CN111024671B - System and method for super-resolution imaging of directional light stimulation structural change - Google Patents
System and method for super-resolution imaging of directional light stimulation structural change Download PDFInfo
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Abstract
The invention provides a system and a method for super-resolution imaging of directional light stimulation structural changes, wherein the system comprises the following components: the system comprises a first laser, a second laser, an image generation module, a directional light stimulation module and a control terminal. According to the invention, the directional optical stimulation module based on the acousto-optic deflector is introduced into the super-resolution imaging system, so that the method can be conveniently used for rapidly and accurately positioning and locally stimulating the sample, and can also be used for monitoring and comparing the super-resolution imaging of nanoscale structural changes of the optical stimulation part and the non-optical stimulation part. The directional light stimulated light source and the super-resolution imaging light source are mutually independent, and the femto-second laser is adopted, so that the emission wavelength is selected in the infrared band, the loss of the sample is minimized, and the spectrum crosstalk between the directional light stimulation and the super-resolution imaging is avoided.
Description
Technical Field
The invention belongs to the technical field of optical microscopy, and particularly relates to a system and a method for super-resolution imaging of directional light stimulation structure change.
Background
Since the microscopic invention, optical microscopy imaging techniques have greatly advanced the art of life science research. However, due to the diffraction effect of the optical element on the light wave by the inherent limited aperture, there is an upper limit on the resolution of the conventional optical microscope, called diffraction limited resolution, i.e., d=0.61 λ/NA, where λ is the wavelength of the light wave and NA is a numerical aperture, typically about 250-300 nm, which is indistinguishable for smaller structures.
Random optical reconstruction microscopy (STORM) utilizes random sparse luminescence and detection of fluorescent molecules, avoids mutual overlapping of single-molecule diffraction spots, and can position the spot center of a single fluorescent molecule through an algorithm to reconstruct a super-resolution structure image of a marked sample, thereby bypassing diffraction limit limitation of a traditional optical microscope, improving resolution to 20-50 nanometers, imaging nano-scale structures in the sample, and providing a powerful tool for deep understanding of the structure and function of the sample. STORM is typically performed using a continuous laser wide field illumination of a fixed sample labeled with an optically switched fluorescent probe to perform superdivision imaging, and thus can be used to study the nanoscale characteristics of specific structures within the sample in specific environments.
The introduction of specific stimuli and the monitoring of changes in cellular structure and function resulting from these stimuli is one of the common methods of studying cellular physiological activities in the field of life sciences, but traditional light stimulation methods generally do not have the ability to direct stimulation. There are also studies on directional stimulation and super-resolution imaging of samples, such as the group of university of Paris Cartesian topics in France, combining Computer Generated Holograms (CGH) with stimulated emission depletion (STED) microscopy for optical stimulation of neurons and monitoring of changes in nanoscale morphology. CGH is used for optical stimulation of specific sites of cells, and STED microscopy is used for super-resolution imaging. Synapses of living cells are labeled with lipophilic organic dyes or fluorescent proteins, dendritic spines are stimulated with CGH directed light, and then morphological changes of the dendritic spines after stimulation are monitored using STED super-resolution imaging. However, the STED in this method requires a high-intensity pulsed laser as the depletion light, which is liable to damage the sample and is disadvantageous for imaging and monitoring living cells.
Accordingly, there is a need for further improvements in the art.
Disclosure of Invention
In view of the above-mentioned shortcomings in the prior art, the present invention aims to provide a system and a method for super-resolution imaging of directional optical stimulus structure changes, which overcome the defect that the existing optical stimulus in the super-resolution imaging technology for optical stimulus of a sample does not have directional stimulus capability, and the sample can be stimulated by combining a hologram with a stimulated emission depletion microscope, but the stimulated emission depletion microscope needs to use high-intensity pulse laser as depletion light when performing super-resolution imaging, so that the sample is easy to be damaged.
A first embodiment of the present disclosure is a system for super-resolution imaging of directional light stimulus structural changes, wherein the system comprises: the system comprises a first laser, a second laser, an image generation module, a directional light stimulation module and a control terminal;
the image generation module is used for irradiating a second laser beam generated by the second laser to a sample to generate a wide-field fluorescent image, and is used for irradiating a third laser beam generated by the second laser to the sample to generate a fluorescent molecule scintillation image when the directional light stimulation module performs directional light stimulation on the sample;
The directional light stimulation module is used for irradiating a first laser beam generated by the first laser onto the sample according to the wide-field fluorescent image to perform directional light stimulation on the sample;
And the control terminal is used for reconstructing a super-resolution image of the sample according to the fluorescent molecule scintillation image.
The system for super-resolution imaging of directional light stimulation structural changes, wherein the directional light stimulation module comprises: a first beam expanding and shaping lens group, a dispersion compensating prism and an acousto-optic deflector;
the first beam expanding and shaping lens group is used for receiving the first laser beam generated by the first laser and carrying out beam expanding and shaping on the first laser beam;
the dispersion compensation prism is used for receiving the first laser beam after the beam expansion and shaping of the first beam expansion and shaping lens group, and pre-correcting the spatial dispersion and the time dispersion of the first laser beam after the beam expansion and shaping;
the acousto-optic deflector is used for receiving the first laser beam after the pre-correction of the dispersion compensation prism and changing the deflection direction of the first laser beam after the pre-correction according to the wide-field fluorescent image to perform directional light stimulation on the sample.
The system for super-resolution imaging of directional light stimulus structure changes, wherein the system further comprises: an objective lens; the image generation module includes: the second beam expanding and shaping lens group, the first tube mirror, the first dichroic mirror, the narrow-band emission filter and the area array detector;
The second beam expanding and shaping lens group is used for receiving the second laser beam and the third laser beam generated by the second laser and carrying out beam expanding and shaping on the second laser beam and the third laser beam;
The first tube mirror and the first dichroic mirror are used for receiving the second laser beam after beam expansion and shaping by the second beam expansion and shaping lens group, guiding the second laser beam after beam expansion and shaping into the objective lens, and irradiating the sample in a parallel light mode to generate wide-field excitation; the third laser beam after beam expansion and shaping is guided into the objective lens and then irradiated onto a sample in a parallel light mode so that fluorescent molecules in the sample enter a scintillation state;
The objective lens is also used for receiving the first laser beam of which the deflection direction is changed by the acousto-optic deflector, converging the first laser beam onto the sample and collecting fluorescent signals generated by fluorescent molecules in the sample entering a scintillation state;
The narrow-band emission filter is used for receiving fluorescent signals generated when fluorescent molecules on the sample collected by the objective lens enter a scintillation state, and filtering crosstalk signals in the fluorescent signals;
The area array detector is used for collecting a wide-field fluorescent image and a fluorescent molecule scintillation image of the sample.
The system for super-resolution imaging of directional light stimulus structure changes, wherein the system further comprises: a third laser;
the third laser is used for generating a fourth laser beam.
The system for super-resolution imaging of the directional light stimulation structural change comprises an image generation module, a first reflecting mirror and a second dichroic mirror, wherein the image generation module comprises a first reflecting mirror and a second reflecting mirror;
the first reflecting mirror is used for receiving a fourth laser beam generated by the third laser and reflecting the fourth laser beam onto the second dichroic mirror;
The second dichroic mirror is used for receiving the fourth laser beam reflected by the first reflecting mirror, and irradiating the fourth laser beam and the third laser beam generated by the second laser onto the sample after combining the fourth laser beam and the third laser beam so that fluorescent molecules in a dark state in the sample enter a scintillation state.
The system for super-resolution imaging of directional light stimulus structure changes, wherein the directional light stimulus module further comprises: a second mirror, a third mirror, a second tube mirror, a third dichroic mirror;
the second reflecting mirror and the third reflecting mirror are used for receiving the first laser beam after the pre-correction of the dispersion compensating prism, adjusting the angle and the height of the first laser beam after the pre-correction, and then irradiating the first laser beam after the pre-correction onto the acousto-optic deflector;
The second tube mirror and the third dichroic mirror are used for receiving the first laser beam of which the deflection direction is changed by the acousto-optic deflector and irradiating the first laser beam of which the deflection direction is changed to the objective lens in a parallel light mode.
The system for super-resolution imaging of the directional light stimulation structural change comprises a control terminal, a second laser, a third laser, a fourth laser, a laser beam control system and a control system.
The control terminal is connected with the acousto-optic deflector and the area array detector, and is used for controlling the acousto-optic deflector to change the deflection direction of the first laser beam and controlling the area array detector to acquire the wide-field fluorescent image and the fluorescent molecule scintillation image.
A second embodiment of the present disclosure is a method of super-resolution imaging of directional light stimulus structural changes, wherein the method comprises:
Irradiating a second laser beam generated by a second laser onto the sample to generate a sample wide-field fluorescent image;
Irradiating a first laser beam generated by a first laser onto the sample according to the sample wide-field fluorescent image to perform directional light stimulation on the sample, and simultaneously irradiating a third laser beam generated by a second laser onto the sample to generate a fluorescent molecule scintillation image;
Reconstructing a super-resolution image of the sample according to the fluorescent molecular scintillation image.
The method for super-resolution imaging of directional light stimulation structural changes, wherein the step of irradiating the first laser beam generated by the first laser onto the sample according to the sample wide-field fluorescent image to perform directional light stimulation on the sample further comprises the following steps:
Selecting a target area needing to be subjected to optical stimulation from the sample wide-field fluorescent image;
And calculating the sound wave frequency corresponding to the pixel coordinates of the target area according to the corresponding relation between the pixel coordinates of the selected target area on the sample wide-field fluorescent image and the sound wave frequency.
The invention has the beneficial effects that the system and the method for super-resolution imaging of the directional light stimulation structure change are provided, and the directional light stimulation module based on the acousto-optic deflector is introduced into the super-resolution imaging system, so that the system and the method can be conveniently used for rapidly and accurately positioning and locally stimulating the sample, and can also be used for super-resolution imaging monitoring and comparison of nanoscale structure change of the light stimulation part and the non-light stimulation part. The directional light stimulated light source and the super-resolution imaging light source are mutually independent, and the femto-second laser is adopted, so that the emission wavelength is selected in the infrared band, the loss of the sample is minimized, and the spectrum crosstalk between the directional light stimulation and the super-resolution imaging is avoided.
Drawings
FIG. 1 is a schematic diagram of a system for super-resolution imaging of directional light stimulus structural changes provided by the present invention;
FIG. 2 is a schematic diagram of scanning addressing of an acousto-optic deflector in a system for super-resolution imaging of directional light stimulus structural changes provided by the present invention;
FIG. 3a is a schematic diagram of a selection procedure of a light stimulation module of the system for super-resolution imaging of directional light stimulation structural changes provided by the present invention;
FIG. 3b is a fluorescence image obtained after the system for performing super-resolution imaging on the structural change of directional light stimulation performs directional stimulation on the mugwort rhizome sample;
FIG. 4a is a wide-field fluorescence image obtained by performing wide-field excitation on mitochondria by the system for performing super-resolution imaging on the structural change of directional light stimulus provided by the invention;
Fig. 4b is a super-resolution image obtained by super-resolution imaging of mitochondria by the system for super-resolution imaging of directional light stimulus structure changes provided by the present invention;
Fig. 5 is a flowchart of a method for super-resolution imaging of a directional light stimulus structure change according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clear and clear, the present invention will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Because the existing light stimulation in the microscopic imaging technology for carrying out light stimulation on the sample does not have directional stimulation capability, the hologram and the stimulated emission depletion microscope are combined to carry out directional light stimulation on the sample, but high-intensity pulse laser is required to be used as depletion light when the stimulated emission depletion microscope carries out super-resolution imaging, so that the sample is easy to damage. In order to solve the above problems, the present invention provides a system for super-resolution imaging of directional light stimulus structural changes, as shown in fig. 1. The system of the invention comprises: a first laser 1, a second laser 2, an image generation module 3, a directional light stimulation module 4 and a control terminal; the image generating module 3 is configured to irradiate a second laser beam generated by the second laser 2 onto a sample 7 to generate a wide-field fluorescent image, and is configured to irradiate a third laser beam generated by the second laser 2 onto the sample 7 to generate a fluorescent molecule scintillation image when the directional light stimulating module 4 stimulates the sample 7 with directional light; the directional light stimulation module 4 is used for irradiating the first laser beam generated by the first laser 1 onto the sample 7 according to the wide-field fluorescent image to perform directional light stimulation on the sample 7; the control terminal is used for reconstructing a super-resolution image of the sample according to the fluorescent molecule scintillation image so as to monitor the nanoscale morphological change of the sample.
In particular, when monitoring the morphology of the nanoscale structures in a sample, the cells in the sample are labeled with fluorescent molecules. The fluorescent molecules used herein should have relatively high photon efficiency while being small in size, and also need to have a light-on characteristic or "scintillation" characteristic, i.e., the fluorescent molecules have on/off states under the same excitation conditions and can specifically bind to a specific structure of a sample. After labeling cells in the sample with fluorescent molecules, a second laser beam with lower power is irradiated onto the sample 7 by the second laser 2, and a wide-field fluorescent image of the sample 7 is acquired. And controlling the first laser beam generated by the first laser 1 to irradiate the sample 7 according to the wide-field fluorescent image of the sample 7, and carrying out directional light stimulation on the sample 7. And then the sample 7 is irradiated by a third laser beam with higher power through the second laser 2, so that fluorescent molecules in the sample 7 enter a scintillation state after being bleached, and a sparse luminescence effect is generated. And finally, collecting a fluorescence scintillation image of the sample, and reconstructing a super-resolution image of the sample after directional light stimulation according to the fluorescence scintillation image so as to monitor the morphological change of the nano-scale structure in the sample caused by light stimulation.
In a specific embodiment, the directional light stimulation module 4 includes a first beam expanding and shaping lens group 41, a dispersion compensating prism 42, and an acousto-optic deflector 45. The first beam expanding and shaping lens group 41 is configured to receive the first laser beam generated by the first laser 1, and to expand and shape the first laser beam; the dispersion compensation prism 42 is configured to receive the first laser beam after beam expansion and shaping by the first beam expansion and shaping lens group 41, and pre-correct the spatial dispersion and the temporal dispersion of the first laser beam after beam expansion and shaping; the acousto-optic deflector 45 is configured to receive the first laser beam pre-corrected by the dispersion compensation prism 42, and change a deflection direction of the pre-corrected first laser beam according to the wide-field fluorescent image to perform directional light stimulation on the sample 7. The dispersion compensation prism 42 performs dispersion compensation on the first laser beam, so as to improve the beam quality, and if the dispersion compensation prism 42 is absent, the first laser beam can cause divergence of a focusing light spot under the dispersion action of the acousto-optic deflector 45, so that the precision of directional light stimulation is affected and signal crosstalk is caused. The dispersion compensating prism 42 may be replaced by a prism pair or a grating pair or the like.
In a specific implementation, the acousto-optic deflector 45 is connected with the control terminal, after the wide-field fluorescent image acquired by the image acquisition module 3 is transmitted to the control terminal, a target area to be stimulated by light is selected on the acquired wide-field fluorescent image through software, and the target area can be single-point or multi-point, or can be single or multiple regular or irregular areas. Then, the obtained pixel coordinate information of the target area is converted into the sound wave frequency loaded on the acousto-optic deflector 45 by using software calibrated in advance, and the deflection direction of the first laser beam pre-calibrated by the dispersion compensation prism 42 can be quickly changed by changing the sound wave frequency loaded on the acousto-optic deflector 45, so that the position of the first laser beam converged on the sample 7 is changed, the sample structure corresponding to the target area is subjected to directional and local optical stimulation, and the aim of directional optical stimulation is fulfilled. Specifically, as shown in fig. 2, in this embodiment, the acousto-optic deflector 45 is a two-dimensional acousto-optic deflector formed by a pair of orthogonal acousto-optic deflectors, and deflection of incident light in two directions X, Y can be achieved by changing the acoustic wave frequencies of the acousto-optic deflectors loaded in two directions X, Y, so as to achieve addressing scanning of any point in the XY plane. The stimulation time and mode can be continuous stimulation or intermittent stimulation, the intensity can be kept unchanged, and the stimulation intensity can be changed, and the stimulation can be realized by setting corresponding parameters in advance on software.
In one embodiment, the system further comprises: an objective lens 6; the image generation module 3 comprises a second beam expanding and shaping lens group 31, a first tube mirror 32, a first dichroic mirror 33, a narrow band emission filter 34, an imaging lens 35 and an area array detector 36. The second beam expanding and shaping lens group 31 is configured to receive the second laser beam and the third laser beam generated by the second laser 2, and to expand and shape the second laser beam and the third laser beam; the first tube mirror 32 and the first dichroic mirror 33 are configured to receive the second laser beam after beam expansion and shaping by the second beam expansion and shaping lens group 31, guide the second laser beam after beam expansion and shaping into the objective lens 6, and irradiate the second laser beam onto the sample 7 in a parallel light mode to excite fluorescent molecules in the sample 7 in a wide field; and the second beam-expanding and shaping lens group 31 is used for receiving the third laser beam after beam expansion and shaping, and the third laser beam after beam expansion and shaping is led into the objective lens 6 and then irradiates the sample 7 in the form of parallel light so that fluorescent molecules in the sample 7 enter a scintillation state; the objective lens 6 is used for receiving the first laser beam of which the deflection direction is changed by the acousto-optic deflector 45 and converging the first laser beam onto the sample 7, and collecting a fluorescent signal generated by fluorescent molecules in the sample 7 entering a scintillation state, in addition to the first dichroic mirror 33 and the second laser beam led in by the objective lens 6 are irradiated onto the sample 7 in the form of parallel light; the narrow-band emission filter 34 is configured to receive a fluorescent signal generated when fluorescent molecules in the sample 7 collected by the objective lens 6 enter a scintillation state, and filter a crosstalk signal in the fluorescent signal; the area array detector 36 is used to collect a wide field fluorescence image and a fluorescent molecule scintillation image of the sample 7.
In specific implementation, when a wide-field fluorescent image of the sample 7 needs to be acquired, a second laser beam is generated by the second laser 2, and is irradiated onto the first tube mirror 32 after being expanded and shaped by the second beam expanding and shaping lens group 31, the second laser beam irradiates fluorescent molecules in the sample 7 in a form of parallel light on the sample 7 after passing through the first tube mirror 32, the first dichroic mirror 33 and the objective lens 6, and then the wide-field fluorescent image of the sample 7 is acquired by the area array detector 36. When a fluorescent molecule scintillation image of the sample 7 needs to be acquired, a third laser beam is generated by the second laser 2, the third laser beam is irradiated onto the first tube mirror 32 after being expanded and shaped by the second beam expanding and shaping lens group 31, the second laser beam is irradiated onto the sample 7 in a parallel light mode after passing through the first tube mirror 32, the first dichroic mirror 33 and the objective lens 6, so that fluorescent molecules in the sample 7 enter a scintillation state, then crosstalk signals in fluorescent signals generated when the fluorescent molecules in the sample 7 enter the scintillation state are filtered by the narrow-band emission filter 34, and the fluorescent molecule scintillation image is acquired by the area array detector 36 after passing through the imaging lens 35. The area array detector 36 is connected to the control terminal, and after the area array detector collects the wide-field fluorescent image and the fluorescent molecule scintillation image, the wide-field fluorescent image and the fluorescent molecule scintillation image are transmitted to the control terminal for processing. The area array detector 36 may be an area array detector with high sensitivity such as an electron multiplying CCD detector or an sCMOS camera.
In a specific embodiment, the system further comprises a third laser 5. The image generation module 3 further comprises a first mirror 37 and a second dichroic mirror 38. The third laser 5 is configured to generate a fourth laser beam to re-activate fluorescent molecules in a dark state in the sample 7 into a scintillation state; the first mirror 37 receives the fourth laser light beam generated by the third laser 4 and reflects the fourth laser light beam onto the second dichroic mirror 38; the second dichroic mirror 38 is configured to receive the fourth laser beam reflected by the first reflecting mirror 37, and irradiate the fourth laser beam onto the sample 7 after combining the fourth laser beam with the third laser beam generated by the second laser 2, so that fluorescent molecules in a dark state in the sample 7 are excited to enter a scintillation state.
In practice, when a fluorescent molecule is excited, the molecule transitions from a ground state to an excited state, fluorescence is generated when the molecule returns from the excited state to the ground state, and intersystem crossing may occur in the excited fluorescent molecule from a singlet state to a triplet state. If a transition of the fluorescent molecule from the singlet state to the triplet state occurs, the fluorescent molecule is in a non-fluorescent state, i.e. a dark state, since the fluorescent molecule no longer participates in the fluorescent emission during the residence time of the triplet state. In order to excite the fluorescent molecules in the dark state to enter the scintillation state again, in this embodiment, a third laser 5 is further provided in the system, the fourth laser beam generated by the third laser 5 is reflected by the first mirror 37 disposed at an angle of 45 ° and then is incident on the second dichroic mirror 38 disposed at an angle of 45 ° as well, and the third laser beam and the fourth laser beam generated by the second laser 2 are combined by the second dichroic mirror 38 and then are irradiated on the sample 7, so that the fluorescent molecules in the dark state in the sample 7 enter the scintillation state, thereby facilitating long-time fluorescent data acquisition.
In a specific embodiment, the directional light stimulation module 4 further comprises a second mirror 43 and a third mirror 44, a scanning lens 46, a second tube mirror 47, a third dichroic mirror 48. The second reflecting mirror 43 and the third reflecting mirror 44 are configured to receive the first laser beam after the pre-correction by the dispersion compensating prism 42, adjust the angle and the height of the first laser beam after the pre-correction, and irradiate the first laser beam after the pre-correction onto the acousto-optic deflector 45; the second tube mirror 47 and the third dichroic mirror 48 are configured to receive the first laser beam after the deflection direction of the acousto-optic deflector 45 is changed, and irradiate the first laser beam after the deflection direction is changed into the objective lens 6 in the form of parallel light. In specific implementation, the first laser beam generated by the first laser 1 changes the deflection direction through the acousto-optic deflector 45, irradiates onto the second tube mirror 47 through the scanning lens 46, irradiates onto the objective lens 6 in the form of parallel light after passing through the third dichroic mirror 48, and the objective lens 6 converges the first laser beam with changed deflection direction into a point to irradiate onto the sample 7.
In specific implementation, the first laser 1 is a pulse laser, and a sapphire femtosecond pulse laser with an output wavelength of 800nm can be selected as a light source for directional light stimulation. The second laser 2 is a continuous light laser, and can select lasers with proper wavelengths, such as continuous light lasers with 642nm, 561nm and 488nm, according to the absorption characteristics of the common fluorescent molecules, or can simultaneously use multiple wavelengths to increase the flexibility of the system, and in this embodiment, a continuous laser with 642nm output wavelength corresponding to the absorption wavelength of the fluorescent molecules is selected. The third laser is an ultraviolet continuous light laser, the wavelength of which is usually ultraviolet, such as a continuous light laser with an output wavelength of 405nm or 375nm, and whether the continuous light laser is needed or not can be selected according to the light-splitting mechanism of fluorescent molecules. The second laser 2 and the third laser 3 are connected to a control terminal for adjusting the power of the second laser beam, the third laser beam and the fourth laser beam generated by the second laser 2 and the third laser 3. When a wide-field fluorescent image of a sample needs to be acquired, the control terminal controls the second laser 2 to generate a second laser beam with lower power; when a fluorescent molecular scintillation image of the sample needs to be acquired, the control terminal controls the second laser 2 to generate a third laser beam with higher power. The power density of the third laser beam generated by the second laser 2 is determined according to the property of fluorescent molecules in the sample 7, and is generally not more than 300W/cm 2, so that the fluorescent molecules enter a scintillation state, sparse luminescence of the fluorescent molecules is realized, but meanwhile, too high excitation power density is required to damage the sample, so that the highest excitation power density which can be borne by the sample and the lowest excitation power density which can be brought into the scintillation state by a probe are generally required to be obtained in advance through pre-experiment, after the fluorescent molecules begin to flash, the area array detector 36 can be controlled to start to collect images, and enough frames of fluorescent molecule scintillation images are recorded. The acquisition rate of the area array detector 36 is typically 30-500Hz. The third laser 5 is controlled by a control program to switch and power according to the need, for some fluorescent molecules, when the fluorescent molecules enter a dark state, the low-power ultraviolet laser can make the fluorescent molecules re-activate to an excited state, which is beneficial to long-time data acquisition, but at the same time, the too high ultraviolet power can damage a coating film on optical devices such as a sample, an objective lens and the like, so that the power of the coating film needs to be strictly controlled.
In specific implementation, whether directional light stimulation and super-resolution imaging are synchronously performed can be determined according to experimental requirements, super-resolution imaging can be performed while directional light stimulation is selected, and super-resolution imaging is performed in the whole process of light stimulation after a target area needing light stimulation is selected, so that the change of a sample nano-scale structure in the whole process can be monitored in real time. Super-resolution imaging can also be performed at different stages of directional light stimulation, so that the method can be suitable for some samples which cannot be subjected to super-resolution imaging for a long time. Different monitoring modes can be set through software control.
In the specific implementation, after a plurality of frames of fluorescent molecule scintillation images are obtained, the fluorescent molecules obtained by positioning each frame of images are overlapped and reconstructed, and then the image with nanoscale super-resolution can be obtained. In the step, we can choose to perform super-resolution imaging only on a smaller area containing the light stimulation part, thereby being beneficial to improving the imaging rate, and can also choose to perform super-resolution imaging on a larger area, thereby being convenient to compare different nanoscale structural changes of the light stimulation part and the non-light stimulation part.
In one embodiment, the system further comprises a bright field illumination light path (not shown) for observing the sample, focusing and capturing a bright field map. After observing the sample aggregation by using the bright field illumination, the control program firstly controls the area array detector 36 to shoot a sample bright field image so as to acquire the outline information of the sample, then controls the second laser 2 to irradiate the sample 7 with lower power after turning off the bright field illumination, and shoots a wide-field fluorescent image by using the area array detector 36. Either the bright field image or the wide field fluorescence image can be used to select the target region for directional light stimulation.
The invention is illustrated in detail by means of the following specific examples: the inventor utilizes the system for carrying out super-resolution imaging on the directional light stimulation structural change to respectively carry out the directional light stimulation and the demonstration experiment of the super-resolution imaging on living cell mitochondria samples marked in a laboratory and the standard sample of the mugwort rhizome of Leica company, wherein the standard sample of the mugwort rhizome can be excited by 800nm pulse laser two-photon, so that the image can directly observe the illumination area of the pulse laser (namely the light stimulation area of the light stimulation experiment).
(1) Directional light stimulation:
Step 1: the first laser 1 is turned on and its wavelength is adjusted to 800nm. The second laser 2 is turned on, and the illumination intensity of the second laser 2 is controlled by a control program of the second laser 2 or an electric filter wheel (not shown in the figure) provided between the second beam expanding and shaping lens groups 31. The controller and module power to the area array detector 36 and the acousto-optic deflector 45 are turned on.
Step 2: the second laser 2 is controlled to reduce photobleaching as much as possible while exciting the sample in a broad field at a lower power. The control area array detector 36 captures a wide-field fluorescent image of the sample in real time, adjusts the objective lens 6 to focus the sample, adjusts the objective table to find a suitable cell area, and collects a wide-field fluorescent image of the sample for program selection of the area to be stimulated by light.
Step 3: the control program converts the coordinates of the selected area into the sound wave frequency of the input acousto-optic deflector 45, so that the incident pulse laser deflects as required, and finally, the incident pulse laser is converged at different positions of the sample through the objective lens 6, thereby realizing directional light stimulation. The results are shown in fig. 3a and 3b, where fig. 3a is a selection procedure of the optical stimulation module and fig. 3b is a fluorescence image recorded with the area array detector 36 after "optical stimulation" of the selected area (the demonstration sample used here is a stationary sample and thus does not actually respond to the stimulation of light). As can be seen from the results, only the selected region is excited by the pulse laser to emit fluorescence, and the visible light stimulation module can conveniently select a specific region and perform optical stimulation of the corresponding region by controlling the acousto-optic deflector 45.
(2) Super-resolution imaging:
Step 1: the sample area is kept not to move, the intensities of the second laser 2 and the third laser 3 are controlled by a control program, the sample is excited in a parallel illumination mode, fluorescent molecules are made to emit light sparsely, and the area array detector 36 is controlled to acquire fluorescent molecule scintillation images, and the result is shown in fig. 4 a.
Step 2: post-processing is performed on fluorescent molecular scintillation images before and after optical stimulation to reconstruct a super-resolution image of the sample, and the result is shown in fig. 4 b.
In a specific embodiment, the present invention further provides a method for super-resolution imaging of a directional light stimulus structure change, as shown in fig. 5, where the method includes the steps of:
S1, irradiating a second laser beam generated by a second laser onto a sample to generate a sample wide-field fluorescent image;
S2, irradiating a first laser beam generated by a first laser onto the sample according to the sample wide-field fluorescent image to perform directional light stimulation on the sample, and simultaneously irradiating a third laser beam generated by a second laser onto the sample to generate a fluorescent molecule scintillation image;
s3, reconstructing a super-resolution image of the sample according to the fluorescent molecule scintillation image.
In a specific embodiment, the step S2 further includes the step of:
r1, selecting a target area needing to be subjected to optical stimulation from the sample wide-field fluorescent image;
r2, calculating the sound wave frequency corresponding to the pixel coordinates of the target area according to the corresponding relation between the pixel coordinates of the selected target area on the sample wide-field fluorescent image and the sound wave frequency.
In summary, the present invention provides a system and a method for super-resolution imaging of directional light stimulus structure changes, where the system includes: the system comprises a first laser, a second laser, an image generation module, a directional light stimulation module and a control terminal. According to the invention, the directional optical stimulation module based on the acousto-optic deflector is introduced into the super-resolution imaging system, so that the method can be conveniently used for rapidly and accurately positioning and locally stimulating the sample, and can also be used for monitoring and comparing the super-resolution imaging of nanoscale structural changes of the optical stimulation part and the non-optical stimulation part. The directional light stimulated light source and the super-resolution imaging light source are mutually independent, and the femto-second laser is adopted, so that the emission wavelength is selected in the infrared band, the loss of the sample is minimized, and the spectrum crosstalk between the directional light stimulation and the super-resolution imaging is avoided.
It is to be understood that the system application of the present invention is not limited to the examples described above, and that modifications and variations may be made by those skilled in the art in light of the above teachings, all of which are intended to be within the scope of the invention as defined in the appended claims.
Claims (6)
1. A system for super-resolution imaging of directional light stimulus structural changes, the system comprising: the system comprises a first laser, a second laser, an image generation module, a directional light stimulation module and a control terminal;
the image generation module is used for irradiating a second laser beam generated by the second laser to a sample to generate a wide-field fluorescent image, and is used for irradiating a third laser beam generated by the second laser to the sample to generate a fluorescent molecule scintillation image when the directional light stimulation module performs directional light stimulation on the sample;
The directional light stimulation module is used for irradiating a first laser beam generated by the first laser onto the sample according to the wide-field fluorescent image to perform directional light stimulation on the sample;
the control terminal is used for reconstructing a super-resolution image of the sample according to the fluorescent molecule scintillation image;
the directional light stimulation module includes: a first beam expanding and shaping lens group, a dispersion compensating prism and an acousto-optic deflector;
the first beam expanding and shaping lens group is used for receiving the first laser beam generated by the first laser and carrying out beam expanding and shaping on the first laser beam;
the dispersion compensation prism is used for receiving the first laser beam after the beam expansion and shaping of the first beam expansion and shaping lens group, and pre-correcting the spatial dispersion and the time dispersion of the first laser beam after the beam expansion and shaping;
the acousto-optic deflector is used for receiving the first laser beam after the pre-correction of the dispersion compensation prism and changing the deflection direction of the first laser beam after the pre-correction according to the wide-field fluorescent image to perform directional light stimulation on the sample;
The image generation module further includes a first mirror and a second dichroic mirror;
the system further comprises: a third laser for generating a fourth laser beam; the first reflecting mirror is used for receiving a fourth laser beam generated by the third laser and reflecting the fourth laser beam onto the second dichroic mirror;
The second dichroic mirror is used for receiving the fourth laser beam reflected by the first reflecting mirror, and irradiating the fourth laser beam and the third laser beam generated by the second laser onto the sample after combining the fourth laser beam and the third laser beam so that fluorescent molecules in a dark state in the sample enter a scintillation state;
The system for super-resolution imaging of the directional light stimulation structural change is characterized in that the directional light stimulation module further comprises: a second mirror, a third mirror, a second tube mirror, a third dichroic mirror;
the second reflecting mirror and the third reflecting mirror are used for receiving the first laser beam after the pre-correction of the dispersion compensating prism, adjusting the angle and the height of the first laser beam after the pre-correction, and then irradiating the first laser beam after the pre-correction onto the acousto-optic deflector;
The second tube mirror and the third dichroic mirror are used for receiving the first laser beam of which the deflection direction is changed by the acousto-optic deflector and irradiating the first laser beam of which the deflection direction is changed to an objective lens in a parallel light mode.
2. The system for super-resolution imaging of a directional light stimulus texture variation of claim 1, wherein the image generation module comprises: the second beam expanding and shaping lens group, the first tube mirror, the first dichroic mirror, the narrow-band emission filter and the area array detector;
The second beam expanding and shaping lens group is used for receiving the second laser beam and the third laser beam generated by the second laser and carrying out beam expanding and shaping on the second laser beam and the third laser beam;
The first tube mirror and the first dichroic mirror are used for receiving the second laser beam after beam expansion and shaping by the second beam expansion and shaping lens group, guiding the second laser beam after beam expansion and shaping into the objective lens, and irradiating the sample in a parallel light mode to generate wide-field excitation; the third laser beam after beam expansion and shaping is guided into the objective lens and then irradiated onto a sample in a parallel light mode so that fluorescent molecules in the sample enter a scintillation state;
The objective lens is also used for receiving the first laser beam of which the deflection direction is changed by the acousto-optic deflector, converging the first laser beam onto the sample and collecting fluorescent signals generated by fluorescent molecules in the sample entering a scintillation state;
The narrow-band emission filter is used for receiving fluorescent signals generated when fluorescent molecules on the sample collected by the objective lens enter a scintillation state, and filtering crosstalk signals in the fluorescent signals;
The area array detector is used for collecting a wide-field fluorescent image and a fluorescent molecule scintillation image of the sample.
3. The system for super-resolution imaging of a directional optical stimulus as recited in claim 1 wherein said control terminal is coupled to said second and third lasers, said control terminal being configured to adjust the power of said second, third and fourth laser beams produced by said second and third lasers.
4. The system for super-resolution imaging of a change in a directional optical stimulus structure of claim 2, wherein the control terminal is connected to the acousto-optic deflector and the area array detector, and the control terminal is configured to control the acousto-optic deflector to change a deflection direction of the first laser beam and to control the area array detector to acquire the wide-field fluorescent image and the fluorescent molecular scintillation image.
5. A method of super-resolution imaging of directional light stimulus texture variations based on the system of super-resolution imaging of directional light stimulus texture variations of any one of claims 1-4, the method comprising:
Irradiating a second laser beam generated by a second laser onto the sample to generate a sample wide-field fluorescent image;
Irradiating a first laser beam generated by a first laser onto the sample according to the sample wide-field fluorescent image to perform directional light stimulation on the sample, and simultaneously irradiating a third laser beam generated by a second laser onto the sample to generate a fluorescent molecule scintillation image;
Reconstructing a super-resolution image of the sample according to the fluorescent molecular scintillation image.
6. The method of super-resolution imaging of a change in a directional optical stimulus structure of claim 5, wherein said step of directing a first laser beam generated by a first laser onto said sample from said sample wide field fluorescence image is preceded by the step of:
Selecting a target area needing to be subjected to optical stimulation from the sample wide-field fluorescent image;
And calculating the sound wave frequency corresponding to the pixel coordinates of the target area according to the corresponding relation between the pixel coordinates of the selected target area on the sample wide-field fluorescent image and the sound wave frequency.
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