CN210834671U - Addressing scanning rapid fluorescence lifetime microscopic imaging system - Google Patents
Addressing scanning rapid fluorescence lifetime microscopic imaging system Download PDFInfo
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- CN210834671U CN210834671U CN201921722247.9U CN201921722247U CN210834671U CN 210834671 U CN210834671 U CN 210834671U CN 201921722247 U CN201921722247 U CN 201921722247U CN 210834671 U CN210834671 U CN 210834671U
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Abstract
The utility model provides a quick fluorescence life microscopic imaging system of addressing scanning, the system includes: the device comprises an illumination light source, a laser, an image generation unit, a data acquisition card, a fluorescence signal generation unit, a fluorescence life acquisition unit and a control terminal. The utility model discloses a pixel coordinate output digital signal and three routes synchronizing signal of target area, through the acoustic wave frequency that corresponds to the acoustic-optic deflector loading digital signal, can make the acoustic-optic deflector deflect the light beam to the sample assigned position fast, realize quick nimble addressing scanning formation of image; the three paths of synchronous signals control the time-dependent single photon counting acquisition card to synchronously store fluorescence lifetime data, and the pixel coordinates of the target area and the external rectangle thereof are utilized to correct the fluorescence lifetime image, so that the addressing scanning rapid fluorescence lifetime imaging of the target area in any shape is realized.
Description
Technical Field
The utility model belongs to the technical field of the optical microscopy, especially, relate to a quick fluorescence life-span microscopic imaging system of addressing scanning.
Background
Since the invention of the microscope, the application of the optical microscopic imaging technology in the biomedical research field is increasingly widespread. Among the optical microscopy imaging technologies, the fluorescence lifetime microscopy (FLIM) technology has recently become favored by researchers. This technique typically employs exogenous fluorescent probes to specifically label the sample and obtain structural and functional information from the sample by probing the fluorescent lifetimes of the probe molecules. The fluorescence lifetime is not affected by the concentration of the probe, the intensity of the excitation light, the photobleaching and other factors during measurement, and can very sensitively reflect the change of the microenvironment where the probe is located, so that the fluorescence lifetime is commonly used for quantitatively measuring a plurality of physical and biochemical parameters in the microenvironment of a sample, such as viscosity, temperature, ion concentration, pH value and the like.
At present, a fluorescence microscopic imaging system for monitoring the fluorescence lifetime adopts a galvanometer scanning technology, the control mode is simple, generally, a grid scanning (sequential scanning) mode is adopted, but mechanical inertia exists during scanning, and the fluorescence microscopic imaging system cannot rapidly move from one point to another point on a sample, so that the imaging speed of the fluorescence lifetime is limited.
Therefore, the prior art is subject to further improvement.
SUMMERY OF THE UTILITY MODEL
In view of the foregoing deficiencies in the prior art, an object of the present invention is to provide a rapid fluorescence lifetime microscopy imaging system for addressing scanning, which overcomes the disadvantages of the prior art that the fluorescence microscopy imaging system adopts a galvanometer scanning technique, has mechanical inertia during scanning, cannot rapidly move to another point from a sample, and has a slow fluorescence lifetime imaging.
The utility model discloses a first embodiment is a microscopic imaging system of quick fluorescence life of addressing scanning, wherein, the system includes:
an illumination source and a laser;
the image generating unit is used for irradiating the illumination light beam generated by the illumination light source to the sample surface to generate a sample bright field map or a fluorescence map;
the data acquisition card is used for outputting a digital signal and three paths of synchronous signals according to the pixel coordinates of the selected target area on the sample bright field diagram or the fluorescence diagram;
the fluorescence signal generating unit is used for projecting the laser beam generated by the laser onto a sample surface according to the digital signal and exciting the target area to generate a fluorescence signal;
the fluorescence life acquisition unit is used for acquiring and processing the fluorescence signals to obtain fluorescence life data of the target area and storing the fluorescence life data into a preset life image file according to the three paths of synchronous signals;
and the control terminal is used for generating a distorted fluorescence life image of the target area according to the fluorescence life data, and correcting the distorted fluorescence life image according to the target area and the coordinates of the rectangular pixels externally connected with the target area to obtain the fluorescence life image of the target area.
The addressing scanning rapid fluorescence lifetime microscopic imaging system comprises a fluorescence signal generating unit and a fluorescence signal generating unit, wherein the fluorescence signal generating unit comprises:
the dispersion compensation prism is used for receiving the laser beam generated by the laser and pre-correcting the spatial dispersion and the time dispersion of the laser beam;
and the acousto-optic deflector is used for receiving the laser beam after being pre-corrected, projecting the laser beam after being pre-corrected onto a sample surface according to the digital signal and exciting the target area to generate a fluorescence signal.
The addressing scanning rapid fluorescence lifetime microscopic imaging system comprises a fluorescence lifetime acquisition unit and a fluorescence lifetime detection unit, wherein the fluorescence lifetime detection unit comprises:
the photomultiplier is used for collecting the fluorescence signal and converting the fluorescence signal into a corresponding electric signal;
and the time-dependent single photon counting acquisition card is used for processing the electric signals converted by the photomultiplier to obtain fluorescence life data and storing the fluorescence life data into a preset life image file according to the three paths of synchronous signals.
The addressing scanning rapid fluorescence lifetime microscopic imaging system is characterized in that the data acquisition card is simultaneously connected with the time-dependent single photon counting acquisition card and the acousto-optic deflector through cables.
The addressing scanning rapid fluorescence lifetime microscopic imaging system is characterized in that the control terminal is simultaneously connected with the data acquisition card, the acousto-optic deflector and the time-dependent single photon counting acquisition card.
The addressing scanning rapid fluorescence lifetime microscopic imaging system, wherein the fluorescence signal generating unit further comprises:
the half-wave plate and the polarization beam splitting prism are used for receiving the laser beam generated by the laser and adjusting the power of the laser beam;
the first beam expanding and shaping lens pair is used for receiving the laser beam after power adjustment and carrying out beam expanding and shaping on the received laser beam;
and the second beam expanding and shaping lens pair is used for receiving the laser beam emitted by the acousto-optic deflector and expanding and shaping the laser beam.
The addressing scanning rapid fluorescence lifetime microscopic imaging system comprises a fluorescence lifetime acquisition unit, a fluorescence lifetime acquisition unit and a fluorescence lifetime detection unit, wherein the fluorescence lifetime acquisition unit comprises:
and the second optical filter is used for receiving the fluorescence signal and filtering the fluorescence signal.
The addressing scanning rapid fluorescence lifetime microscopic imaging system, wherein the system further comprises:
the beam splitter is used for splitting the fluorescence signal output by the second optical filter;
and the image recording module is used for receiving the fluorescence signal split by the beam splitter and recording a fluorescence intensity image of the fluorescence signal.
The addressing scanning rapid fluorescence lifetime microscopic imaging system comprises an image recording module and a scanning module, wherein the image recording module comprises:
the third reflector is used for receiving the fluorescence signal split by the beam splitter and reflecting the fluorescence signal;
and the area array detector is used for receiving the fluorescence signals reflected by the third reflector and recording fluorescence intensity images of the fluorescence signals.
The addressing scanning rapid fluorescence lifetime microscopic imaging system is characterized in that the illumination light source is one of a halogen lamp, an LED lamp, a mercury lamp or a xenon lamp; the laser is a femtosecond pulse laser.
The beneficial effects are that, the utility model provides an addressing scanning fast fluorescence life microscopic imaging system, through the pixel coordinate output digital signal and three routes synchronizing signal of target area, through the acoustic wave frequency that the loading digital signal corresponds to the acousto-optic deflector, can make the acousto-optic deflector deflect the light beam to the sample assigned position fast, realize the quick nimble addressing scanning imaging; the three paths of synchronous signals control the time-dependent single photon counting acquisition card to synchronously store fluorescence life data, and pixel coordinates of the target area and an external rectangle of the target area are used for correcting a fluorescence life image, so that the addressing scanning rapid fluorescence life imaging of the target area in any shape is realized.
Drawings
Fig. 1 is a schematic structural diagram of an addressing scanning fast fluorescence lifetime microscopy imaging system provided by the present invention;
FIG. 2 is a schematic diagram of the digital signal and three-way synchronization signal generation for regular shape target area addressing scanning fluorescence lifetime imaging provided by the present invention;
FIG. 3 is a schematic diagram of the selected region shape analysis and three-way synchronization signal generation for irregular-shaped target region address scan fluorescence lifetime imaging provided by the present invention;
FIG. 4 is a schematic diagram of the control terminal correcting a distorted fluorescence lifetime image of a single irregular target area or a plurality of irregular target areas that continuously change in the Y direction;
FIG. 5 is a schematic diagram of the control terminal correcting distorted fluorescent lifetime images of a plurality of irregular target areas that vary discontinuously in the Y-direction;
fig. 6 is a fluorescence lifetime image obtained by the addressing scanning rapid fluorescence lifetime microscopic imaging system provided by the utility model measuring the lily of the valley rhizome standard sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention will be described in further detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Because the fluorescence microscopic imaging system in the prior art adopts a galvanometer scanning technology, mechanical inertia exists during scanning, and the fluorescence microscopic imaging system cannot rapidly move from one point to another point on a sample, so that the imaging speed of the fluorescence life is limited. In order to solve the above problem, the present invention provides an addressing scanning fast fluorescence lifetime microscopic imaging system, as shown in fig. 1. The utility model discloses a system includes: an illumination light source 1 and a laser 2; an image generating unit 3 for irradiating the illumination beam generated by the illumination light source 1 onto a sample surface 9 to generate a sample bright field map or a fluorescence map; the data acquisition card 6 is used for outputting a digital signal and three paths of synchronous signals according to the pixel coordinates of the selected target area on the sample bright field diagram or the fluorescence diagram; the fluorescence signal generating unit 4 is used for projecting the laser beam generated by the laser 2 onto a sample surface 9 according to the digital signal and exciting the target area to generate a fluorescence signal; a fluorescence lifetime collecting unit 5 for collecting and processing the fluorescence signal to obtain fluorescence lifetime data of the target area, and storing the fluorescence lifetime data in a preset lifetime image file according to the three paths of synchronous signals; and the control terminal 7 is used for generating a distorted fluorescence lifetime image of the target area according to the fluorescence lifetime data, and correcting the distorted fluorescence lifetime image according to the target area and the coordinates of the rectangular pixels externally connected with the target area to obtain the fluorescence lifetime image of the target area.
In the specific implementation process, when a fluorescence lifetime image of a sample to be detected needs to be acquired, the sample to be detected is placed on the sample stage, and an illumination light beam generated by the illumination light source 1 is received by the image generation unit 2 and is irradiated on the sample surface 9 to generate a sample bright field image or a fluorescence image. Then, a bright field image or a fluorescence image of the sample is shot, the shot image is stored in the control terminal 7 to select a target area needing fluorescence imaging, the control terminal 7 can automatically acquire the pixel coordinates of the target area according to the target area selected by a user, and the data acquisition card 6 is controlled to output corresponding digital signals and three paths of synchronous signals according to the pixel coordinates of the target area. Then, the data acquisition card 6 transmits the digital signal to the fluorescence signal generating unit 4, and the fluorescence signal generating unit 4 projects the laser beam generated by the laser 2 to a target area on the sample surface 9 according to the received digital information, so as to excite the target area to generate a fluorescence signal. Meanwhile, the data acquisition card 6 transmits the three paths of synchronous signals to the fluorescence life acquisition unit 5, and after the fluorescence life acquisition unit 5 acquires and processes the fluorescence signals to obtain fluorescence life data of the target area, the fluorescence life data of the target area is stored in a preset life image file according to the three paths of synchronous signals. And finally, the control terminal 7 reads fluorescence life data in the life image file, obtains a fluorescence life value of each pixel of the target area through negative exponential fitting, generates a distorted fluorescence life image of the target area, and corrects the distorted fluorescence life image to obtain a correct fluorescence life image of the target area. The utility model scans the target area according to the pixel coordinate of the target area, and has accurate positioning and high scanning speed; the three paths of synchronous signals generated according to the shape analysis of the target area store the life data and correct the generated fluorescence life image, so that the fluorescence life imaging of any shape and any shape selected area can be realized, and the imaging speed is high.
In one embodiment, when the illumination light source 1 is a halogen lamp or an LED lamp, the image generation unit 3 generates a sample bright field map; when the illumination light source 1 is a mercury lamp or a xenon lamp, the image generation unit 3 generates a wide-field fluorescence map. The image generating unit 3 comprises a first condenser lens 31 for receiving the illumination light beam generated by the illumination light source 1 and condensing the illumination light beam; a first reflecting mirror 32 for receiving the illumination light beam collected by the first condenser lens 31 and reflecting the illumination light beam; a second condenser lens 33 for receiving the illumination beam reflected by the first reflection surface 32 and condensing the illumination beam; a first filter 34 for receiving the illumination beam collected by the second condenser 33 and filtering the illumination beam. In specific implementation, an illumination beam generated by the illumination light source 1 is condensed by the first condenser 31 and then irradiated onto the surface of the first reflector 32, is reflected by the first reflector 32 onto the second condenser 33 to be condensed and then irradiated onto the first filter 34, and is filtered by the first filter 34 and then irradiated onto the sample surface 9 to generate a sample bright field image or a wide field fluorescence image.
In a specific embodiment, after the bright field map or the wide field fluorescence map of the sample is acquired, the bright field map or the wide field fluorescence map of the sample can be generated by shooting with a CCD detector, a CMOS camera or other area array detectors with higher sensitivity. In a specific embodiment, a CCD detector is used when a sample bright field image or a fluorescence image is captured, and if the sample bright field image is obtained, the CCD exposure time is not too high, and is generally controlled to be about 10ms, so as to avoid overexposure.
In a specific embodiment, after the shooting is finished, the obtained picture is stored in the control terminal 7, and then a target region to be subjected to fluorescence lifetime imaging can be manually selected by a user, wherein the shape of the target region can be a regular shape (such as a rectangle), or a preset specific irregular shape (such as a ring shape, a fan shape, and the like) or any other irregular shape defined by the user; of course, the target area may be a continuous area or a plurality of discontinuous areas. After the target area is selected, the control terminal 7 automatically positions and stores pixel coordinate information of the target area, and simultaneously automatically analyzes the shape of the target area to generate an external rectangle and store the pixel coordinate information of the external rectangle, in a specific embodiment, the pixel coordinate information is arranged according to the sequence that X coordinates are sequentially increased and then Y coordinates are sequentially increased, the stored pixel coordinate information is used for outputting a digital signal and three paths of synchronous signals by a subsequent data acquisition card 6, and the stored pixel coordinate information of the external rectangle is used for subsequently correcting a distorted fluorescence life image.
In one embodiment, the laser 2 is a femtosecond pulsed laser, and in one embodiment is a tunable sapphire femtosecond pulsed laser. The fluorescence signal generating unit 4 comprises a half-wave plate 41 and a polarization beam splitter prism 42, wherein the half-wave plate 41 is used for receiving the laser beam generated by the laser 2 and adjusting the power of the laser beam; a second mirror 43 for reflecting the received laser beam; a first beam expanding and shaping lens pair 44 for receiving the laser beam reflected by the second reflecting mirror 43 and performing beam expanding and shaping on the laser beam; a dispersion compensation prism 45 for receiving the laser beam expanded and shaped by the first expanded beam shaping lens pair 44 and correcting the spatial dispersion and the temporal dispersion of the laser beam; an acousto-optic deflector (AOD)46 for receiving the laser beam corrected by the dispersion compensation prism 45 and deflecting the laser beam according to the digital signal output by the data acquisition card 6; a second beam expanding and shaping lens pair 47 for receiving the laser beam emitted from the acousto-optic deflector (AOD)46 and performing beam expanding and shaping on the laser beam; a dichroic mirror 48 for receiving the laser beam expanded and shaped by the second expanded beam shaping lens pair 47 and reflecting the laser beam to the sample surface 9; and an objective lens 49 for receiving the laser beam reflected by the dichroic mirror 48, focusing and irradiating the laser beam on the sample surface 9 to excite the sample at the focal point to generate fluorescence, and collecting the fluorescence signal generated at the focal point of the sample surface 9. In specific implementation, a laser beam emitted by the laser 2 is subjected to power adjustment through the half-wave plate 41 and the polarization beam splitter prism 42, then reflected to the first beam expanding and shaping lens 44 through the reflector 43 for beam expanding and shaping, and then subjected to correction of spatial dispersion and temporal dispersion through the dispersion compensation prism 45, so as to improve the subsequent fluorescence imaging quality. The corrected laser beam is raised by the two reflectors 43 and then irradiated into a pair of acousto-optic deflectors (AODs) 46 which are orthogonally arranged, and after being expanded and shaped by a second expanded beam shaping lens 47, the laser beam is irradiated onto the sample surface 9 by a dichroic mirror 48 to generate a fluorescent signal.
In an embodiment, the control terminal 7 is connected to the data acquisition card 6 by a cable, and the linear correspondence between the pixel coordinate and the acoustic frequency is pre-stored in the control terminal 7. After the pixel coordinates of the target area are obtained by the control terminal 7, the acoustic wave frequency of each pixel coordinate in the target area is calculated one by one according to the linear corresponding relation between the pixel coordinates and the acoustic wave frequency, and the acoustic wave frequency corresponding to each pixel coordinate is transmitted to the data acquisition card 6. The data acquisition card 6 outputs a corresponding digital signal according to each pixel coordinate, and converts the digital signal into a corresponding acoustic signal to be loaded onto an acousto-optic deflector (AOD)46 to scan according to the pixel coordinate of the target area. In one embodiment, the digital signal output by the data acquisition card 6 is a 32-bit digital signal, wherein the low 16-bit digital signal controls the scanning of the acousto-optic deflector (AOD)46 in the X direction, and the high 16-bit digital signal controls the scanning of the acousto-optic deflector (AOD)46 in the Y direction. Each pixel location corresponds to a particular acoustic frequency, i.e., to a particular digital signal, so that the AOD can rapidly deflect the beam to a specified location on the sample by outputting the corresponding 32-bit digital signal.
In a specific embodiment, the fluorescence lifetime collecting unit 5 includes a second optical filter 51 for receiving and filtering the fluorescence signal; a photo-electric multiplier-timer (PMT)53 for receiving the fluorescence signal filtered by the second filter 51 and converting the received fluorescence signal into an electrical signal; and a time-dependent single photon counting acquisition card (tcspcard) 54 for processing the electrical signal converted by the photoelectric multiplying and intensifying (PMT)53 to obtain fluorescence lifetime data of the target region, and storing the fluorescence lifetime data in a preset lifetime image file according to the three-way synchronization signal. In specific implementation, the photo multiplying and light amplifying (PMT)53 is connected to a time dependent single photon counting and collecting card (tcspcard) 54 through a cable, a fluorescence signal generated in a target area passes through an objective lens 49 and a dichroic mirror 48, then irradiates onto a second optical filter 51 for filtering, is collected and converted into an electrical signal through the photo multiplying and light amplifying (PMT)53, and the electrical signal is processed by the time dependent single photon counting and collecting card (tcspcard) 54 to obtain a time-dependent distribution situation of fluorescence photons of each pixel in the target area, i.e., photon number-time distribution, which is referred to as "fluorescence lifetime information" for short. Of course, in this embodiment, the photomultiplier tube (PMT)53 may be replaced with a spot detector such as an Avalanche Photo Diode (APD).
In a specific embodiment, the addressing scanning rapid fluorescence lifetime microscopy imaging system further comprises a beam splitter 10 for splitting the fluorescence signal output by the second optical filter 51; part of the fluorescence signal split by the beam splitter 10 is used for acquiring fluorescence lifetime information by the fluorescence lifetime acquisition unit 5, and the other part is recorded by the image recording module 8. The image recording module 8 includes a third reflector 81 for receiving the fluorescence signal split by the beam splitter 10 and reflecting the fluorescence signal; a lens 83 for receiving the fluorescence signal reflected by the third mirror 81 and converging the fluorescence signal; and an area array detector 82 for receiving the fluorescence signal converged by the lens 83 and recording a fluorescence intensity image of the fluorescence signal. In specific implementation, the fluorescence signal filtered by the second optical filter 51 is split into two beams by the beam splitter 10, one beam irradiates the photoelectric multiplier Photo (PMT)53, and the other beam irradiates the third reflector 81 and is detected by the area array detector 82, and the fluorescence signal intensity is recorded for verifying the accuracy of the fluorescence lifetime data stored by the time-dependent single photon counting acquisition card 54. Of course, similar to the area array detector used in the previous step of taking the bright field pattern or the fluorescence pattern of the sample, the area array detector 82 in this embodiment may be a CCD detector, or may be a CMOS camera or other area array detector with higher sensitivity. The invention can use two area array detectors, one for shooting the bright field image or fluorescence image of the sample and the other for recording the fluorescence signal intensity. The area array detector 82 in the image recording module 8 can also be used to shoot the bright field pattern or the fluorescence pattern of the sample and record the fluorescence signal intensity, and the above embodiments are all within the protection scope of the present invention.
In one embodiment, when the area array detector 82 is used to capture a bright field image or a fluorescence image of a sample, the microscope objective 49 needs to be focused before capturing so that the structure of the sample is clear. The image generating unit 2 generates a sample bright field image or a wide field fluorescence image, the beam splitter 10 is set to be in an idle state through the objective lens 49 in the fluorescence signal generating unit 4, the dichroic mirror 48 and the second optical filter 51 in the fluorescence life collecting unit 5, the recording module 8 shoots the sample and stores the shot image in the control terminal 7. When the illumination light source 1 is a halogen lamp or an LED lamp, the dichroic mirror 48 and the optical filter 51 are set to be in an empty state for recording a sample bright field pattern; when the illumination light source 1 is a mercury lamp or a xenon lamp, the dichroic mirror 48 and the second filter 51 select an appropriate wavelength band according to the emission wavelength of fluorescence excited by the sample, and the appropriate wavelength band is used for recording a wide-field fluorescence map.
In a specific embodiment, the data acquisition card 6 outputs a digital signal according to the acquired pixel coordinates of the target area, and outputs three corresponding synchronous signals according to the pixel coordinates of the target area. The three paths of synchronous signals comprise pixel signals, line signals and frame signals, and the three paths of synchronous signals are analog pulse signals. The line signal and the frame signal are only output when the number of the pixel signals reaches a certain number, the occurrence of the line signal depends on the number of pixels of each line in the target area, the frame signal depends on the total number of pixels of the selected area, and the rising edge positions of the multiple output signals are consistent when the multiple output signals occur simultaneously. The data acquisition card 6 is connected with the time-dependent single photon counting acquisition card (tcspcard) 54 and the acousto-optic deflector (AOD)46 through cables, and simultaneously outputs three paths of synchronous signals and digital signals to the time-dependent single photon counting acquisition card (tcspcard) 54 and the acousto-optic deflector (AOD)46 respectively for synchronization between the two.
In a specific implementation, when the acousto-optic deflector (AOD)46 scans the first pixel of each frame, the data acquisition card 6 simultaneously outputs a pixel signal, a line signal and a frame signal, when the acousto-optic deflector (AOD)46 scans the first pixel of each frame, the data acquisition card 6 simultaneously outputs a pixel signal and a line signal, when the acousto-optic deflector (AOD)46 scans the first pixel of each frame, the data acquisition card 6 only outputs a pixel signal, as shown in fig. 2, the left side is a rectangular target area with a pixel size of 5 × 2, and the right side is three corresponding synchronous signals, when the acousto-optic deflector (AOD)46 scans other pixels of each frame, i.e., the first pixel of each row, the data acquisition card 6 simultaneously outputs a pixel signal, a line signal and a frame signal, then the acousto-optic deflector (AOD)46 scans the second pixel to the fifth pixel of the first row, the acousto-optic deflector (AOD)46 starts scanning the first pixel of the first row, the acousto-optic deflector (AOD) 46) simultaneously outputs a pixel signal, the acousto-optic deflector (AOD) 46) scans the first pixel of the second row to the fifth pixel of the fifth pixel, and the acousto-optic deflector (AOD) outputs a pixel scanning pixel, and the data acquisition card data scanning cycle, and the acousto-optic deflector (AOD) outputs a data scanning signal, and the data acquisition card 46 repeatedly.
In a specific embodiment, the control terminal 7 is connected to the data acquisition card 6, the acousto-optic deflector 46, the time-dependent single photon counting acquisition card 54, and the area array detector 82 for capturing the bright field pattern and the fluorescence pattern of the sample, and is used for controlling the synchronous signal output, the addressing scanning, the fluorescence signal output, the fluorescence lifetime data storage, and the like.
In a specific embodiment, the control terminal 7 creates a lifetime image file with size N × M according to the pixel coordinates of the target area, the time-dependent single photon counting and collecting card 54 processes the electrical signal to obtain fluorescence lifetime data, and stores the fluorescence lifetime data in the lifetime image file according to the three synchronous signals output by the data collecting card 6, that is, the three synchronous signals, i.e., pixel, line, and frame, respectively control the switching between pixel and pixel, line and line, and frame when the fluorescence lifetime data is stored in the lifetime image file, for example, as illustrated in fig. 2, the control terminal 7 creates a lifetime image file with size 5 × 2 according to a rectangular target area with size 5 × 2 pixel, when the data collecting card 6 simultaneously outputs the pixel signal, the line signal, and the frame signal, the time-dependent single photon counting and collecting card 54 stores the obtained fluorescence lifetime data in the first pixel in the lifetime image file, then the data collecting card 6 outputs only the pixel signal, the time-dependent single photon counting and collecting card 54 sequentially stores the obtained fluorescence lifetime data in the same row as the other pixels in the same row, and then switches the fluorescence lifetime data to the next row data of the fluorescence lifetime image file, thereby realizing the three fluorescent lifetime data storage by the three fluorescent lifetime image file.
In one embodiment, the fluorescence lifetime value requires a sufficient number of fluorescence lifetime data to be accumulated. When the acousto-optic deflector (AOD)46 finishes scanning all pixels in the target area, the acousto-optic deflector (AOD)46 starts scanning from the first pixel of the target area again to continue accumulating the fluorescence lifetime data in each pixel, so that the fluorescence lifetime data in each pixel is enough for calculating the fluorescence lifetime value, and at this time, the three synchronous signals of pixel, line and frame are output simultaneously, wherein the frame signal triggers the storage of the lifetime data to switch from the last pixel of the lifetime image file to the first pixel, and the lifetime data is accumulated and added to the first pixel, and the fluorescence lifetime information (i.e. photon number-time distribution) in the first pixel is updated. And (4) circulating the fluorescence life data storage sequence according to the rule until the acquisition is finished, and automatically stacking the fluorescence life information acquired by the multi-frame acquisition to form the fluorescence life information of the target area.
In specific implementation, when the shape of the target region is a regular rectangle as shown in fig. 2, the fluorescence signal of the target region obtained from the fluorescence lifetime data stored in the three paths of synchronous signals is completely correct, and no later reconstruction is required. When the shape of the target area is an irregular figure as shown in fig. 3, the control terminal 7 controls the data acquisition card 6 to output pixel signals, line signals and frame signals corresponding to the irregular figure according to the comparison result of the circumscribed rectangular pixel coordinate and the pixel coordinate of the target area obtained by analyzing the shape of the target area, at this time, the number of the pixel signals between every two line signals depends on the number of pixels of each line, for the irregular figure, the line pixels no longer appear in a regular periodic rule, when the time-dependent single photon counting acquisition card 54 stores the fluorescence lifetime data in the lifetime image file according to the three-way synchronous signal, when the storage of the fluorescence lifetime data still realizes the switching from one line to the next line when the line signals and the pixel signals arrive at the same time, i.e. the fluorescence lifetime data is still stored according to the line, but the number of pixels stored in each line is different, and simultaneously, because each line is stored from the first pixel of the lifetime image file, although the finally obtained fluorescence lifetime image is still a distorted fluorescence lifetime image, the number of pixels in each row is consistent with that in the correct fluorescence lifetime image, only the relative position is deviated, and correct fluorescence lifetime data can be obtained only by further shifting the pixels in each row in the X direction and the Y direction through the control terminal 7, so that the operation is simple.
In a specific embodiment, as shown in fig. 4, after the control terminal 7 acquires the target area in the foregoing steps, the shape of the target area is analyzed to generate a circumscribed rectangle, and pixel coordinates of the target area and the circumscribed rectangle are acquired. After generating a distorted fluorescence lifetime image according to the fluorescence lifetime data stored in the lifetime image file, the control terminal 7 further marks the overlapping part of the circumscribed rectangular area of the target area and the target area as 1, and marks the non-overlapping part of the circumscribed rectangular area of the target area and the target area as 0, so as to form a binary matrix. And then determining the relative position of each pixel in each row of the selected target area by taking the first pixel coordinate in each row of the circumscribed rectangle as a reference, namely finally needing to reconstruct the pixel arrangement of the achieved correct fluorescence lifetime image. And then, carrying out subtraction operation on the positions of the pixels in each row in the correct fluorescence lifetime image and the positions of the corresponding pixels in the distorted fluorescence lifetime image to obtain a shift matrix in the X direction, wherein the matrix contains the position distortion amount information of each pixel in the distorted lifetime image, namely the correct storage position can be reached by shifting each pixel to the right by how many pixels. After the binarization matrix and the shift matrix are obtained, the two matrices are successively acted on each pixel of the image file, and then the distorted fluorescence lifetime image can be corrected.
In specific implementation, for a single arbitrary-shaped region and a plurality of arbitrary-shaped regions that are continuously distributed in the Y direction, the correction of the fluorescence lifetime image may be performed by correctly shifting pixels in the X direction as shown in fig. 4. On the other hand, in the case where a plurality of arbitrary shapes are not continuously distributed in the Y direction, after the shift in the X direction, it is necessary to shift the discrete distribution regions in the Y direction according to the intervals in the Y direction. As shown in fig. 5, a plurality of target regions having arbitrary shapes but not continuous in the Y direction are shifted in the X direction and the Y direction to obtain an accurate fluorescence lifetime image. The invention can extend the shape of the imaging area from a single rectangle to one or more arbitrary shapes by closely connecting the output three paths of synchronous signals with the shape of the selected target area and then by means of simple post-image processing, namely, arranging the pixels according to the shape characteristics of the target area again, thereby greatly expanding the flexibility of imaging and improving the imaging speed to a certain extent.
In a specific embodiment, the inventor utilizes the utility model to provide an addressing scanning rapid fluorescence lifetime microscopic imaging system carries out fluorescence imaging to lily of the valley rhizome standard sample, the fluorescence lifetime image that obtains is as shown in fig. 6, fig. 6(a) is for selecting a plurality of different regions that need to carry out fluorescence lifetime imaging from lily of the valley rhizome standard sample, fig. 6(b) is the fluorescence intensity image that area array detector 82 shot, fig. 6(c) is the fluorescence lifetime image that time-dependent single photon counting acquisition card (tcspcard) 54 obtained according to three routes synchronizing signal storage fluorescence lifetime data, can see serious distortion, fig. 6(d) obtains the correct fluorescence lifetime image for the fluorescence lifetime image of correcting the distortion, can see that it is unanimous with the fluorescence intensity image that area array detector 82 shot, it is accurate to explain the correction effect.
To sum up, the utility model provides an addressing scanning quick fluorescence life microscopic imaging system, the system includes: the device comprises an illumination light source, a laser, an image generation unit, a data acquisition card, a fluorescence signal generation unit, a fluorescence life acquisition unit and a control terminal. The utility model discloses a pixel coordinate output digital signal and three routes synchronizing signal of target area, through the acoustic wave frequency that corresponds to the acoustic-optic deflector loading digital signal, can make the acoustic-optic deflector deflect the light beam to the sample assigned position fast, realize quick nimble addressing scanning formation of image; the three paths of synchronous signals control the time-dependent single photon counting acquisition card to synchronously store fluorescence lifetime data, and the pixel coordinates of the target area and the external rectangle thereof are utilized to correct the fluorescence lifetime image, so that the addressing scanning rapid fluorescence lifetime imaging of the target area in any shape is realized.
It should be understood that the application of the system of the present invention is not limited to the above examples, and that modifications and variations can be made by those skilled in the art in light of the above teachings, and all such modifications and variations are intended to fall within the scope of the appended claims.
Claims (10)
1. An address scan fast fluorescence lifetime microscopy imaging system, the system comprising:
an illumination source and a laser;
the image generating unit is used for irradiating the illumination light beam generated by the illumination light source to the sample surface to generate a sample bright field map or a fluorescence map;
the data acquisition card is used for outputting a digital signal and three paths of synchronous signals according to the pixel coordinates of the selected target area on the sample bright field diagram or the fluorescence diagram;
the fluorescence signal generating unit is used for projecting the laser beam generated by the laser onto a sample surface according to the digital signal and exciting the target area to generate a fluorescence signal;
the fluorescence life acquisition unit is used for acquiring and processing the fluorescence signals to obtain fluorescence life data of the target area and storing the fluorescence life data into a preset life image file according to the three paths of synchronous signals;
and the control terminal is used for generating a distorted fluorescence life image of the target area according to the fluorescence life data, and correcting the distorted fluorescence life image according to the target area and the coordinates of the rectangular pixels externally connected with the target area to obtain the fluorescence life image of the target area.
2. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 1, wherein the fluorescence signal generation unit comprises:
the dispersion compensation prism is used for receiving the laser beam generated by the laser and pre-correcting the spatial dispersion and the time dispersion of the laser beam;
and the acousto-optic deflector is used for receiving the laser beam after being pre-corrected, projecting the laser beam after being pre-corrected onto a sample surface according to the digital signal and exciting the target area to generate a fluorescence signal.
3. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 2, wherein the fluorescence lifetime acquisition unit comprises:
the photomultiplier is used for collecting the fluorescence signal and converting the fluorescence signal into a corresponding electric signal;
and the time-dependent single photon counting acquisition card is used for processing the electric signals converted by the photomultiplier to obtain fluorescence life data and storing the fluorescence life data into a preset life image file according to the three paths of synchronous signals.
4. The addressing scanning rapid fluorescence lifetime microscopy imaging system of claim 3, wherein said data acquisition card is connected to said time dependent single photon counting acquisition card and said acousto-optic deflector simultaneously by a cable.
5. The addressing scanning rapid fluorescence lifetime microscopy imaging system according to claim 3, wherein said control terminal is connected to said data acquisition card, acousto-optic deflector and time dependent single photon counting acquisition card simultaneously.
6. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 2, wherein the fluorescence signal generation unit further comprises:
the half-wave plate and the polarization beam splitting prism are used for receiving the laser beam generated by the laser and adjusting the power of the laser beam;
the first beam expanding and shaping lens pair is used for receiving the laser beam after power adjustment and carrying out beam expanding and shaping on the received laser beam;
and the second beam expanding and shaping lens pair is used for receiving the laser beam emitted by the acousto-optic deflector and expanding and shaping the laser beam.
7. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 3, wherein the fluorescence lifetime acquisition unit further comprises:
and the second optical filter is used for receiving the fluorescence signal and filtering the fluorescence signal.
8. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 7, further comprising:
the beam splitter is used for splitting the fluorescence signal output by the second optical filter;
and the image recording module is used for receiving the fluorescence signal split by the beam splitter and recording a fluorescence intensity image of the fluorescence signal.
9. The address scanning rapid fluorescence lifetime microscopy imaging system of claim 8, wherein said image recording module comprises:
the third reflector is used for receiving the fluorescence signal split by the beam splitter and reflecting the fluorescence signal;
and the area array detector is used for receiving the fluorescence signals reflected by the third reflector and recording fluorescence intensity images of the fluorescence signals.
10. The addressing scanning rapid fluorescence lifetime microscopy imaging system of claim 1, wherein the illumination source is one of a halogen lamp, an LED lamp, a mercury lamp, or a xenon lamp; the laser is a femtosecond pulse laser.
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| CN113504634A (en) * | 2021-06-30 | 2021-10-15 | 华南师范大学 | Intelligent high-speed scanning method and device for point scanning long-life fluorescence microscopic imaging |
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| CN113504634A (en) * | 2021-06-30 | 2021-10-15 | 华南师范大学 | Intelligent high-speed scanning method and device for point scanning long-life fluorescence microscopic imaging |
| CN113504634B (en) * | 2021-06-30 | 2023-01-03 | 华南师范大学 | Intelligent high-speed scanning method and device for point scanning long-life fluorescence microscopic imaging |
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