CN116297352A - Quality evaluation and calibration method for confocal fluorescence microscope - Google Patents
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Abstract
The invention discloses a quality evaluation and calibration method of a confocal fluorescence microscope, which comprises the steps of shooting a fluorescent microsphere test piece by using the confocal fluorescence microscope, screening, fitting the screened fluorescent microsphere by using a least square method to obtain a point spread function of a system, evaluating the quality of the confocal fluorescence microscope system by measuring the full width at half maximum value of the point spread function of the system, and calibrating the confocal fluorescence microscope according to the shape of the obtained point spread function of the system.
Description
Technical Field
The invention relates to a performance evaluation and calibration technology of a microscope, in particular to a quality evaluation and calibration method of a confocal fluorescence microscope.
Background
The confocal fluorescence microscope is a fluorescence microscope capable of directly carrying out three-dimensional visualization on thicker samples at the tissue and cell level, and is one of the most advanced cell biomedical analysis instruments in recent times. The method can observe fixed cells and tissue slices, can dynamically observe and detect the structures, molecules and ions of living cells in real time, and can obtain images with resolution up to hundreds of nanometers, so that the method is widely applied to the fields of medicine, biology and the like. And the high-quality confocal image is required to be obtained, which is essential for monitoring the performance of a confocal fluorescence microscope system.
The Point Spread Function (PSF), which describes the response of an imaging system to a point source or point object, is widely used to measure the imaging quality of confocal fluorescence microscopy systems. Where the PSF of a microscope can be simply measured by imaging a point object, while nanoscale fluorescent microbeads can be approximated as point objects in a confocal fluorescent microscope, so that confocal fluorescent microscope system performance can be assessed by fluorescent microbeads.
Confocal fluorescence microscopy requires periodic scheduling of performance tests in order to generate high quality confocal images, and such daily monitoring requires specific methods and metrics. However, the existing method lacks a specific method and index for measuring the performance of the confocal fluorescence microscope system, and is difficult to intuitively perform daily metering evaluation on the stability of the confocal fluorescence microscope. It is therefore desirable to provide a simple and effective method of quality monitoring and calibration of confocal fluorescence microscopes.
Disclosure of Invention
The invention aims to solve the technical problem of providing a quality evaluation and calibration method for a confocal fluorescence microscope, which can better monitor and evaluate the quality of the confocal fluorescence microscope system in daily life and can help a user to calibrate the confocal fluorescence microscope better after detecting the problem.
The technical scheme adopted for solving the technical problems is as follows: a quality evaluation and calibration method of a confocal fluorescence microscope comprises the following steps:
s1: shooting the fluorescent microsphere test piece by using a confocal fluorescent microscope;
s2: screening the shot fluorescent microsphere images;
s3: averaging the screened microspheres to inhibit noise, and then using a Gaussian kernel function to perform least square fitting to obtain a point spread function of the system;
s4: the transverse half-width and axial half-width experimental values of the system point spread function are obtained through measurement, and compared with theoretical values to evaluate the quality of the confocal fluorescence microscope system;
s5: based on the resulting system Point Spread Function (PSF) shape, the confocal fluorescence microscope was calibrated by the following method:
compared with the prior art, the method for evaluating and calibrating the quality of the confocal fluorescence microscope based on the fluorescent microsphere fitting point diffusion function has the advantages that a more accurate and reliable result can be obtained even under the low-illumination condition, and the obtained experimental value of the full width at half maximum of the transverse and axial point diffusion functions is compared with the theoretical value, so that the actual resolution of the confocal fluorescence microscope can be well quantified, and the method plays a good role in evaluating the quality of the confocal fluorescence microscope. And the confocal fluorescence microscope is correctly calibrated through the shape of the point spread function, so that the subsequent acquisition of the high-resolution confocal sample image is facilitated. Meanwhile, the point spread function of the confocal fluorescence microscope system can be applied to post-processing software of confocal microscopic images.
Further, the specific method for shooting the fluorescent microsphere test piece in the step S1 is as follows:
s1.1, selecting a fluorescent microsphere test piece: the mixed microsphere test pieces with different colors are selected, the size of the fluorescent microsphere is smaller than one half of the theoretical resolution of a confocal fluorescent microscope system, and for an immersion objective lens with NA > =0.6, the size of the fluorescent microsphere is smaller than 175 nm. The refractive index of the sealing agent after curing is consistent with that of the microscope lens; the wavelength of the laser is selected from the wavelength matched with the fluorescent microsphere dye;
s1.2 adjusting instrument: preheating a laser for at least 1 hour, cleaning an objective lens, removing all differential interference microscopy elements, adjusting a correction ring, calibrating a pinhole position by using a uniform fluorescent plastic sheet matched with a selected microsphere dye to ensure that the maximum brightness in a field of view is centered, calibrating to obtain a laser power of 8 mu W, a detector gain of 500V, a scaling factor of 1-2, a scanning speed of 7-9 microseconds per pixel, setting a color lookup table LUT to ensure that the visual difference of signals is as obvious as possible, ensuring that a condenser lens and the objective lens are both focused on the same focal plane, aligning the laser and performing imaging test by using a transmission photodetector, ensuring that the focusing of the microscope is stable, and the ambient temperature of the system is stable;
s1.3, shooting fluorescent microsphere images: the confocal fluorescence microscope image acquisition is set to scan 1024 x 1024 pixel image frames unidirectionally at a scanning speed of 5-25 mu s for each pixel, the scaling factor is set to 2-3, and the bit number per pixel is set to 8 or 12 or 16; setting the gain of a PMT of a detector to 600-750V, and then adjusting the gain of the PMT and the laser power to ensure that the average light intensity of the microsphere is 75% of the maximum light intensity in the image; using a continuous scan mode to ensure that there are no saturated pixels within the image; the digital gain is set to 1; the pinhole is set to be 1 Airy unit; the number of pixels is set in software to ensure that the image pixel size is at least less than one third of the resolution of the objective lens, according to the magnification of the system, where the lateral and axial pixel sizes of the confocal fluorescence microscope are given by the following formulas:
Step S2, screening the shot fluorescent microsphere images, wherein the specific method is as follows:
adjusting the focal plane, calculating the signal-to-noise ratio of each fluorescent microsphere and surrounding background signals, selecting 20-30 fluorescent microspheres with the signal-to-noise ratios ranked at the front, ensuring that the selected fluorescent microspheres are close to the central visual field, and screening the darkest group of fluorescent microspheres in the image for analysis, wherein the mutual distance is not less than 15 microns.
When screening fluorescent microspheres, 10-20 darkest microspheres in an image are selected from 20-30 fluorescent microspheres for marking and pre-screening, then the pre-screened fluorescent microspheres are subjected to binarization treatment, the fluorescent microspheres are confirmed to be regular and reasonable in shape and not aggregated, the optimal 5-10 fluorescent microspheres are selected again, a field of view with uniform size is drawn according to 20 times of the diameter of each of the screened fluorescent microspheres, the microspheres are ensured to be in the center of the drawn field of view, and signals of a plurality of drawn fields of view are averaged.
The experimental values of the full width at half maximum of the lateral and axial directions of the system point spread function obtained by measurement in step S4 are the full width at half maximum values of the x, y, z axes of the pointing spread function.
In the description of the present invention, it should also be noted that the "axial direction" describes the direction in which light propagates along the confocal fluorescence microscope, corresponding to the z-axis of the confocal fluorescence microscope; "lateral" describes the direction of light diffusion along the confocal fluorescence microscope, corresponding to the xy-axis plane of the confocal fluorescence microscope.
Drawings
FIG. 1 is a flow chart of a method for quality assessment and calibration of a confocal fluorescence microscope of the present invention;
FIG. 2 is a schematic diagram of a selected fluorescent microsphere marked with a rectangular frame in an example of a first embodiment of the present invention;
FIG. 3 shows a point spread function obtained by Gaussian fitting according to the selected microspheres in example 1 of the first embodiment of the present invention, and the display system is well calibrated;
fig. 4 is a point spread function of an example of an objective lens fitting with coma, featuring an asymmetry in the XY-axis image and curvature in the XZ and YZ-axis images, according to an embodiment of the present invention.
Detailed Description
In order that the manner in which the invention may be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to the embodiments, some of which are illustrated in the appended drawings.
Embodiment one: a quality evaluation and calibration method of a confocal fluorescence microscope comprises the following steps:
s1: shooting a fluorescent microsphere test piece by using a confocal fluorescent microscope, wherein the specific method comprises the following steps of:
s1.1, selecting a fluorescent microsphere test piece: the mixed microsphere test pieces with different colors are selected, the size of the fluorescent microsphere is smaller than one half of the theoretical resolution of a confocal fluorescent microscope system, and for an immersion objective lens with NA > =0.6, the size of the fluorescent microsphere is smaller than 175 nm. The refractive index of the sealing agent after curing is consistent with that of the microscope lens; the wavelength of the laser is selected from the wavelength matched with the fluorescent microsphere dye;
s1.2 adjusting instrument: preheating a laser for at least 1 hour, cleaning an objective lens, removing all differential interference microscopy elements, adjusting a correction ring, calibrating a pinhole position by using a uniform fluorescent plastic sheet matched with a selected microsphere dye to ensure that the maximum brightness in a field of view is centered, calibrating to obtain a laser power of 8 mu W, a detector gain of 500V, a scaling factor of 1-2, a scanning speed of 7-9 microseconds per pixel, setting a color lookup table LUT to ensure that the visual difference of signals is as obvious as possible, ensuring that a condenser lens and the objective lens are both focused on the same focal plane, aligning the laser and performing imaging test by using a transmission photodetector, ensuring that the focusing of the microscope is stable, and the ambient temperature of the system is stable;
s1.3, shooting fluorescent microsphere images: the confocal fluorescence microscope image acquisition is set to scan 1024 x 1024 pixel image frames unidirectionally at a scanning speed of 5-25 mu s for each pixel, the scaling factor is set to 2-3, and the bit number per pixel is set to 8 or 12 or 16; setting the gain of a PMT of a detector to 600-750V, and then adjusting the gain of the PMT and the laser power to ensure that the average light intensity of the microsphere is 75% of the maximum light intensity in the image; using a continuous scan mode to ensure that there are no saturated pixels within the image; the digital gain is set to 1; the pinhole is set to be 1 Airy unit; the number of pixels is set in software to ensure that the image pixel size is at least less than one third of the resolution of the objective lens, according to the magnification of the system, where the lateral and axial pixel sizes of the confocal fluorescence microscope are given by the following formulas:
s2: screening the shot fluorescent microsphere images; adjusting focal plane, calculating signal-to-noise ratio of each fluorescent microsphere and surrounding background signals, selecting 20-30 fluorescent microspheres with the signal-to-noise ratio ranked at the front, ensuring that the selected fluorescent microspheres are close to a central view field and have a mutual distance of not less than 15 microns, screening the darkest 10-20 microspheres in an image from the 20-30 fluorescent microspheres for marking and pre-screening, performing binarization treatment on the pre-screened fluorescent microspheres, confirming that the fluorescent microspheres are regular and reasonable in shape and have no aggregation, selecting the optimal 5-10 fluorescent microspheres again, drawing a view field with uniform size according to 20 times of the diameters of the microspheres for the screened fluorescent microspheres, ensuring that the microspheres are at the center of the drawn view field, and averaging signals of a plurality of drawn view fields;
s3: averaging the screened microspheres to inhibit noise, and then using a Gaussian kernel function to perform least square fitting to obtain a point spread function of the system;
s4: the full width at half maximum value of the x, y and z axes of the system point spread function is obtained through measurement, and compared with a theoretical value to evaluate the quality of the confocal fluorescence microscope system; the experimental values of the full width at half maximum of the lateral and axial directions of the system point spread function obtained by measurement in step S4 are the pointing spread function.
S5: based on the resulting system Point Spread Function (PSF) shape, the confocal fluorescence microscope was calibrated by the following method:
further description will be given below by way of a specific example:
in this example, fluorescent microspheres from Alexa Fluor company having a diameter of 100nm and an excitation wavelength of 488nm were used; the NA of the confocal fluorescence microscope objective lens is 1.4, the oil immersion refractive index is 1.5, and the magnification is 63.
The acquisition of the confocal fluorescence microscope in this example is set to: obtaining 1024 x 1024 pixel image frame (pixel dwell time is 5-25 μs per pixel) at medium scanning speed in line scanning mode, scaling factor is 2-3, and bit number per pixel is 16; the digital gain is set to 1; the pinhole is set to be 1 Airy unit; the pixel size meets the nyquist sampling frequency requirement (the image pixel size is less than half the resolution of the objective).
The 5 fluorescent microspheres screened at the center of the field of view are marked with rectangular frames as shown in fig. 2.
The view of PSF fitted by fluorescent microspheres after screening and denoising treatment on three planes XY, XZ and YZ is shown in figure 3, the scale is 5 μm, the shape is symmetrical, and the expected is met, so that the confocal fluorescent microscope is considered to be unnecessary to calibrate.
Table 1 shows the FWHM of the PSF in x, y and z axes obtained by the method, because the actual PSF is approximated by a Gaussian function, we use the full width at half maximum as the actual resolution of the system. The theoretical resolution in the table is calculated according to the Rayleigh criterion. Due to the size of the fluorescent microsphere, the signal to noise ratio of the detector, the actual measurement resolution and the theoretical resolution of the system can be different due to the influence of factors such as a fitting method.
When the ratio of the experimental resolution to the theoretical resolution is smaller than 1.5 in the transverse direction and smaller than 2 in the axial direction, the system is considered to have good working performance. The ratio of the experimental resolution to the theoretical resolution is within the allowable range, and the performance and the quality of the confocal fluorescence microscope can be considered to be better.
TABLE 1
| Experimental value FWHM | Theoretical resolution | |
| X | 0.192μm | 0.139μm |
| Y | 0.240μm | 0.139μm |
| Z | 0.635μm | 0.349μm |
Claims (5)
1. The quality evaluation and calibration method of the confocal fluorescence microscope is characterized by comprising the following steps of:
s1: shooting the fluorescent microsphere test piece by using a confocal fluorescent microscope;
s2: screening the shot fluorescent microsphere images;
s3: averaging the screened microspheres to inhibit noise, and then using a Gaussian kernel function to perform least square fitting to obtain a point spread function of the system;
s4: the transverse half-width and axial half-width experimental values of the system point spread function are obtained through measurement, and compared with theoretical values to evaluate the quality of the confocal fluorescence microscope system;
s5: based on the resulting system Point Spread Function (PSF) shape, the confocal fluorescence microscope was calibrated by the following method:
2. the method for evaluating and calibrating the quality of the confocal fluorescence microscope according to claim 1, wherein the specific method for photographing the fluorescent microsphere test piece in the step S1 is as follows:
s1.1, selecting a fluorescent microsphere test piece: the mixed microsphere test pieces with different colors are selected, the size of the fluorescent microsphere is smaller than one half of the theoretical resolution of a confocal fluorescent microscope system, and for an immersion objective lens with NA > =0.6, the size of the fluorescent microsphere is smaller than 175 nm. The refractive index of the sealing agent after curing is consistent with that of the microscope lens; the wavelength of the laser is selected from the wavelength matched with the fluorescent microsphere dye;
s1.2 adjusting instrument: preheating a laser for at least 1 hour, cleaning an objective lens, removing all differential interference microscopy elements, adjusting a correction ring, calibrating a pinhole position by using a uniform fluorescent plastic sheet matched with a selected microsphere dye to ensure that the maximum brightness in a field of view is centered, calibrating to obtain a laser power of 8 mu W, a detector gain of 500V, a scaling factor of 1-2, a scanning speed of 7-9 microseconds per pixel, setting a color lookup table LUT to ensure that the visual difference of signals is as obvious as possible, ensuring that a condenser lens and the objective lens are both focused on the same focal plane, aligning the laser and performing imaging test by using a transmission photodetector, ensuring that the focusing of the microscope is stable, and the ambient temperature of the system is stable;
s1.3, shooting fluorescent microsphere images: the confocal fluorescence microscope image acquisition is set to scan 1024 x 1024 pixel image frames unidirectionally at a scanning speed of 5-25 mu s for each pixel, the scaling factor is set to 2-3, and the bit number per pixel is set to 8 or 12 or 16; setting the gain of a PMT of a detector to 600-750V, and then adjusting the gain of the PMT and the laser power to ensure that the average light intensity of the microsphere is 75% of the maximum light intensity in the image; using a continuous scan mode to ensure that there are no saturated pixels within the image; the digital gain is set to 1; the pinhole is set to be 1 Airy unit; the number of pixels is set in software to ensure that the image pixel size is at least less than one third of the resolution of the objective lens, according to the magnification of the system, where the lateral and axial pixel sizes of the confocal fluorescence microscope are given by the following formulas:
3. The method for evaluating and calibrating the quality of the confocal fluorescence microscope according to claim 1, wherein the step S2 is to screen the photographed images of the fluorescent microspheres, and the specific method is as follows:
adjusting the focal plane, calculating the signal-to-noise ratio of each fluorescent microsphere and surrounding background signals, selecting 20-30 fluorescent microspheres with the signal-to-noise ratios ranked at the front, ensuring that the selected fluorescent microspheres are close to the central visual field, and screening the darkest group of fluorescent microspheres in the image for analysis, wherein the mutual distance is not less than 15 microns.
4. The method for evaluating and calibrating the quality of a confocal fluorescence microscope according to claim 3, wherein the fluorescent microspheres are screened, the darkest 10-20 microspheres in an image are selected from 20-30 fluorescent microspheres for marking and pre-screening, then the pre-screened fluorescent microspheres are subjected to binarization treatment, the fluorescent microspheres are confirmed to be regular and reasonable in shape and not aggregated, the optimal 5-10 fluorescent microspheres are selected again, a field of view with uniform size is drawn according to 20 times of the diameter of the microspheres for the screened fluorescent microspheres, the microspheres are ensured to be in the center of the drawn field of view, and signals of a plurality of drawn fields of view are averaged.
5. The method according to claim 1, wherein the experimental values of the full width at half maximum of the lateral and axial directions of the system point spread function obtained by measurement in step S4 are the full width at half maximum values of the x, y, z axes of the pointing spread function.
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