阅读说明：本技术 基于变频激光的扫描成像系统及其方法 (Scanning imaging system based on variable frequency laser and method thereof ) 是由 赵磊 于 2019-09-21 设计创作，主要内容包括：本发明公开了一种基于变频激光的扫描成像系统,包括：数字信号发生器(1)、激光器(2)、激光扫描装置(3)、显微镜模块(4)、共聚焦探测模块(5)和数字采集卡(6)。同时,本发明还公开了一种基于变频激光的扫描成像方法。本发明可提高荧光图像的空间分辨率,同时可获得成像样品的荧光寿命数据,为物质种类的分析提供时间维度的信号。(The invention discloses a scanning imaging system based on variable frequency laser, which comprises: the device comprises a digital signal generator (1), a laser (2), a laser scanning device (3), a microscope module (4), a confocal detection module (5) and a digital acquisition card (6). Meanwhile, the invention also discloses a scanning imaging method based on the variable frequency laser. The invention can improve the spatial resolution of the fluorescence image, can obtain the fluorescence life data of the imaging sample, and provides a time dimension signal for the analysis of the substance type.)

1. A variable frequency laser-based scanning imaging system, comprising: the device comprises a digital signal generator (1), a laser (2), a laser scanning device (3), a microscope module (4), a confocal detection module (5) and a digital acquisition card (6); laser scanning device (3) include respectively along X axle and two sets of scanning modules of Y axle setting, each scanning module all includes: a scanning galvanometer (31), a scanning lens (32), and a tube lens (33); the microscope module (4) comprises a dichroic mirror (41), a microscope objective (42) and a sample (43); the digital signal generator (1) is internally provided with three analog signals and a digital pulse signal (1a), wherein the three analog signals are a variable frequency signal (1b) and two sawtooth wave signals (1c, 1 d); the frequency conversion signal (1b) drives a laser (2) to generate frequency conversion intensity laser; two sawtooth wave signals (1c, 1d) are used as scanning signals of an X axis and a Y axis to drive two galvanometers (31) in the laser scanning device; the digital pulse signal (1a) is used for driving the digital acquisition card (6) to start data acquisition so as to realize synchronous detection; when the three analog signals are sent out, the system is in frequency conversion laser scanning, the sample is excited to emit frequency conversion intensity fluorescence, the fluorescence signal is detected by the confocal detection module (5), and is converted into an electric signal to be synchronously collected by the digital collection card (6).

2. The variable frequency laser-based scanning imaging system of claim 1, wherein: the mirror surface angle of the scanning galvanometer (31) is changed under the driving of an electric signal, so that the emergent angle of incident laser is changed, and the back focus of the scanning lens can be superposed with the middle point of the mirror surface of the scanning galvanometer; the back focus of the tube lens can coincide with the front focus of the scanning lens, and the scanning lens and the tube lens jointly conjugate the midpoint of the scanning galvanometer to the entrance pupil of the next module.

3. The variable frequency laser-based scanning imaging system of claim 1, wherein: and the connecting line from the back focus to the front focus of the scanning galvanometer (31), the scanning lens (32) and the tube lens (33) is superposed with the light transmission direction.

4. The variable frequency laser-based scanning imaging system of claim 1, wherein: microscope module (4) include dichroic mirror (41), microscope objective (42) and sample (43), laser passes through laser scanning device after, by the dichroic mirror reflection, reach microscope objective, microscope objective's entrance pupil and the coincidence of the preceding focus of the pipe lens of last level, under the drive of two way sawtooth wave signals, laser scanning device realizes the high-speed scanning of laser angle, above-mentioned laser passes through behind the microscope objective, focus on the sample face, its focus laser point realizes two-dimentional grid point scanning, excite the fluorescence signal of M N discrete region on the sample, fluorescence signal is reverse to pass through microscope objective, and then see through dichroic mirror module, get into confocal detection module.

5. The variable frequency laser-based scanning imaging system of claim 1, wherein: the confocal detection module (5) comprises a reflector (51), an achromatic lens (52), a confocal small hole (53) and a high-speed detector (54), wherein a fluorescence signal passing through the dichroic mirror passes through the achromatic lens and then is focused at the confocal small hole, and the confocal small hole is coincided with the front focus of the achromatic lens.

6. A scanning imaging method based on variable frequency laser comprises the following steps:

s1: setting three analog signals and a digital pulse signal in a digital signal generator, wherein the three analog signals are a variable frequency signal and two sawtooth wave signals respectively;

the laser is driven by a frequency conversion signal to generate laser with frequency conversion intensity, and the frequency conversion signal is defined as F (i) ═ sin [ omega (i) × i + ψ]WhereinWhereinDenotes taking integers, i and k are integers greater than 0, Feq and coe are rational numbers greater than 0, ψ is a rational number greater than 0;

two paths of sawtooth wave signals are used as scanning signals of an X axis and a Y axis to drive two galvanometers in a laser scanning device, and the sawtooth wave signals for driving the X axis and the Y axis are respectively defined asAnda is a rational number which is larger than 0 and represents scanning amplitude, M is the number of pixel points which are scanned transversely on an x axis, N is the number of pixel points which are scanned longitudinally on a y axis, i is an integer which is larger than 0, the number of pixel points which scan a pair of two-dimensional images completely is MxN, the generation frequency of a sawtooth wave signal is defined to be OmHz, Om is an integer which is larger than 0, the time which is required for scanning a pair of two-dimensional images completely is MxN/Om seconds, and a digital pulse signal is used for driving a digital acquisition card to start data acquisition so as to realize synchronous detection;

s2: when the three analog signals are sent out, the system is in frequency conversion laser scanning, a sample is excited to emit frequency conversion intensity fluorescence, the fluorescence signals are detected by a confocal detection module and converted into electric signals to be synchronously collected by a digital collection card, the sampling frequency of the digital collection card is ImHz, Im is P times of Om, both Im and P are integers larger than 0, the photoelectric signals collected by the digital collection card are defined as a one-dimensional matrix Dig, the dimension of the matrix is MXNxP, and pix ═ yj-1 (yj-1) xM + xi is defined, then P elements of Dig [ (pix-1) xP +1: pixxP ] represent the fluorescence light intensity of the (xi, yj) th pixel, xi is an integer with the value range from 1 to M, yj is an integer with the value range from 1 to N, and the dark area of the matrix Dig is searched to obtain an area with low darkness;

s3: fourier transform is carried out on fluorescence intensity data in each pixel of the matrix Dig, intensity and phase information is extracted, and differential expansion is carried out by combining a region with low darkness;

s4: and outputting a high-precision space image which simultaneously contains the fluorescence lifetime information of each pixel point.

7. The method of claim 6, wherein: in step S2, the dark area search is performed by first performing segmentation integration on the digital signal Dig stored in the digital acquisition card to obtain a new one-dimensional matrix Dig _2, the dimension of the matrix is M multiplied by N, and the definition mode of the matrix elements isThe value range of i is 1 to M × N, a Threshold is set (Threshold is a number greater than 0), all elements in Dig _2 are traversed by using for loop, the index numbers corresponding to the elements smaller than Threshold are recorded in a matrix Dark, and the number of the elements smaller than Threshold in Dig _2 is recorded, defined as Dark _ num, which is an integer greater than 0 and is also the dimension of the matrix Dark.

8. The method of claim 6, wherein: in step S3, the fourier transform is performed by performing fourier transform on the fluorescence intensity data in each pixel of the matrix Dig to obtain a new matrix Dig _ FFT, the data dimension of the Dig _ FFT is the same as that of Dig, the transform method is Dig _ FFT [ (b-1) × P +1: b × P ] ═ FFT (Dig [ (b-1) × P +1: b × P ]), and the FFT is a classical fast discrete fourier transform.

9. The method of claim 6, wherein: in step S3, the intensity and phase information is extracted for the data in each pixel, and for the b-th pixel, Dig _ FFT [ (b-1). times.P +1: b.times.P ] is applied]The maximum value of the P numerical values is taken as the intensity data of the pixel point and is stored into a two-dimensional array Amp [ xi, yj]In whichxi is b-yj × M, and for the b-th pixel, the phase information is from Dig [ (b-1) × P +1: b × P]In (1), using for loop, traverse Dig [ (b-1). times.P +1: b.times.P]Of which the index values are 1 to P, the index value Ipeak of the first extreme point is recorded, and if the extreme value is the maximum value, the phase is determinedIf the extreme value is the minimum value,the phase is then the phase of,wherein

10. The method of claim 6, wherein: in step S3, the differential expansion is to perform differential calculation based on the Dark area search, the fourier transform and the intensity information extraction, to newly create a new differential matrix DiffA, where the dimension of the matrix is mxn, the initial values of the elements in the matrix are all 0, to extract the index values in the matrix Dark, and to convert each index value into a two-dimensional image pixel index value, where the conversion formula isxi＝Dark[i]-yj × M, where i is an integer ranging from 1 to Dark _ num, to Dark field pixel Amp [ xi, yj]As a reference point, DiffA [ xi, yj]＝Amp[xi,yj]The adjacent pixel points are differentiated with the reference point in sequence, DiffA [ xi +1, yj]＝Amp[xi+1,yj]-DiffA[xi,yj]，DiffA[xi,yj+1]＝Amp[xi,yj+1]-DiffA[xi,yj]，DiffA[xi-1,yj]＝Amp[xi-1,yj]-DiffA[xi,yj]，DiffA[xi,yj-1]＝Amp[xi,yj-1]-DiffA[xi,yj]After the differential expansion, DiffA adds 4 nonzero elements, and further repeats the differential process by using the 4 nonzero elements as reference points to realize the differential expansion.

Technical Field

The invention relates to a scanning imaging system, in particular to a scanning imaging system based on variable frequency laser and a method thereof, belonging to the technical field of optical imaging.

Background

In recent years, optical imaging tools have become more and more prominent in scientific research. To be able to achieve higher resolution, a number of novel microscopic imaging methods emerge. The confocal laser microscope system becomes the first choice of scientific researchers due to good imaging resolution, three-dimensional imaging capability and outstanding imaging mode expansion capability.

In the confocal laser microscope system, resolution enhancement is an important issue, and currently, the spatial resolution enhancement of confocal laser scanning is achieved by using dual-beam dissipative excitation, spatial frequency modulation excitation and other modes, however, even though various innovative methods are proposed, the resolution of the confocal laser microscope still does not meet the final requirements of researchers. The pursuit of spatial resolution for confocal laser microscopy is endless.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a scanning imaging method based on variable frequency laser and a system thereof, and further improves the spatial resolution of a laser confocal microscope.

Therefore, the invention adopts the following technical scheme:

a variable frequency laser-based scanning imaging system, comprising: the device comprises a digital signal generator (1), a laser (2), a laser scanning device (3), a microscope module (4), a confocal detection module (5) and a digital acquisition card (6);

laser scanning device (3) are including two sets of scanning modules and a speculum that sets up along X axle and Y axle respectively, and each scanning module all includes: a scanning galvanometer (31), a scanning lens (32), and a tube lens (33); a mirror (34) is disposed behind the rear tube lens.

The microscope module (4) comprises a dichroic mirror (41), a microscope objective (42) and a sample (43);

the digital signal generator (1) is internally provided with three analog signals and a digital pulse signal (1a), wherein the three analog signals are a variable frequency signal (1b) and two sawtooth wave signals (1c, 1 d); the frequency conversion signal (1b) drives a laser (2) to generate frequency conversion intensity laser; two sawtooth wave signals (1c, 1d) are used as scanning signals of an X axis and a Y axis to drive two galvanometers (31) in the laser scanning device; the digital pulse signal (1a) is used for driving the digital acquisition card (6) to start data acquisition so as to realize synchronous detection; when the three analog signals are sent out, the system is in frequency conversion laser scanning, the sample is excited to emit frequency conversion intensity fluorescence, the fluorescence signal is detected by the confocal detection module (5), and is converted into an electric signal to be synchronously collected by the digital collection card (6).

Furthermore, under the drive of an electric signal, the mirror surface angle of the scanning galvanometer (31) is changed, so that the emergent angle of incident laser is changed, and the back focus of the scanning lens can coincide with the middle point of the mirror surface of the scanning galvanometer; the back focus of the tube lens can coincide with the front focus of the scanning lens, and the scanning lens and the tube lens jointly conjugate the midpoint of the scanning galvanometer to the entrance pupil of the next module.

Furthermore, a connecting line from the back focal point to the front focal point of the scanning galvanometer (31), the scanning lens (32) and the tube lens (33) is superposed with the light transmission direction.

Furthermore, the microscope module (4) comprises a dichroic mirror (41), a microscope objective (42) and a sample (43), laser is reflected by the dichroic mirror after passing through the laser scanning device and reaches the microscope objective, an entrance pupil of the microscope objective coincides with a front focus of a tube lens of the previous stage, the laser scanning device realizes high-speed scanning of laser angles under the drive of two sawtooth wave signals, the laser is focused on a sample surface after passing through the microscope objective, a focused laser point of the microscope laser realizes two-dimensional grid point scanning, fluorescent signals of M multiplied by N discrete areas on the sample are excited, and the fluorescent signals reversely pass through the microscope objective and then penetrate through the dichroic mirror module to enter the confocal detection module.

Furthermore, the confocal detection module (5) comprises a reflector (51), an achromatic lens (52), a confocal pinhole (53) and a high-speed detector (54), wherein a fluorescence signal passing through the dichroic mirror passes through the achromatic lens and then is focused at the confocal pinhole, and the confocal pinhole is overlapped with the front focus of the achromatic lens. After the fluorescence signal passes through the confocal small hole, spatial filtering is realized, the fluorescence signal is collected by a high-speed detector tightly attached to the confocal small hole, and the high-speed detector converts the optical signal into an electric signal and outputs the electric signal to a digital acquisition card.

In another aspect of the present invention, a scanning imaging method based on variable frequency laser is provided, which includes the following steps:

s1: setting three analog signals and a digital pulse signal in a digital signal generator, wherein the three analog signals are a variable frequency signal and two sawtooth wave signals respectively;

the laser is driven by a frequency conversion signal to generate laser with frequency conversion intensity, and the frequency conversion signal is defined as F (i) ═ sin [ omega (i) × i + ψ]WhereinWhereinDenotes taking integers, i and k are integers greater than 0, Feq and coe are rational numbers greater than 0, ψ is a rational number greater than 0;

two paths of sawtooth wave signals are used as scanning signals of an X axis and a Y axis to drive two galvanometers in a laser scanning device, and the sawtooth wave signals for driving the X axis and the Y axis are respectively defined asAnda is a rational number which is larger than 0 and represents scanning amplitude, M is the number of pixel points which are scanned transversely on an x axis, N is the number of pixel points which are scanned longitudinally on a y axis, i is an integer which is larger than 0, the number of pixel points which scan a pair of two-dimensional images completely is MxN, the generation frequency of a sawtooth wave signal is defined to be OmHz, Om is an integer which is larger than 0, the time which is required for scanning a pair of two-dimensional images completely is MxN/Om seconds, and a digital pulse signal is used for driving a digital acquisition card to start data acquisition so as to realize synchronous detection;

s2: when the three analog signals are sent out, the system is in frequency conversion laser scanning, a sample is excited to emit frequency conversion intensity fluorescence, the fluorescence signals are detected by a confocal detection module and converted into electric signals to be synchronously collected by a digital collection card, the sampling frequency of the digital collection card is ImHz, Im is P times of Om, both Im and P are integers larger than 0, the photoelectric signals collected by the digital collection card are defined as a one-dimensional matrix Dig, the dimension of the matrix is MXNxP, and pix ═ yj-1 (yj-1) xM + xi is defined, then P elements of Dig [ (pix-1) xP +1: pixxP ] represent the fluorescence light intensity of the (xi, yj) th pixel, xi is an integer with the value range from 1 to M, yj is an integer with the value range from 1 to N, and the dark area of the matrix Dig is searched to obtain an area with low darkness;

s3: fourier transform is carried out on fluorescence intensity data in each pixel of the matrix Dig, intensity and phase information is extracted, and differential expansion is carried out by combining a region with low darkness;

s4: and outputting a high-precision space image which simultaneously contains the fluorescence lifetime information of each pixel point.

Further, in step S2, the dark area search is performed by first performing a segmentation integration on the digital signal Dig stored in the digital acquisition card to obtain a new one-dimensional matrix Dig _2, where the dimension of the matrix is mxn, and the definition of the matrix elements is thatThe value range of i is 1 to M × N, a Threshold is set (Threshold is a number greater than 0), all elements in Dig _2 are traversed by using for loop, the index numbers corresponding to the elements smaller than Threshold are recorded in a matrix Dark, and the number of the elements smaller than Threshold in Dig _2 is recorded, defined as Dark _ num, which is an integer greater than 0 and is also the dimension of the matrix Dark.

Further, in step S3, the fourier transform is performed by performing fourier transform on the fluorescence intensity data in each pixel of the matrix Dig to obtain a new matrix Dig _ FFT, the data dimension of the Dig _ FFT is the same as Dig, the transform method is Dig _ FFT [ (b-1) × P +1: b × P ] ═ FFT (Dig [ (b-1) × P +1: b × P ]), and the FFT is a classical fast discrete fourier transform.

Further, in step S3, the intensity and phase information is extracted for the data in each pixel, and for the b-th pixel, Dig _ FFT [ (b-1). times.P +1: b.times.P ] is applied]The maximum value of the P numerical values is taken as the intensity data of the pixel point and is stored into a two-dimensional array Amp [ xi, yj]In whichxi is b-yj × M, and for the b-th pixel, the phase information is from Dig [ (b-1) × P +1: b × P]In (1), using for loop, traverse Dig [ (b-1). times.P +1: b.times.P]Of which the index values are 1 to P, the index value Ipeak of the first extreme point is recorded, and if the extreme value is the maximum value, the phase is determinedIf the extreme value is the minimum value, the phase is,wherein

Further, in step S3, the differential expansion is to perform differential calculation based on the Dark area search, the fourier transform and the intensity information extraction, to newly create a new differential matrix DiffA, where the dimension of the matrix is mxn, the initial values of the elements in the matrix are all 0, extract the index values in the matrix Dark, convert each index value into a two-dimensional image pixel index value, and convert the conversion formula intoxi＝Dark[i]-yj × M, where i is an integer ranging from 1 to Dark _ num, to Dark field pixel Amp [ xi, yj]As a reference point, DiffA [ xi, yj]＝Amp[xi,yj]The adjacent pixel points are differentiated with the reference point in sequence, DiffA [ xi +1, yj]＝Amp[xi+1,yj]-DiffA[xi,yj]，DiffA[xi,yj+1]＝Amp[xi,yj+1]-DiffA[xi,yj]，DiffA[xi-1,yj]＝Amp[xi-1,yj]-DiffA[xi,yj]，DiffA[xi,yj-1]＝Amp[xi,yj-1]-DiffA[xi,yj]After the differential expansion, DiffA adds 4 nonzero elements, and further repeats the differential process by using the 4 nonzero elements as reference points to realize the differential expansion.

The invention has the beneficial effects that:

the invention realizes the output of the laser with variable frequency intensity by driving the laser with variable frequency signals, completes the detection of the variable frequency fluorescent signals at a sample by combining a laser scanning device, a microscope module, a confocal detection module and a digital acquisition card, processes the variable frequency fluorescent signals in the time dimension, searches for reference pixels through dark area search, analyzes data through Fourier transform, extracts intensity and phase information, and improves the spatial resolution of a fluorescent image through the difference with the reference pixels. In addition, the scanning imaging method provided by the invention can also obtain fluorescence lifetime data of an imaging sample and provide a time-dimension signal for analysis of substance types.

Drawings

FIG. 1 is a schematic view of example 1 of the present invention;

FIG. 2 is a flow chart of a process for detecting fluorescence data according to the present invention;

FIG. 3 is a schematic diagram of differential expansion in accordance with the present invention;

in the figure, 1 is a digital signal generator, 1a is a digital pulse signal, 1b is a variable frequency signal, and 1c and 1d are sawtooth wave signals respectively; 2 is a laser; 3, a laser scanning device, 31, 32, 33, 31a, 32a, 33b, 32b, 33b, 34, 31a, 32a, 33b, 31 b; 4 is a microscope module, 41 is a dichroic mirror, 42 is a microscope objective, and 43 is a sample; 5 is a focusing detection module, 51 is a reflector, 52 is an achromatic lens, 53 is a confocal pinhole, and 54 is a high-speed detector; and 6, a digital acquisition card.

Detailed Description

In order to make the public more clearly understand the technical spirit and the advantages of the present invention, the applicant shall make the following detailed description by way of example, but the description of the example is not intended to limit the technical scope of the present invention, and any equivalent changes made according to the present inventive concept, which are only in form and not substantial, shall be regarded as the technical scope of the present invention.