A super-resolution microscopy method to reduce photobleaching

Abstract

The present invention discloses a super-resolution microscopy method for reducing photobleaching. In the present invention, light beams emitted by two lasers are synchronously projected onto a fluorescent sample, and one of the light beams is phase modulated to form a high-intensity annular spot on the sample surface; while the other light beam maintains a solid spot state with low light intensity. The two light beams are modulated with different frequencies by a time modulation device, and the modulation contrast of the annular light beam is adjusted in real time; in addition, optical phase-locked detection technology is used at the detection end to replace the expensive phase-locked hardware equipment in the traditional method, and the signal is converted from the spatial domain to the frequency domain while obtaining the same spatial resolution. The method requires lower optical power, thereby alleviating the effects of the imaging system on the photobleaching and phototoxicity of the sample. The method does not require any changes to the dye, has a simple design, is easy to operate, and is universal for high-resolution imaging of biological samples.

Description

Technical Field

[0001] The invention belongs to the field of super-resolution microscopic imaging and relates to a super-resolution microscopic method for reducing photobleaching. Background Art

[0002] Due to the influence of diffraction in the optical system, the resolution that can be achieved by conventional far-field optical microscopy methods is limited. According to the Abbe diffraction limit theory, the size of the spot formed by the beam after being focused by the microscope objective is expressed in terms of the full width at half maximum: ,in is the working wavelength of the microscope, is the numerical aperture of the microscope objective used. Therefore, the limiting resolution of conventional far-field optical microscopes is generally limited to about half a wavelength.

[0003] In order to break through the limitation of optical diffraction limit and improve the resolution of microscopic system, researchers have proposed a variety of super-resolution optical microscopy methods, among which fluorescence excitation has received great attention. In the past two decades, more and more researchers have devoted themselves to the development of optical nano-resolution microscopy technology. The most typical representative is stimulated emission depletion microscopy (STED) proposed by Stefan Hell in 1994. At present, STED has been successfully applied to biological or cell structures, such as cell membranes, vesicles, endosomes, viruses and other microstructures, and has solved many difficult problems that have troubled life scientists for many years. As the first truly widely recognized far-field super-resolution microscopy technology, STED has made great progress and achieved unprecedented breakthroughs in spatial resolution, sensitivity, temporal resolution, imaging depth, etc. In addition, based on STED research, many derivative super-resolution imaging technologies based on dual-beam illumination have been produced, such as saturated absorption competition imaging, ground state depletion microscopy, reversible saturated optical linear fluorescence transition microscopy, etc.

[0004] The above super-resolution microscopy techniques are determined by the optical power of the loaded inhibitory light. In actual operation, the inhibitory light power cannot be increased infinitely, because this will cause serious photobleaching and photodamage to the fluorescent sample, greatly shortening the observable time and being unfavorable for long-term biological imaging. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a super-resolution microscopy method for reducing photobleaching.

[0006] The method of the present invention specifically comprises:

[0007] S1. Introduce two laser beams, regulate their power and polarization, and realize dual-beam illumination;

[0008] S2. The two laser beams are spatially intensity modulated by a phase modulation device;

[0009] S3. Temporally modulate the two laser beams through a temporal modulation device and optimize the modulation contrast;

[0010] S4. By optical phase-locked detection, the signal is moved from the spatial domain to the frequency domain and the desired signal is demodulated;

[0011] S5. Obtain the optimal illumination intensity to generate fluorescence excitation for the sample and achieve super-resolution imaging with low photobleaching.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects:

[0013] (1) No high-power illumination is required to achieve the same resolution imaging;

[0014] (2) Algorithms are used to replace hardware, reducing device complexity and saving costs. In addition, the lack of a phase-locked amplifier also diversifies the choice of detectors, and avalanche detectors (APDs) with lower dark counts can be used to achieve higher signal-to-noise ratio imaging.

[0015] (3) The method has wide application potential and can be extended to super-resolution imaging devices based on dual-beam illumination. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the workflow diagram of this embodiment;

[0017] Figure 2 A schematic diagram of the optical path of the stimulated emission depletion super-resolution microscopy device for reducing photobleaching in this embodiment;

[0018] Figure 3 Schematic diagram of the definition of modulation contrast in this embodiment;

[0019] Figure 4 is the corresponding relationship between the intensity of loss light and the full width at half maximum of imaging under different modulation contrasts in the embodiment;

[0020] Figure 5 The cell imaging image of STED (right) using the method of reducing photobleaching in this example is compared with the traditional STED (left). DETAILED DESCRIPTION

[0021] The present application is further described in detail below in conjunction with the accompanying drawings.

[0022] like Figure 1 As shown, a super-resolution microscopy method for reducing photobleaching in an embodiment of the present application comprises the following steps:

[0023] S1. Introduce two laser beams, regulate their power and polarization, and realize dual-beam illumination;

[0024] S2. The two laser beams are spatially intensity modulated by a phase modulation device;

[0025] S3. Temporally modulate the two laser beams through a temporal modulation device and optimize the modulation contrast;

[0026] S4. Use optical phase-locked detection technology to move the signal from the spatial domain to the frequency domain and demodulate the desired signal;

[0027] S5. Obtain the optimal illumination intensity to generate fluorescence excitation for the sample and achieve super-resolution imaging with low photobleaching.

[0028] According to the above records, the embodiments of the present application can effectively alleviate the photobleaching phenomenon generated during the imaging process. At the illumination end, dual-beam illumination is used, and modulation signals are loaded on both beams to make the modulation contrast adjustable; at the detection end, optical phase-locked detection technology is used to convert the signal from the spatial domain to the frequency domain and demodulate the required signal. By optimizing the modulation contrast, a lower illumination light power can be obtained, greatly reducing the impact of photobleaching.

[0029] Furthermore, in step S1, the power and polarization control comprises the following steps:

[0030] S101. Adjust one of the laser beams to a low power laser with a power of I s , adjust its polarization to vertically polarized light.

[0031] S102. Adjust another laser beam to make it a high-power laser with a power of I d , adjust its polarization to horizontal polarized light.

[0032] S103. Combine the two laser beams and adjust them into circular polarized light through a polarizer, and irradiate them onto the sample surface.

[0033] Furthermore, in step S2, the spatial intensity modulation is to modulate the wavefront of the high-power laser into vortex light through a phase modulation device.

[0034] Furthermore, in step S3, the time modulation is to load sinusoidal wave modulation f on the two laser beams respectively. 1 and f 2 , so that its light intensity changes periodically with time.

[0035] Further, in step S3, the modulation contrast is , where I max and I min They are the minimum and maximum light intensity of the excitation light or the loss light respectively; and the modulation contrasts loaded by the two beams of light are different, namely CM1 (corresponding to f1) and CM2 (corresponding to f2).

[0036] Furthermore, in step S4, the optical phase-locked detection technology includes the following steps:

[0037] S401. Independent variable is I s , I d , f 1 , f 2 , C.M. 1 , C.M. 2 , calculate the fluorescence intensity .

[0038] S402. I fluor Through Fourier transform, we can transfer to the frequency domain and obtain the values ​​of each frequency component, including ξ(f 1 ),ξ(f 2 ), ξ(f 1 + f 2 ), ξ(f 1 + f 2 )wait.

[0039] S403. Through differential processing, such as ξ(f 1 )-ξ(f 1 + f 2 ),ξ(f 1 )-ξ(f 2 ) or ξ(f 1 )-ξ(f 1 -f 2 ).

[0040] S404. Through the inverse Fourier transform, the differential result is transferred to the spatial domain to obtain the fluorescence quantity with low background noise and improved resolution , and obtain the corresponding imaging half-maximum FWHM.

[0041] Furthermore, in step S4, the optical phase-locked detection technology can also be calculated using a gradient descent method. , in the case of minimum FWHM, I d The value of does not need to be too large to achieve the effect of reducing photobleaching.

[0042] like Figure 2As shown, this embodiment also provides a super-resolution microscopy device based on stimulated emission loss, including: a low-power laser 1, a first single-mode optical fiber 2, a first collimating lens 3, a first reflector 4, a second reflector 5, a λ / 2 wave plate 6, a first polarization beam splitter 7, a third reflector 8, a fourth reflector 9, a second polarization beam splitter 10, a first dichroic mirror 11, a high-power laser 12, a second single-mode optical fiber 13, a second collimating lens 14, a Glan prism 15, a 0~2π vortex position Phase plate 16, first λ / 4 wave plate 17, second dichroic mirror 18, dielectric reflector 19, second λ / 4 wave plate 20, scanning galvanometer 21, reflector 22, scanning lens 23, field lens 24, objective lens 25, sample stage 26, bandpass filter 27, focusing lens 28, pinhole 29, multimode optical fiber 30, avalanche detector 31, acousto-optic modulator 32, RF driver 33, first signal generator 34, electro-optic modulator 35, second signal generator 36, host 37.

[0043] Among them, the first single-mode optical fiber 2, the first collimating lens 3, the first reflector 4, the second reflector 5, the λ / 2 wave plate 6, the first polarization beam splitter 7, the third reflector 8, the acousto-optic modulator 32, the fourth reflector 9, and the second polarization beam splitter 10 are sequentially located on the optical axis of the outgoing light beam of the low-power laser 1; the second single-mode optical fiber 13, the second collimating lens 14, the Glan prism 15, the 0~2π vortex phase plate 16, and the first λ / 4 wave plate 17 are sequentially located on the optical axis of the outgoing light beam of the high-power laser 12.

[0044] The dichroic mirror 18 is located on the optical axis of the light beam modulated by the 0~2π vortex phase plate 16 .

[0045] The bandpass filter 20 and the scanning galvanometer 21 are sequentially located on the optical axis after being deflected by the dielectric reflector 19 .

[0046] The scanning lens 23 , the field lens 24 , the microscope objective lens 25 , and the sample stage 26 are sequentially located on the optical axis of the light beam emitted by the scanning galvanometer 21 .

[0047] Among them, the bandpass filter 27 , the focusing lens 28 , the pinhole 29 , the multimode optical fiber 30 , and the avalanche detector 31 are sequentially located on the optical axis of the transmitted light beam after passing through the dichroic mirror 11 ; the pinhole 29 is located at the focal plane of the focusing lens 28 .

[0048] The controller (not shown in the figure) is connected to the scanning galvanometer 21 and is used to load and control the scanning of the scanning galvanometer 21 .

[0049] In the above device, the numerical aperture NA of the microscope objective lens 25 is 1.4; the diameter of the pinhole 29 used is 0.6 Airy disk diameter.

[0050] use Figure 2The method for reducing photobleaching achieved by the device shown is as follows:

[0051] The signal light emitted by the sample to be tested is collected by the microscope objective 25, and then passes through the field lens 24, the scanning lens 23, the reflector 22, the scanning galvanometer 21, the dielectric reflector 19, the dichroic mirror 18, and the dichroic mirror 11. The signal light beam is filtered out of stray light by the bandpass filter 27, and then focused by the focusing lens 28 and spatially filtered by the pinhole 29, and finally received by the avalanche detector 31.

[0052] The output frequency of the signal generator 34 is f 1 When the modulated light beam is focused on the sample by the microscope, it forms a pulse with a frequency of f 1 The solid spot has a light intensity of I s The size of the solid spot is the same as the size of the diffraction spot formed by focusing the illumination beam used in conventional optical microscopy. The output frequency of the signal generator 36 is f 2 , when the modulation function of the 0~2π vortex phase plate is When the Debye integral is calculated, the light spot formed on the sample after the modulated light beam is focused by the microscope objective lens is a pulse with a frequency of f 2 The annular spot has a light intensity of I d . The frequency function of the loaded sinusoid, f 1 (t) and f 2 (t), can be expressed as follows:

[0053]

[0054]

[0055] The modulation contrast of the annular spot is adjusted in real time by the signal generator, and the modulation contrast is defined as , where I max and I min are the minimum and maximum intensity of the excitation light or loss light, respectively. The modulation contrast of the solid spot and the annular spot is CM 1 and CM 2 (like Figure 3 as shown).

[0056] Through the above parameters, based on the molecular energy level formula, the fluorescence intensity can be calculated ;Will Through Fourier transform, we can transfer to the frequency domain and obtain the values ​​of each frequency component, including ξ(f 1 ), ξ(f 2 ), ξ(f 1 + f 2 ), ξ(f 1 + f 2) etc. Through differential processing, such as ξ(f 1 )-ξ(f 1 + f 2 ),ξ(f 1 )-ξ(f 2 ) or ξ(f 1 )-ξ(f 1 -f 2 ), the obtained value is transferred to the spatial domain through inverse Fourier transform, and the fluorescence value with low background noise can be obtained , and obtain the corresponding resolution-enhanced imaging half-width FWHM. The gradient descent method is used to calculate , in the case of minimum FWHM, I d The value of does not need to be too large to achieve the effect of reducing photobleaching.

[0057] By adjusting the modulation contrast of the two laser beams, the required loss light intensity can be effectively reduced. Figure 4 The relationship between the required loss light intensity and the obtained imaging resolution (full width at half maximum) under different modulation contrast conditions is shown in the figure. 2 At a light intensity of 100 MW / cm, a resolution of 40 nanometers can be achieved; in traditional STED, 100 MW / cm is required. 2 The light intensity can only obtain a resolution of 40 nanometers, and the illumination light intensity only needs to be 40% of the original. This application can effectively reduce the loss of light intensity, thereby reducing the photobleaching effect. Figure 5 It is a comparative experiment. Figure 5 The left side of the middle shows the situation after long-term irradiation of biological samples by traditional STED. The central irradiated area (50 microns × 50 microns) has no fluorescent signal. Figure 5 The middle right side is the result of using this method, in which the central irradiated area can still emit fluorescence.

[0058] The terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or also includes elements inherent to such process, method, article, or apparatus.

[0059] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of application involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the aforementioned application concept. For example, the above features are replaced with (but not limited to) technical features with similar functions applied in the present application.

Claims

1. A super-resolution microscopy method for reducing photobleaching, characterized in that: The following steps are involved: S1. Introduce two laser beams, regulate their power and polarization, and realize dual-beam illumination; S2. The two laser beams are spatially intensity modulated by a phase modulation device; S3. Temporally modulate the two laser beams through a temporal modulation device and optimize the modulation contrast; S4. By optical phase-locked detection, the signal is moved from the spatial domain to the frequency domain and the desired signal is demodulated; S5. Obtain the optimal illumination intensity to generate fluorescence excitation for the sample and achieve super-resolution imaging with low photobleaching; The power and polarization control comprises the following steps: S101. Adjust one of the laser beams to a low power laser with a power of I s , adjust its polarization to vertically polarized light; S102. Adjust another laser beam to make it a high-power laser with a power of I d , adjust its polarization to horizontal polarized light; S103. Combining the two laser beams, adjusting them to circular polarization through a polarizer, and irradiating them onto the sample surface; The spatial intensity modulation is to modulate the wavefront of high-power laser into vortex light through a phase modulation device; The time modulation is to load the two laser beams with sinusoidal wave modulation f1 and f2 respectively, so that their light intensity changes periodically with time; The modulation contrast is , where I min is the minimum intensity of the excitation light or loss light, I max is the maximum light intensity of the excitation light or loss light; the modulation contrasts loaded by the two laser beams are different, namely CM1 and CM2, where CM1 corresponds to the sinusoidal modulation f1 and CM2 corresponds to the sinusoidal modulation f2; The optical phase-locked detection comprises the following steps: S401. I s , I d , f1, f2, CM1, CM2 are considered as independent variables and the fluorescence intensity is calculated ; S402. The fluorescence intensity Through Fourier transform, it is transferred to the frequency domain to obtain the value of each frequency component; S403. Performing differential processing on the values ​​of each frequency component; S404. Through inverse Fourier transform, the difference result is transferred to the spatial domain to obtain the fluorescence amount with low background noise .

2. The method according to claim 1, characterized in that In S404 , the corresponding imaging full width at half maximum FWHM with improved resolution is also obtained.

3. The method according to claim 2, characterized in that It also includes the use of gradient descent to calculate , when the imaging half-maximum full width FWHM is minimum, I d The value of does not need to be too large to achieve the effect of reducing photobleaching.

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