscope of claim 1, wherein the laser beam of the first wavelength range is used to excite fluorescence of the first wavelength range and the laser beam of the second wavelength range is used to excite fluorescence of the second wavelength range.
7. The tiled light sheet microscope of claim 1, further comprising a light splitting optical member configured to split the mixed fluorescence of different colors collected by the detection objective into fluorescence of two color ranges.
8. The tiled light sheet microscope of claim 7, wherein the beam splitting optical member comprises a half mirror.
9. The tiled light sheet microscope of claim 1, wherein the illumination path includes symmetrical first and second illumination paths on either side of the imaging chamber, the excitation objective including a first excitation objective at an end of the first illumination path and a second excitation objective at an end of the second illumination path.
10. The tiled light sheet microscope of claim 7, further comprising:
the first optical filter, the first barrel lens and the first detection camera are used for filtering the separated fluorescence of the first color, and then the fluorescence is further imaged to the first detection camera by the first barrel lens; and
the first barrel lens and the second barrel lens have the same focal length.
11. The tiled light sheet microscope of claim 10, wherein the first spatial light modulator assembly, the second spatial light modulator assembly, the first detection camera, and the second detection camera operate in a synchronized manner.
12. A method of imaging a sample, the method comprising:
generating a first laser beam of a first wavelength range;
generating a second laser beam of a second wavelength range;
performing first optical modulation on the first laser beam;
performing a second optical modulation on the second laser beam;
combining the first laser beam after the first optical modulation and the second laser beam after the second optical modulation;
directing the combined laser beam onto an illumination path and generating first and second excitation light sheets corresponding to the first and second wavelength ranges by scanning the laser beam, wherein the first and second excitation light sheets are independently controlled and spatially separated, respectively;
tiling the first excitation light sheet and the second excitation light sheet along a propagation direction of excitation light to illuminate the sample; and
collecting fluorescence emitted by the sample.
13. The imaging method of claim 12, wherein the first and second laser beams are simultaneously enabled and continuously generated.
14. The imaging method of claim 12, wherein the first excitation light sheet and the second excitation light sheet are tiled independently.
15. The imaging method of claim 12, wherein the independent tiling of the first excitation light sheet and the second excitation light sheet is such that individual excitation light sheets corresponding to different wavelength ranges remain spatially separated from each other after tiling.
16. Imaging method according to claim 12, characterized in that the combination of the first optically modulated first laser beam and the second optically modulated second laser beam is achieved by a semi-transparent semi-reflective treatment and/or a polarization beam splitting treatment.
17. The imaging method of claim 12, wherein the fluorescence emitted by the sample comprises fluorescence in a first wavelength range that is excited by an excitation light sheet generated by the laser beam in the first wavelength range and fluorescence in a second wavelength range that is excited by an excitation light sheet generated by the laser beam in the second wavelength range.
18. The imaging method of claim 12, further comprising: the collected mixed fluorescence of different colors is separated into fluorescence of two color ranges.
19. The imaging method of claim 12, wherein the first optical modulation and the second optical modulation are performed simultaneously.
20. The imaging method according to claim 12, wherein the sample is a biological tissue subjected to a transparentization treatment or a biological tissue which has been transparent in a natural state.
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| CN105044897A (en) * | 2015-07-07 | 2015-11-11 | 中国科学院上海高等研究院 | Rapid random optical reconstruction imaging system and method based on sparse constraint |
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