CN115437131A

CN115437131A - Method for three-dimensional imaging of biological sample and light sheet microscope system

Info

Publication number: CN115437131A
Application number: CN202110619532.3A
Authority: CN
Inventors: 高亮; 付志飞; 冯瑞丽; 谢炅芳
Original assignee: Westlake University
Current assignee: Westlake University
Priority date: 2021-06-03
Filing date: 2021-06-03
Publication date: 2022-12-06
Also published as: WO2022252838A1

Abstract

The present disclosure relates to a method for three-dimensional imaging of a biological sample using a light sheet microscope and a light sheet microscope system, the method comprising: using a photo-fluorescent protein molecule as a fluorescent probe in a biological sample, imaging each set of sub-regions sequentially by: illuminating the set of subregions with a control laser such that the light-operated fluorescent protein molecules in the set of subregions are capable of emitting fluorescence under illumination by the excitation light; exciting the photo-control fluorescent protein molecules in the set of sub-regions using an excitation light sheet formed by a light sheet microscope based on an excitation laser to emit fluorescence; imaging the set of sub-regions with the emitted fluorescence; and splicing the imaging results of the sub-regions of each group to obtain a three-dimensional imaging result of the biological sample. The invention utilizes the light control conversion characteristic of the light control fluorescent protein to solve the light bleaching problem when a light sheet microscope carries out high-resolution three-dimensional imaging on a large-volume transparent biological sample.

Description

Method for three-dimensional imaging of biological sample and light sheet microscope system

Technical Field

The present disclosure relates to a precision optical instrument and a method of using the same, and more particularly, to a method of three-dimensionally imaging a biological sample using a light sheet microscope and a light sheet microscope system.

Background

High-resolution three-dimensional fluorescence imaging of biological tissues is an effective means for acquiring three-dimensional structures of the biological tissues and researching biological problems such as gene expression, cell morphology, cell distribution and the like on the scale of subcellular, cellular and tissue. However, due to the opacity of biological tissue, the traditional approach of sectioning, two-dimensional imaging, and three-dimensional reconstruction of biological tissue has been the only means to obtain high resolution three-dimensional structures of biological tissue. However, the slice imaging reconstruction technology for biological tissues has low imaging efficiency, great sample preprocessing difficulty and complex data reconstruction, so that the technology is difficult to be widely applied.

The biological tissue transparentization technology breaks through the main obstacle of preventing the fluorescent microscope imaging technology from being applied to high-resolution three-dimensional imaging of biological tissues, so that the three-dimensional fluorescent microscope imaging technology at the front edge can be used for efficiently acquiring three-dimensional structure information of various biological tissue cell levels and subcellular levels, and scientific research personnel can be helped to better know the structures and functions of the biological tissues and organs. Among various three-dimensional fluorescence microscope imaging technologies, the light sheet microscope technology has the characteristics of high speed, high resolution and high signal-to-noise ratio, and is very suitable for three-dimensional imaging of transparent biological tissues. By combining with the biological tissue transparentization technology, the optical section microscope uses the optical section method to replace a physical section method in the traditional tissue imaging technology, and the speed and the resolution of the high-resolution three-dimensional fluorescence imaging of the biological tissue are greatly improved.

The imaging of the large-volume transparent biological sample by the light sheet microscope is realized by sequentially carrying out three-dimensional imaging on a series of subareas of the sample and splicing the three-dimensional imaging results of all the subareas. The resolution of the light sheet microscope imaging the entire sample is equivalent to the resolution of the light sheet microscope imaging each sub-area in three dimensions. Three-dimensional imaging of a large volume of a transparentized biological sample by a light sheet microscope is accomplished by three-dimensional imaging of a series of subregions. As shown in fig. 1 (a), the spatial range of each sub-region is large, the number of sub-regions is small, and the imaging resolution is low. As shown in fig. 1 (B), the spatial range of each sub-region is small, the number of sub-regions is large, and the imaging resolution is high. When a light sheet microscope is used to three-dimensionally image a sample in this way, although only the subregion located in the center of the excitation light sheet is imaged, the other subregions located in the propagation path of the excitation light are simultaneously illuminated by the excitation light sheet. Therefore, when the light-sheet microscope is used for three-dimensional imaging of the sub-region imaged first, the sub-region imaged later is also repeatedly irradiated by the excitation light sheet, so that the fluorescence labeling probe in the sub-region imaged later is quenched before the light-sheet microscope is used for three-dimensional imaging of the sub-region, photobleaching of a sample is caused, and the light-sheet microscope cannot obtain a three-dimensional image with a sufficient signal-to-noise ratio in the sub-region imaged later. When the ROI1-ROI4 sub-regions are imaged, the excitation light simultaneously irradiates the non-imaged sub-regions ROI5-ROI16, as shown in FIG. 2 (A). When imaging the ROI5-ROI8, the excitation light simultaneously irradiates the non-imaged sub-regions ROI9-ROI12, as shown in FIG. 2 (B). When the sub-regions ROI9 to ROI12 are imaged, the excitation light is simultaneously irradiated to the non-imaged sub-regions ROI13 to ROI16 as shown in FIG. 2 (C). As shown in fig. 2 (D), the non-imaged subareas ROI13-ROI16 have been irradiated with excitation light several times before imaging, causing premature quenching of the fluorescent probes of the area, thereby causing a more serious photo-bleaching problem, so that the light sheet microscope cannot complete three-dimensional imaging of all subareas, i.e., the entire sample, with a desired resolution.

Furthermore, as the resolution of the light sheet microscope imaging the sample increases, the volumetric extent of each sub-region decreases. Thus, higher resolution three-dimensional imaging of the same sample needs to be accomplished by three-dimensional imaging of a greater number of smaller volumetric sub-regions, and thus, more severe photobleaching problems are encountered. Therefore, the photobleaching problems encountered with light sheet microscopes for high resolution three-dimensional imaging of large volumes of transparentized biological samples limit the highest resolution achievable with light sheet microscopes for three-dimensional imaging of large volumes of transparentized biological samples.

Disclosure of Invention

The method and the system aim to provide a method for three-dimensional imaging of a biological sample by using a light-sheet microscope, and solve the photobleaching problem when the light-sheet microscope performs high-resolution three-dimensional imaging on a large-volume transparent biological sample by using the light control conversion characteristic of light control fluorescent protein.

In a first aspect, embodiments of the present disclosure provide a method for three-dimensional imaging of a biological sample with a light sheet microscope, the method comprising: using a light-controlled fluorescent protein molecule as a fluorescent probe in the biological sample, wherein the light-controlled fluorescent protein molecule can emit fluorescence under the irradiation of exciting light after the irradiation of control laser; dividing the biological sample into a plurality of groups of sub-regions, so that each group of sub-regions is distributed on a corresponding path; imaging each set of sub-regions in sequence by: illuminating the set of subregions with the control laser light such that the light control fluorescent protein molecules in the set of subregions are capable of emitting fluorescence under illumination by the excitation light; exciting the photo-control fluorescent protein molecules in the set of sub-regions using an excitation light sheet formed by the light sheet microscope based on an excitation laser to emit fluorescence; imaging the set of sub-regions with the emitted fluorescence; and splicing the imaging results of the sub-regions of each group to obtain a three-dimensional imaging result of the biological sample.

In a second aspect, embodiments of the present disclosure provide a light sheet microscopy system for three-dimensional imaging of a biological sample using a photo-fluorescent protein molecule as a fluorescent probe in the biological sample, the light sheet microscopy system comprising: a first laser configured to: emitting control laser to irradiate the biological sample, so that the light-operated fluorescent protein molecules can emit fluorescence under the irradiation of exciting light after the irradiation of the control laser; a second laser configured to: emitting excitation laser; a light sheet microscope body configured to: forming an excitation light sheet to irradiate a biological sample based on the excitation laser so that the light-operated fluorescent protein molecules in the biological sample can emit fluorescence under irradiation of the excitation light; an imaging member configured to: receiving fluorescence from a biological sample and imaging the biological sample using the received fluorescence; and a processor configured to: dividing the biological sample into a plurality of groups of sub-regions, so that each group of sub-regions is distributed on a corresponding path; controlling the first laser, the second laser, the light sheet microscope body and the imaging component to sequentially image each group of subareas by the following steps: controlling the first laser to emit the control laser to illuminate the set of sub-regions; controlling the light sheet microscope body to irradiate the set of sub-regions of the biological sample with an excitation light sheet formed based on the excitation laser; controlling the imaging means to image the set of sub-regions with fluorescence from the set of sub-regions of the biological sample; and splicing the imaging results of the sub-regions of each group to obtain a three-dimensional imaging result of the biological sample.

By using the method for three-dimensional imaging of a biological sample by using a light sheet microscope and the light sheet microscope system according to various embodiments of the present disclosure, the light control conversion characteristic of the light control fluorescent protein can be used to solve the light bleaching problem when the light sheet microscope is used for high-resolution three-dimensional imaging of a large-volume transparent biological sample, so that three-dimensional imaging of the biological sample is realized.

Drawings

In the drawings, which are not necessarily drawn to scale, like reference numerals may describe similar components in different views. Like reference numerals having letter suffixes or different letter suffixes may represent different instances of similar components. The drawings illustrate various embodiments generally by way of example, and not by way of limitation, and together with the description and claims serve to explain the disclosed embodiments. Such embodiments are illustrative, and are not intended to be exhaustive or exclusive embodiments of the present apparatus or method.

Fig. 1 (a) -1 (B) show schematic diagrams of imaging a biological sample with a light sheet microscope according to an embodiment of the present disclosure.

Fig. 2 (a) -2 (D) show schematic diagrams of the occurrence of photobleaching when imaging a biological sample using a light sheet microscope.

Fig. 3 shows a flow diagram of a method for three-dimensional imaging of a biological sample with a light sheet microscope according to an embodiment of the present disclosure.

FIG. 4 shows a schematic diagram of the operation of light-activated fluorescent protein molecules.

Fig. 5 (a) -5 (I) show schematic diagrams of imaging a sub-region of a biological sample using light activated fluorescent protein molecules with a light sheet microscope, according to embodiments of the present disclosure.

FIG. 6 shows a schematic diagram of the operation of light-converting fluorescent protein molecules.

Fig. 7 (a) -7 (I) show schematic diagrams of imaging a sub-region of a biological sample with a light-sheet microscope using light-converted fluorescent protein molecules, according to embodiments of the present disclosure.

FIG. 8 shows a schematic diagram of the operation of the photoswitch fluorescent protein molecule.

Fig. 9 (a) -9 (I) show schematic diagrams of imaging a sub-region of a biological sample with a light-sheet microscope using light-switched fluorescent protein molecules, according to embodiments of the present disclosure.

Fig. 10 shows a schematic block diagram of a light sheet microscope system according to an embodiment of the present disclosure.

Fig. 11 shows a schematic structural diagram of an example of a light sheet microscope system according to an embodiment of the present disclosure.

Detailed Description

For a better understanding of the technical aspects of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings. Embodiments of the present disclosure are described in further detail below with reference to the figures and the detailed description, but the present disclosure is not limited thereto.

The use of "first," "second," and similar terms in this disclosure are not intended to indicate any order, quantity, or importance, but rather are used for distinction. The word "comprising" or "comprises", and the like, means that the element preceding the word covers the element listed after the word, and does not exclude the possibility that other elements are also covered.

Embodiments of the present disclosure provide a method for three-dimensional imaging of a biological sample using a light sheet microscope, as shown in fig. 3, the three-dimensional imaging method starts in step 301, in which a biological sample is labeled using a photo-fluorescent protein molecule as a fluorescent probe, wherein the photo-fluorescent protein molecule can emit fluorescence under irradiation of excitation light after irradiation of control laser light. The biological sample may be divided into a plurality of sets of sub-regions at step 302, such that each set of sub-regions is distributed on a corresponding path. Next, for each set of sub-regions, imaging may be performed sequentially through steps 303a-303c as follows. In step 303a, the set of sub-regions is illuminated with the control laser light, so that the photo-fluorescent protein molecules in the set of sub-regions can emit fluorescence under illumination of the excitation light. In step 303b, the photo-luminescent protein molecules in the set of sub-regions are excited to emit fluorescence using an excitation light sheet formed by the light sheet microscope based on an excitation laser. In step 303c, the set of sub-regions is imaged with the emitted fluorescence. Next, after the imaging of each set of sub-regions is completed, the imaging results of each set of sub-regions may be stitched to obtain a three-dimensional imaging result of the biological sample in step 304.

By the three-dimensional imaging method, when the sub-region is required to be imaged, the irradiation of the laser is controlled to enable the light-controlled fluorescent protein molecule serving as the fluorescent probe to emit fluorescence in response to the irradiation of the exciting light, so that the problem that in the traditional three-dimensional imaging method, the sub-region of the post-imaging is irradiated by the exciting light for multiple times before being imaged, so that the fluorescent probe is quenched in advance to cause photobleaching of the sub-region of the post-imaging is solved. The embodiment provides the three-dimensional imaging method, so that a satisfactory signal-to-noise ratio can be obtained in the imaging process by using the light sheet microscope, particularly when a post-imaging subregion is imaged, and the three-dimensional imaging method of the embodiment can be applied to high-resolution three-dimensional imaging of a large-volume transparent biological sample.

In some embodiments, the biological sample is naturally optically transparent, or is treated by a biological tissue transparentization technique or a swelling technique, thereby having certain optically transparent properties. Dividing the biological sample into a plurality of groups of sub-regions may divide the biological sample into a plurality of groups of sub-regions in rows and columns, such that each group of sub-regions is located on each column, such that each group of sub-regions is distributed on a corresponding path, and each obtained path is non-overlapping and linear. Therefore, each group of sub-regions can be traversed according to the planned path so as to realize efficient imaging of each group of sub-regions, and the non-overlapping design of the path can further reduce the early quenching of the fluorescent probe before the imaging of the sub-regions.

In some embodiments, the photo-control fluorescent protein molecule is a photo-activated fluorescent protein molecule which does not emit fluorescence before being irradiated by the control laser and is activated to a state capable of emitting fluorescence under irradiation by the control laser after being irradiated by the control laser, as shown in fig. 4. In some embodiments, the light-activated fluorescent protein molecule can be PA-GFP. In the specific operation process, purple laser with the wavelength ranging from 350nm to 450nm can be used as control laser, the excitation laser used for forming the excitation light sheet of the light sheet microscope is blue laser, and the light-activated fluorescent protein molecules emit green fluorescence under the irradiation of the excitation light sheet after being activated.

Next, the method of three-dimensional imaging of a biological sample using a light sheet microscope of this example will be exemplified by taking a light activated fluorescent protein molecule as an example of a light controlled fluorescent protein molecule.

After the biological sample is subjected to transparentization treatment, the light-sheet microscope performs three-dimensional imaging of all the subregions, namely the whole sample, by respectively performing fluorescence activation and imaging on each column of subregions. Because the light activated fluorescent protein molecule of each subregion is activated to enter an activated state only before the subregion is three-dimensionally imaged, green fluorescence can be emitted, and the photobleaching problem can be effectively avoided. Taking the imaging of biological samples labeled with light activated fluorescent protein shown in FIGS. 5 (A) -5 (I) as an example, light sheet microscopy completes three-dimensional imaging of the entire sample, i.e., ROI1 through ROI16 sub-regions, as follows.

As shown in fig. 5 (a), the light-activated protein molecules of all subregions were not activated before imaging, and thus did not emit fluorescence.

As shown in fig. 5 (B), a first array of sub-regions (ROI 1-ROI 4) of the sample may be irradiated with a control laser having a wavelength of 405nm to activate the light-activated fluorescent protein molecules in these sub-regions. After the irradiation of the control laser with 405nm, the light activated fluorescent protein molecules in the first column sub-region are activated from a non-fluorescence state to an activation state capable of emitting green fluorescence.

Then, as shown in fig. 5 (C), the first column sub-regions (ROI 1 to ROI 4) can be sequentially subjected to three-dimensional imaging by exciting the light-activated fluorescent protein molecules capable of emitting green fluorescence in the activated state using an excitation light sheet formed based on excitation light having a wavelength of 488 nm.

Then, the aforementioned steps can be repeated, the activation of the light-activated fluorescent protein molecules in the remaining sub-regions is completed column by column, and the light-activated fluorescent protein molecules in the activated state are used for three-dimensional imaging.

For example, as shown in fig. 5 (D) -5 (E), a controlled laser with a wavelength of 405nm may be used to irradiate a second column of sub-regions (ROI 5-ROI 8) of the biological sample, activating the light-activated fluorescent protein in the second column of sub-regions. The excitation light sheet formed based on the excitation light with the wavelength of 488nm is used for exciting the light-activated fluorescent protein molecules which can emit green fluorescence and are in the activated state, so that the three-dimensional imaging can be carried out on the second column of sub-regions (ROI 5-ROI 8) in sequence.

Next, as shown in FIG. 5 (F) -FIG. 5 (G), the third column subregion (ROI 9-ROI 12) of the biological sample is irradiated with a control laser having a wavelength of 405nm to activate the light-activated fluorescent protein in the third column subregion. The light-activated fluorescent protein molecules which can emit green fluorescence in the activated state are excited using an excitation light sheet formed based on excitation light having a wavelength of 488nm, so that the third column sub-region (ROI 9-ROI 12) can be sequentially subjected to three-dimensional imaging.

Then, as shown in FIGS. 5 (H) -5 (I), the fourth row of sub-region (ROI 13-ROI 16) of the biological sample is irradiated with a control laser having a wavelength of 405nm to activate the light-activated fluorescent protein in the fourth row of sub-region. The fourth column of sub-regions (ROI 13-ROI 16) can be sequentially subjected to three-dimensional imaging by exciting the light-activated fluorescent protein molecules capable of emitting green fluorescence in an activated state using an excitation light sheet formed based on excitation light having a wavelength of 488 nm.

Finally, the obtained three-dimensional imaging results of the ROI1 to the ROI16 sub-regions can be spliced to obtain the three-dimensional imaging result of the whole biological sample.

According to the invention, the light-activated fluorescent protein molecules are used as the light-operated fluorescent protein molecules, and the method for three-dimensional imaging of the biological sample by using the light sheet microscope effectively solves the photobleaching problem when the light sheet microscope is used for high-resolution three-dimensional imaging of the large-volume transparentized biological sample, and realizes high-resolution imaging of the large-volume transparentized biological sample.

In some embodiments, the photo-control fluorescent protein molecule may also employ a light-converting fluorescent protein molecule that emits a first fluorescent light in a first wavelength range under irradiation of a first excitation light before irradiation of a control laser and converts to a second fluorescent light in a second wavelength range under irradiation of a second excitation light after irradiation of the control laser. In some embodiments, the first excitation light and the second excitation light may have different wavelength ranges, and the first wavelength range and the second wavelength range may be different. As shown in fig. 6, the light-converting fluorescent protein molecule may emit fluorescence when excited before light conversion, and the wavelength range in which the light-converting fluorescent protein absorbs the excitation light and the wavelength range in which the fluorescence is emitted may be converted to another wavelength range by irradiation with violet light, thereby emitting fluorescent proteins of different color fluorescence. The light-converting fluorescent protein molecule may be mEOS, and a violet laser having a wavelength ranging from 350nm to 450nm may be used in a specific operation process. In some embodiments, the control laser may be a violet laser, the first fluorescence may be a green fluorescence and the second fluorescence may be a red fluorescence, and the excitation laser used by the light sheet microscope to form the second excitation light may be a yellow-green laser.

In some embodiments, prior to the light-converting fluorescent protein molecule converting: the biological sample may also be irradiated with first excitation light to emit first fluorescence in a first wavelength range; imaging the biological sample with the emitted first fluorescence. The light-converting fluorescent protein may be excited to emit fluorescence before being converted, and after being converted, the excited fluorescent protein generally emits fluorescence of a longer wavelength than before being converted. Therefore, the light conversion protein can be stimulated to emit fluorescence before conversion, and the structure of the sample can be conveniently observed and imaged before three-dimensional imaging by using the light conversion fluorescent protein.

Hereinafter, a method of three-dimensional imaging of a biological sample using a light sheet microscope will be exemplified by taking a light-converting fluorescent protein molecule as an example of a light-controlling fluorescent protein molecule.

After the biological sample is subjected to transparentization treatment by using the light conversion fluorescent protein for labeling, the light sheet microscope completes three-dimensional imaging of all the subareas, namely the whole sample by respectively carrying out fluorescence conversion and imaging on each column of subareas. Since the light-converting fluorescent protein molecules of each sub-region are converted into the used fluorescent color channel for imaging only before the sub-region is three-dimensionally imaged. Therefore, the photobleaching problem can be effectively solved by using the converted fluorescence color channel. Taking the imaging of biological samples labeled with light activated fluorescent protein shown in FIGS. 7 (A) -7 (I) as an example, light sheet microscopy performed the three-dimensional imaging of the entire sample, i.e., ROI1 through ROI16 sub-regions, as follows.

As shown in fig. 7 (a), all the light-converting protein molecules in the subregions are not converted, and the light-converting fluorescent protein molecules before conversion are excited to emit green fluorescence.

As shown in fig. 7 (B), a first sequence of sub-regions (ROI 1-ROI 4) of the sample may be irradiated with a control laser having a wavelength of 405nm to convert the light-converted fluorescent protein molecules in these sub-regions. After the irradiation of the control laser with 405nm, the light conversion fluorescent protein molecules in the first column of sub-regions emit green fluorescence after being excited, and then emit red fluorescence after being excited.

Then, as shown in fig. 7 (B), the first column sub-region (ROI 1 to ROI 4) can be sequentially subjected to three-dimensional imaging by exciting the fluorescence-converted light-converting fluorescent protein molecule capable of emitting red fluorescence, which has completed the fluorescence conversion, using an excitation light sheet formed based on the excitation light having the wavelength of 561 nm.

Then, the foregoing steps may be repeated to complete the conversion of the light-converting fluorescent protein molecules of the remaining sub-regions column by column, and the converted light-converting fluorescent protein molecules are used for three-dimensional imaging.

For example, as shown in fig. 7 (D) -7 (E), a second column of sub-regions (ROI 5-ROI 8) of the biological sample may be irradiated with a control laser having a wavelength of 405nm, so that the light-converting fluorescent protein in these sub-regions may be subjected to fluorescence conversion.

And exciting the light conversion fluorescent protein molecules which complete fluorescence conversion and can emit red fluorescence by using an excitation light sheet formed by excitation light with the wavelength of 561nm, and sequentially carrying out three-dimensional imaging on the second column of sub-regions (ROI 5-ROI 8).

Next, as shown in fig. 7 (F) to 7 (G), the third column subregion (ROI 9 to ROI 12) of the biological sample is irradiated with the control laser having a wavelength of 405nm, and the light-converted fluorescent protein in the third column subregion is subjected to fluorescence conversion.

The light-converted fluorescent protein molecules that have completed fluorescence conversion and can emit red fluorescence are excited using an excitation light sheet formed based on excitation light having a wavelength of 561nm, and the third column subregions (ROI 9-ROI 12) can be sequentially subjected to three-dimensional imaging.

Next, as shown in fig. 7 (H) to 7 (I), the fourth column of sub-regions (ROI 13 to ROI 16) of the sample was irradiated with the control laser having a wavelength of 405nm, and the light-converted fluorescent protein in the fourth column of sub-regions was subjected to fluorescence conversion.

The fourth column of sub-regions (ROI 13-ROI 16) can be sequentially subjected to three-dimensional imaging by exciting the fluorescence-converted light-converting fluorescent protein molecules capable of emitting red fluorescence, which have undergone fluorescence conversion, using an excitation light sheet formed based on excitation light having a wavelength of 561 nm.

And finally, splicing the obtained three-dimensional imaging results of the ROI1 to the ROI16 sub-regions to obtain the three-dimensional imaging result of the whole biological sample.

According to the invention, the light-conversion fluorescent protein molecule is used as the light-operated fluorescent protein molecule, and the method for three-dimensional imaging of the biological sample by using the light sheet microscope effectively solves the photobleaching problem when the light sheet microscope is used for high-resolution three-dimensional imaging of the large-volume transparentized biological sample, and realizes high-resolution imaging of the large-volume transparentized biological sample.

In some embodiments, the photoswitch fluorescent protein molecule is a photoswitch fluorescent protein molecule that is in an off state that does not emit fluorescent light prior to illumination by the control laser, enters an on state that is capable of emitting fluorescent light under illumination by the excitation light after illumination by the control laser, and returns to the off state that does not emit fluorescent light after emitting fluorescent light. As shown in fig. 8, the photoswitch fluorescent protein molecule is a photoswitch fluorescent protein that does not emit fluorescence before being turned on by light, and can emit fluorescence when turned on by ultraviolet light, whereas when excited by excitation light, the photoswitch fluorescent protein in the on state enters the off state after emitting fluorescence. Photoswitch fluorescent protein molecules allow repeated light on, excitation, and light off operations, switching between on and off states multiple times. Therefore, a sample labeled with the photoswitch fluorescent protein can be repeatedly turned on and off during the imaging process, thereby allowing a more flexible imaging mode. In one embodiment, the photoswitch fluorescent protein molecule may be Dronpa. Violet laser light, which may have a wavelength in the range of 350nm to 450nm, may be used during a particular operation.

The method for three-dimensional imaging of a biological sample using a light sheet microscope of this example is illustrated below with a photoswitch fluorescent protein molecule as the photoswitch fluorescent protein molecule.

After the biological sample is subjected to transparentization treatment, the light sheet microscope completes three-dimensional imaging of all the subregions, namely the whole biological sample, by respectively carrying out fluorescence turn-on and imaging on each column of subregions. The photoswitch fluorescent protein molecules of each subregion are in an open state only before and when the subregion is three-dimensionally imaged, and can emit fluorescence. Therefore, the photobleaching problem can be effectively solved by using the photoswitch fluorescent protein molecule. Taking the three-dimensional imaging of the ROI1 to ROI16 sub-regions of the samples shown in fig. 9 (a) -9 (I) as an example, the light sheet microscope performs the three-dimensional imaging of the entire sample, i.e., the ROI1 to ROI16 sub-regions, as follows.

As shown in fig. 9 (a), before the light switch fluorescent protein molecules are turned on, the light switch fluorescent protein molecules in all the subregions are in the off state, and the light switch fluorescent protein molecules before being turned on do not emit fluorescence even if excited.

As shown in fig. 9 (B), a first column of sub-regions (ROI 1-ROI 4) of the sample can be illuminated with a control laser having a wavelength of 405nm, turning on the photoswitch fluorescent protein molecules in the first column of sub-regions. After the irradiation of the control laser with 405nm, the photoswitch fluorescent protein molecules in the first column of sub-regions are switched from non-fluorescence emission to fluorescence protein molecules capable of emitting green fluorescence after excitation. Because the photoswitch protein enters the off state that the fluorescence can not be emitted after the fluorescence is emitted, the control laser with the wavelength of 405nm can be used for repeatedly irradiating the first column of subareas in the imaging process, so that the photoswitch protein of the imaged subarea is always in the on state and used for three-dimensional imaging of the subarea.

Then, as shown in fig. 9 (C), the photoswitch fluorescent protein molecules that are already in the on state can be excited using an excitation sheet formed based on excitation light having a wavelength of 488nm, and three-dimensional imaging can be sequentially performed on the first row of sub-regions (ROI 1-ROI 4).

Then, the above steps can be repeated, the photoswitch fluorescent protein molecules of the remaining subregions are opened column by column, and the photoswitch fluorescent protein molecules in the opened state are used for three-dimensional imaging of the corresponding subregions.

For example, as shown in fig. 9 (D) -9 (E), the second column of sub-regions (ROI 5-ROI 8) of the sample can be repeatedly irradiated with a control laser with a wavelength of 405nm, turning on the light switch fluorescent protein in the ROI5-ROI8 sub-region.

The second column of sub-regions (ROI 5-ROI 8) can be sequentially subjected to three-dimensional imaging by exciting the light-switch fluorescent protein molecules which are turned on and can emit green fluorescence by using an excitation light sheet formed based on excitation light with the wavelength of 488 nm.

Then, as shown in FIGS. 9 (F) to 9 (G), the third column subregion (ROI 9-ROI 12) of the sample can be repeatedly irradiated with a control laser having a wavelength of 405nm, and the light-switch fluorescent protein in the ROI9-ROI12 subregion is turned on.

The third column of sub-regions (ROI 9-ROI 12) can be sequentially subjected to three-dimensional imaging by exciting the photoswitch fluorescent protein molecule that has been turned on and can emit green fluorescent light using an excitation light sheet formed based on excitation light having a wavelength of 488 nm.

Subsequently, as shown in FIGS. 9 (H) to 9 (I), the fourth column subregion (ROI 13-ROI 6) of the sample was repeatedly irradiated with a control laser beam having a wavelength of 405nm, and the light-switch fluorescent protein in the ROI13-ROI16 subregion was turned on.

The fourth column of sub-regions (ROI 13-ROI 16) can be sequentially subjected to three-dimensional imaging by exciting the light-switch fluorescent protein molecules which are turned on and can emit green fluorescence by using an excitation light sheet formed based on excitation light with the wavelength of 488 nm.

According to the method, the photoswitch fluorescent protein molecule is used as the photoswitch fluorescent protein molecule, and the light microscope is used for carrying out three-dimensional imaging on the biological sample, so that the photobleaching problem when the light microscope carries out high-resolution three-dimensional imaging on the large-volume transparent biological sample is effectively solved, and the high-resolution imaging on the large-volume transparent biological sample is realized. The light switch fluorescent protein in this example does not fluoresce before being turned on, can fluoresce green or other colors after being turned on, and returns to an off state after being excited and fluoresced. The closed photoswitch protein may be turned on and off again and the cycling operation may be performed multiple times until the photoswitch fluorescent protein quenches. Therefore, the operation of opening and closing the photoswitch fluorescent protein can be repeatedly carried out in the specific operation process, so that the imaging quality is effectively improved.

Fig. 10 shows a schematic block diagram of a light sheet microscope system for three-dimensional imaging of a biological sample using light-controlled fluorescent protein molecules as fluorescent probes in the biological sample, according to an embodiment of the present disclosure. As shown in fig. 10, the light sheet microscope system may include a first laser 1011, a second laser 1008, a light sheet microscope body 1000, an imaging component 1010, and a processor 1012. The first laser 1011 may be configured to emit a control laser to illuminate the biological sample such that the photo-fluorescent protein molecules are capable of emitting fluorescence under illumination by the excitation light after illumination by the control laser. The second laser 1008 may be configured to emit excitation laser light. The light sheet microscope body 1000 may be configured to form an excitation light sheet to illuminate a biological sample based on the excitation laser light, so that the photo-fluorescent protein molecules in the biological sample can emit fluorescence under illumination of the excitation light. The imaging member 1010 may be configured to: fluorescence from a biological sample is received, and the biological sample is imaged using the received fluorescence. The processor 1012 may be configured to: dividing the biological sample into a plurality of groups of sub-regions, so that each group of sub-regions is distributed on a corresponding path; the first laser 1011, the second laser 1008, the light sheet microscope main body and the imaging component 1010 are controlled to sequentially image each group of sub-areas in the following steps. For example, the processor 1012 may be configured to control the first laser 1011 to emit the control laser to illuminate the set of sub-regions. The processor 1012 may be configured to control the light sheet microscope body to illuminate the set of sub-regions of the biological sample with an excitation light sheet formed based on the excitation laser. The processor 1012 may be configured to control the imaging component 1010 to image the set of sub-regions with fluorescence from the set of sub-regions of the biological sample. And, the processor 1012 may be configured to stitch the imaging results of the sets of sub-regions to obtain a three-dimensional imaging result of the biological sample.

In embodiments consistent with the present disclosure, the processor 1012 may be a processing device including more than one general-purpose processing device, such as a microprocessor, central Processing Unit (CPU), graphics Processing Unit (GPU), or the like. More specifically, the processor may be a Complex Instruction Set Computing (CISC) microprocessor, reduced Instruction Set Computing (RISC) microprocessor, very Long Instruction Word (VLIW) microprocessor, processor running other instruction sets, or processors running a combination of instruction sets. The processor may also be one or more special-purpose processing devices such as an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), a Digital Signal Processor (DSP), a system on a chip (SoC), or the like. The processor 1012 may be communicatively coupled to a memory and configured to execute computer-executable instructions stored thereon to perform methods such as three-dimensional imaging of biological samples according to embodiments of the present disclosure.

In some embodiments, the memory/storage may be a non-transitory computer-readable medium, such as read-only memory (ROM), random-access memory (RAM), phase-change random-access memory (PRAM), static random-access memory (SRAM), dynamic random-access memory (DRAM), electrically erasable programmable read-only memory (EEPROM), other types of random-access memory (RAM), flash disks or other forms of flash memory, caches, registers, static memory, compact disk read-only memory (CD-ROM), digital Versatile Disks (DVD) or other optical storage, tape cassettes or other magnetic storage devices, or any other possible non-transitory medium that is used to store information or instructions that can be accessed by a computer device, and so forth.

The biological sample in this example is a biological sample that is naturally optically transparent, or has been processed by a biological tissue transparentization technique or a swelling technique so as to have certain optically transparent properties. Dividing the biological sample into a plurality of groups of sub-regions may divide the biological sample into a plurality of groups of sub-regions in rows and columns, such that each group of sub-regions is located on each column, such that each group of sub-regions is distributed on a corresponding path, and each obtained path is non-overlapping and linear.

In some embodiments, the photo-control fluorescent protein molecule may be a light-activated fluorescent protein molecule that does not emit fluorescence before irradiation of the control laser light and is activated to a state capable of emitting fluorescence under irradiation of the excitation light after irradiation of the control laser light. In some embodiments, the control laser may be a violet laser, the excitation laser may be a blue laser, and the light-activated fluorescent protein molecules may emit green fluorescence upon activation under irradiation of the excitation sheet.

In some embodiments, the photo-control fluorescent protein molecule may be a photo-converted fluorescent protein molecule that emits a first fluorescent light in a first wavelength range under irradiation of a first excitation light before irradiation of a control laser and is converted to a second fluorescent light in a second wavelength range under irradiation of a second excitation light after irradiation of the control laser. Wherein the first excitation light and the second excitation light may have different wavelength ranges, and the first wavelength range and the second wavelength range may be different. In some embodiments, the control laser is a violet laser, the first fluorescence is green fluorescence and the second fluorescence is red fluorescence, and the excitation laser used by the light sheet microscope to generate the second excitation light is a yellow-green laser. In some embodiments, the processor may be further configured to, prior to the light-converting fluorescent protein molecule converting: controlling the light sheet microscope body to irradiate the biological sample with first exciting light so that the biological sample emits first fluorescence in a first wavelength range; and controlling the imaging means to image the biological sample with the emitted first fluorescence.

In some embodiments, the photo-control fluorescent protein molecule may be a photo-switch fluorescent protein molecule that is in an off state where no fluorescence is emitted before irradiation of the control laser, enters an on state where fluorescence is emitted under irradiation of the excitation light after irradiation of the control laser, and returns to the off state where no fluorescence is emitted after emission of the fluorescence. In some embodiments, the processor may be further configured to control the first laser to repeatedly irradiate the set of sub-regions with the control laser until imaging of the set of sub-regions is completed. In some embodiments, the control laser may be a violet laser, the excitation laser may be a blue laser, and the photoswitch fluorescent protein molecule emits green fluorescence under illumination of the excitation sheet in an on state.

In some embodiments, the processor 1012 may be configured to perform a sequential processing of the respective sub-regions using any one of light activated fluorescent protein molecules, light converted fluorescent protein molecules, light switched fluorescent protein molecules as fluorescent probes, including but not limited to a sequential imaging process of the respective sub-regions according to various embodiments of the present disclosure.

Light sheet microscope systems according to embodiments of the present disclosure may take various configurations, as shown in fig. 11. It should be understood, however, that the optical sheet microscope system according to the embodiments of the present disclosure is not limited thereto, as long as the corresponding microscope components can be controlled to perform the imaging process for each set of sub-regions via the processor 1012.

For example, as shown in fig. 11, the light-sheet microscope body may include beam expanding collimator lenses L1 and L2, a binary SLM assembly 1001, an optical slit 1009, at least a first pair of relay lenses L3 and L4, a first galvanometer 1002, at least a second pair of relay lenses L5 and L6 or L7 and L8, and

excitation objective lenses

1004 and 1007.

The second laser 1008 is configured to generate a laser beam, e.g., a combined laser beam having excitation wavelengths of 488nm and 561 nm. The beam expanding and collimating lenses L1 and L2 may be configured to expand and collimate the laser beam from the laser generating assembly 1008, e.g., to expand the combined laser beam to about 7mm 1/e ² The beam diameter, the focal length of the beam expanding and collimating lens L1, such as, but not limited to, 30mm and the focal length of the beam expanding and collimating lens L2, such as, but not limited to, 250mm, and transmits the expanded laser beam to the binary SLM assembly 1001.

The binary SLM assembly 1001 can include a binary SLM 10011 and be configured to phase modulate the expanded laser beam. Accordingly, the phase map loaded into the binary SLM 10011 is a binary phase map, which can be obtained by binarizing the corresponding successive phase maps. In addition to the binary SLM 10011 (e.g., a binary SLM 10011 with dimensions 1280 x 1024), the binary SLM assembly 1001 can also include a polarization splitting prism 10013, a half wave plate 10012 for splitting, filtering and phase modulating the expanded laser beam. The modulated laser beam can be focused onto an optical slit 1009 to block unwanted laser diffraction orders generated by the binary SLM assembly 1001, thereby enhancing the imaging effect. The binary SLM assembly 1001 may be conjugated to the first galvanometer 1002 via the at least a first pair of relay lenses L3 and L4, with lens L3 having a focal length such as, but not limited to, 300mm and lens L4 having a focal length such as, but not limited to, 175mm. The first galvanometer 1002 may direct illumination light onto one of two

symmetric illumination paths

1003 and 1006 by deflecting the galvanometer angle. In a lattice light sheet microscope, the first galvanometer 1002 may also create a virtual excitation light sheet by scanning a laser beam to achieve illumination of a biological sample.

The first galvanometer 1002 may be conjugated to the entrance pupil of the corresponding

excitation objective

1004 or 1007 via the at least a second pair of relay lenses L5 and L6 or L7 and L8, wherein the focal lengths of the lenses L5 and L7 are, for example and without limitation, 150mm, and the focal lengths of the lenses L6 and L8 are, for example and without limitation, 250mm, so that the sample can be illuminated from two opposite directions, thereby minimizing the transmission distance of the excitation light in the sample, correspondingly reducing the attenuation of the excitation light, and achieving better imaging effect. In some embodiments, second excitation objective 1007 and first excitation objective 1004 may have different Numerical Apertures (NA), for example, MY5X-802 and MY5X-803 of Mitutoyo corporation may both be used as first excitation objective 1004 and second excitation objective 1007.

The following description will be made using light-activated fluorescent protein molecules as fluorescent probes using a light sheet microscope system shown in FIG. 11 and following the flow of FIGS. 5 (A) -5 (I) as an example. However, it should be understood that the process can be adjusted according to the type of the photo-control fluorescent protein molecule used as the fluorescent probe, for example, a photo-conversion fluorescent protein molecule is used as the photo-control fluorescent protein molecule, and a photo-switch fluorescent protein molecule is used as the photo-control fluorescent protein molecule. The adjustment can be performed according to the specific structure of the light-sheet microscope system to adapt to the actual situation, which is not described herein. Further, although the imaging procedure is described with a sub-area group in a row-column configuration as an example, other configurations of the sub-area group may be adopted, and the control may be adjusted based on a variation of the imaging procedure, which is not described herein.

In some embodiments, the photo-control fluorescent protein molecule is a light-activated fluorescent protein molecule that does not emit fluorescence before irradiation of the control laser light and is activated to a state capable of emitting fluorescence under irradiation of the excitation light after irradiation of the control laser light.

In some embodiments, a violet laser with a wavelength in the range of 350nm-450nm may be used as the control laser. The light-activated fluorescent protein molecules do not emit fluorescence before light activation, can be activated from a state of not emitting fluorescence to a state of emitting fluorescence by irradiation with violet light, and can emit fluorescence under irradiation with excitation light after being activated. In some embodiments, the light-activated fluorescent protein molecule can be PA-GFP.

The specific operation flow may include: placing the processed biological sample in the imaging chamber 1005, and controlling the first laser 1011, the second laser 1008, the light sheet microscope body and the imaging means 1010 by the processor 1012, sequentially imaging each set of sub-regions of the biological sample by:

as shown in FIG. 5 (A), all the subregions of the light-activated protein molecules are not activated and thus do not emit fluorescence.

As shown in fig. 5 (B), the processor 1012 may control the first laser 1011 to emit a control laser having a wavelength of 405nm to irradiate the first sub-regions (ROI 1-ROI 4) of the sample, so that the light-activated fluorescent protein molecules in these sub-regions may be activated. After the irradiation of the control laser with 405nm, the light activated fluorescent protein molecules in the first sub-region are activated from a non-fluorescent state to an activated state capable of emitting green fluorescence.

Then, as shown in FIG. 5 (C), processor 1012 may control second laser 1008 to emit excitation light having a wavelength of 488nm to form the excitation patch.

The processor 1012 may control the light sheet microscope body to excite the light-activated fluorescent protein molecules that may emit green fluorescence in an activated state using the excitation light sheet.

The imaging means 1010 sequentially three-dimensionally images the first sub-regions (ROI 1-ROI 4).

For example, as shown in fig. 5 (D) -5 (E), the processor 1012 may control the first laser 1011 to emit control laser light with a wavelength of 405nm to irradiate a second column of sub-regions (ROI 5-ROI 8) of the biological sample, and activate the light-activated fluorescent protein in the second column of sub-regions. The processor 1012 may control the light sheet microscope body to excite the light-activated fluorescent protein molecules capable of emitting green fluorescence in the activated state using the excitation light sheet formed based on the excitation light having the wavelength of 488nm, so that the three-dimensional imaging may be sequentially performed on the second column of sub-regions (ROI 5 to ROI 8).

Next, as shown in fig. 5 (F) -5 (G), the processor 1012 can control the first laser 1011 to emit control laser light with a wavelength of 405nm to irradiate the third column sub-region (ROI 9-ROI 12) of the biological sample, and activate the light-activated fluorescent protein in the third column sub-region. The processor 1012 may control the light sheet microscope body to excite the light-activated fluorescent protein molecules capable of emitting green fluorescence in the activated state using the excitation light sheet formed based on the excitation light having the wavelength of 488nm, so that the three-dimensional imaging may be sequentially performed on the third column sub-regions (ROI 9-ROI 12).

Next, as shown in fig. 5 (H) -5 (I), the processor 1012 can control the first laser 1011 to emit control laser light with a wavelength of 405nm to irradiate a fourth row of sub-regions (ROI 13-ROI 16) of the biological sample, and activate the light-activated fluorescent protein in the fourth row of sub-regions. The processor 1012 may control the light sheet microscope body to excite the light-activated fluorescent protein molecules capable of emitting green fluorescence in the activated state by using the excitation light sheet formed based on the excitation light with the wavelength of 488nm, and may sequentially perform three-dimensional imaging on the fourth column of sub-regions (ROI 13-ROI 16).

The processor 1012 concatenates the obtained three-dimensional imaging results of the sub-regions to obtain a three-dimensional imaging result of the entire biological sample.

With the light sheet microscope system of the present example, a satisfactory signal-to-noise ratio is obtained during the imaging process, especially when the post-imaging sub-area is imaged, so that the three-dimensional imaging system of the present embodiment can be applied to high-resolution three-dimensional imaging of a large-volume transparent biological sample.

Moreover, although exemplary embodiments have been described herein, the scope thereof includes any and all embodiments based on the disclosure with equivalent elements, modifications, omissions, combinations (e.g., of various embodiments across), adaptations or alterations. The elements of the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive. It is intended, therefore, that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims and their full scope of equivalents.

The above description is intended to be illustrative and not restrictive. For example, the above-described examples (or one or more versions 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 foregoing detailed description, various features may be grouped together to streamline the disclosure. This should not be interpreted as an intention that a disclosed feature not claimed is essential to any claim. Rather, the subject matter of the present disclosure may lie in less than all 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 the embodiments can be combined with each other 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 equivalents may be made thereto by those skilled in the art within the spirit and scope of the present disclosure, and such modifications and equivalents should be considered to be within the scope of the present invention.

Claims

1. A method for three-dimensional imaging of a biological sample using a light sheet microscope, the method comprising:

using a light-controlled fluorescent protein molecule as a fluorescent probe in the biological sample, wherein the light-controlled fluorescent protein molecule can emit fluorescence under the irradiation of exciting light after the irradiation of control laser;

dividing the biological sample into a plurality of groups of sub-regions, so that each group of sub-regions is distributed on a corresponding path; imaging each set of sub-regions in sequence by: illuminating the set of subregions with the control laser light such that the light-operated fluorescent protein molecules in the set of subregions are capable of emitting fluorescence under illumination by excitation light; exciting the photo-control fluorescent protein molecules in the set of sub-regions using an excitation light sheet formed by the light sheet microscope based on an excitation laser to emit fluorescence; imaging the set of sub-regions with the emitted fluorescence; and

and splicing the imaging results of the sub-regions of each group to obtain a three-dimensional imaging result of the biological sample.

2. The method according to claim 1, wherein the photo-control fluorescent protein molecule is a photo-activated fluorescent protein molecule which does not emit fluorescence before irradiation of the control laser light and is activated to a state capable of emitting fluorescence under irradiation of the excitation light after irradiation of the control laser light.

3. The method of claim 2, wherein the control laser is a violet laser, the excitation laser used by the light sheet microscope to form the excitation light sheet is a blue laser, and the light-activated fluorescent protein molecules emit green fluorescence under irradiation of the excitation light sheet after activation.

4. The method of claim 1, wherein the light control fluorescent protein molecule is a light-converting fluorescent protein molecule that emits a first fluorescent light in a first wavelength range under irradiation of a first excitation light before irradiation of a control laser light and converts to a second fluorescent light in a second wavelength range under irradiation of a second excitation light after irradiation of the control laser light, wherein the first excitation light and the second excitation light are different in wavelength range and the first wavelength range and the second wavelength range are different.

5. The method of claim 4, wherein the control laser is a violet laser, the first fluorescence is a green fluorescence and the second fluorescence is a red fluorescence, and the excitation laser used by the light sheet microscope to generate the second excitation light is a yellow-green laser.

6. The method of claim 4, further comprising, prior to the converting the light-converting fluorescent protein molecule: irradiating the biological sample with first excitation light to emit first fluorescence in a first wavelength range; imaging the biological sample with the emitted first fluorescence.

7. The method of claim 1, wherein the light-operated fluorescent protein molecule is a light-operated switch fluorescent protein molecule, which is in an off state where no fluorescence is emitted before the irradiation of the control laser light, enters an on state where fluorescence is emitted under the irradiation of the excitation light after the irradiation of the control laser light, and returns to the off state where no fluorescence is emitted after the fluorescence is emitted.

8. The method of claim 7, wherein illuminating the set of sub-regions with the control laser further comprises repeatedly illuminating the set of sub-regions with the control laser until imaging of the set of sub-regions is completed.

9. The method according to claim 7 or 8, wherein the control laser is a violet laser, the excitation laser used by the light sheet microscope to form the excitation light sheet is a blue laser, and the photoswitch fluorescent protein molecule emits green fluorescence under irradiation of the excitation light sheet in an on state.

10. The method of claim 1, wherein the paths do not overlap and are linear.

11. The method of claim 1, wherein dividing the biological sample into a plurality of sets of sub-regions further comprises dividing the biological sample into a plurality of sets of sub-regions in rows and columns such that each set of sub-regions is located on each column.

12. The method according to claim 1, wherein the biological sample is naturally optically transparent or has been subjected to a biological tissue transparentization technique or a swelling technique to have an optically transparent property.

13. A light sheet microscope system for three-dimensional imaging of a biological sample using photo-controlled fluorescent protein molecules as fluorescent probes in the biological sample, comprising:

a first laser configured to: emitting control laser to irradiate the biological sample, so that the light-operated fluorescent protein molecules can emit fluorescence under the irradiation of exciting light after the irradiation of the control laser;

a second laser configured to: emitting excitation laser;

a light sheet microscope body configured to: forming an excitation light sheet based on the excitation laser to irradiate a biological sample so that the optically controlled fluorescent protein molecules in the biological sample can emit fluorescence under irradiation of the excitation light;

an imaging member configured to: receiving fluorescence from a biological sample and imaging the biological sample using the received fluorescence; and

a processor configured to:

dividing the biological sample into a plurality of groups of sub-regions, so that each group of sub-regions is distributed on a corresponding path;

controlling the first laser, the second laser, the light sheet microscope body and the imaging component to sequentially image each group of subareas by the following steps: controlling the first laser to emit the control laser to illuminate the set of sub-regions; controlling the light sheet microscope body to irradiate the set of sub-regions of the biological sample with an excitation light sheet formed based on the excitation laser; controlling the imaging means to image the set of sub-regions with fluorescence from the set of sub-regions of the biological sample; and

14. The light sheet microscope system of claim 13, wherein the light control fluorescent protein molecules are light activated fluorescent protein molecules that do not emit fluorescence before irradiation of the control laser light and are activated to a state capable of emitting fluorescence under irradiation of the excitation light after irradiation of the control laser light.

15. The light sheet microscope system of claim 13, wherein the control laser is a violet laser, the excitation laser is a blue laser, and the light activated fluorescent protein molecules emit green fluorescence upon activation under illumination by the excitation light sheet.

16. The light sheet microscope system of claim 13, wherein the light control fluorescent protein molecules are light conversion fluorescent protein molecules that emit first fluorescent light in a first wavelength range under irradiation of first excitation light before irradiation of control laser light and convert to second fluorescent light in a second wavelength range under irradiation of second excitation light after irradiation of the control laser light, wherein the first excitation light and the second excitation light are different in wavelength range and the first wavelength range and the second wavelength range are different.

17. The light sheet microscope system according to claim 16, wherein the control laser is a violet laser, the first fluorescence is a green fluorescence and the second fluorescence is a red fluorescence, and the excitation laser used by the light sheet microscope to form the second excitation light is a yellow-green laser.

18. The light sheet microscope system of claim 16, further comprising the processor further configured to, prior to the light-converting fluorescent protein molecule converting: controlling the light sheet microscope body to irradiate the biological sample with first exciting light so that the biological sample emits first fluorescence in a first wavelength range; and controlling the imaging means to image the biological sample using the emitted first fluorescence.

19. The light sheet microscope system of claim 13, wherein the light control fluorescent protein molecules are light switch fluorescent protein molecules which are in an off state of not emitting fluorescent light before irradiation of the control laser light, enter an on state of being capable of emitting fluorescent light under irradiation of the excitation light after irradiation of the control laser light, and return to the off state of not emitting fluorescent light after emission of fluorescent light.

20. The light sheet microscope system of claim 19, wherein the processor is further configured to: controlling the first laser to repeatedly irradiate the set of sub-regions with the control laser until imaging of the set of sub-regions is completed.

21. The light sheet microscope system of claim 19 or 20, wherein the control laser is a violet laser, the excitation laser is a blue laser, and the light switch fluorescent protein molecules emit green fluorescence under illumination of the excitation light sheet in an on state.

22. The light sheet microscope system of claim 13, wherein the paths are non-overlapping and linear.

23. The light sheet microscope system of claim 13, wherein dividing the biological sample into a plurality of sets of sub-regions further comprises dividing the biological sample into a plurality of sets of sub-regions in rows and columns such that each set of sub-regions is located on each column.

24. The light sheet microscope system of claim 13, wherein the biological sample is a biological sample that is optically transparent in nature or has been processed by a biological tissue transparentization technique or a bulking technique to have certain optically transparent properties.