Super-resolution microscopic imaging method for removing Poisson background noise

Abstract

The invention discloses a super-resolution microscopic imaging method for removing Poisson background noise. According to the invention, switching modulation of different frequencies is carried out on the signal light and the suppression light split by the illumination light source at the same time, and the frequency of the signal light is read by the detection end, so that noise interference caused by the suppression light is removed. The method aims at most point scanning super-resolution microscopy methods, such as stimulated radiation depletion microscopy, saturated absorption competition microscopy, fluorescence differential microscopy and the like. The method can be applied to the fields of biomedical imaging and material science requiring high signal-to-noise ratio and high contrast.

Description

Technical Field

[0001] The invention belongs to the field of super-resolution, and in particular relates to a super-resolution microscopy method for realizing a resolution beyond the diffraction limit in a far field by utilizing the principle of fluorescence saturation. Background Art

[0002] Optical far-field fluorescence microscopy plays an important role in life science research due to its advantages such as non-invasiveness and specificity. However, its resolution is limited to approximately half the illumination wavelength due to the diffraction limit. The past two decades have witnessed the emergence of super-resolution optical microscopy, which successfully circumvents this diffraction limit and improves the resolution to the nanoscale, such as stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), structured illumination microscopy (SIM), and stimulated emission depletion microscopy (STED).

[0003] However, super-resolution methods are still limited in many ways to the biological community. For example, the STED mode suffers from photobleaching due to the necessary ultra-high power consumption and requires special fluorescent dyes. Recently, saturation effects have been used to achieve super-resolution imaging, including saturation excitation microscopy (SAX), excited state saturation microscopy (ESSat), nonlinear focal mode modulation microscopy (NFOMM), etc. Saturated absorption competition microscopy (SAC) is a newly proposed super-resolution technique under saturation excitation conditions, which extracts nonlinearities containing high spatial frequency components through lock-in detection. SAC has advantages in both fluorescence or non-fluorescence imaging. It only requires a single laser source, and other advantages include not being limited by the choice of fluorescent dyes, especially in pulsed mode, combined with anti-bleaching inorganic fluorescent probes, it can achieve high lateral spatial resolution of sub-hundred nanometers.

[0004] In conventional SACs, fluorescence excited by an unmodulated hollow beam is usually regarded as a direct current (DC) component of a lock-in amplifier (LIA) and is therefore filtered out. However, in actual experiments, due to the unmodulation of various devices, such as lasers or light modulation devices, DC and unrelated fluorescence signals cannot be completely demodulated by the LIA. Therefore, this DC portion will be partially extracted and mixed with the effective fluorescence from real beam illumination, and its residual signal follows a Poisson distribution, which cannot be removed by conventional methods. So far, many methods have been proposed to overcome this background noise, including gated detection, temporal modulation, etc. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for removing Poisson background noise in super-resolution imaging.

[0006] The present invention comprises the following steps:

[0007] Step 1). Adjust the collimation of the laser output by the laser;

[0008] Step 2). Split the collimated laser beam into two linearly polarized beams through a beam splitter, namely parallel polarized light and vertical polarized light;

[0009] Step 3). Phase modulating the vertically polarized light;

[0010] Step 4). The split parallel polarized light and vertical polarized light are modulated into two sequential lights with different preset frequencies by a frequency modulation device;

[0011] Step 5). Combining the sequential light into a beam of light through a beam combining device;

[0012] Step 6). Project the combined beam onto the sample surface and perform point scanning on the sample;

[0013] Step 7). Receive the signal light emitted by the sample during the scanning process, and demodulate the received signal light to obtain a super-resolution image with Poisson background noise removed.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) A background signal removal scheme for saturation excitation competition microscopy was proposed;

[0016] (2) The imaging quality has been improved;

[0017] (3) The device is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic structural diagram of the super-resolution microscopy device of this embodiment;

[0019] Figure 2 This is a schematic diagram of the background noise removal in this embodiment;

[0020] Figure 3 This is a diagram showing the effect of removing background signals in this embodiment. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings, but the present invention is not limited thereto.

[0022] The embodiment of the present application provides a method for removing Poisson background noise in super-resolution imaging to improve the signal-to-noise ratio. By applying two different frequencies of temporal modulation to the real light beam and the hollow excitation light beam, the fluorescence introduced by the real light beam and the hollow light beam can be separated, and the speckle-like noise can be significantly reduced during the lock-in demodulation process. The specific steps are:

[0023] 1. Adjust the collimation of the laser output by the laser to ensure that the cross-sectional size of the laser spot remains fixed.

[0024] 2. The collimated laser beam is split into two linearly polarized beams through a beam splitter, namely parallel polarized light and vertical polarized light.

[0025] 3. Phase modulating the vertically polarized light.

[0026] 4. The split parallel polarized light and vertical polarized light are modulated into two sequential lights with different preset frequencies through a frequency modulation device.

[0027] 5. The parallel polarized light and the vertical polarized light that have been phase modulated and frequency modulated are combined into one beam through a beam combining device.

[0028] 6. Project the combined beam onto the sample surface and perform point scanning on the sample.

[0029] 7. Receive the signal light emitted by the sample during the scanning process, and perform demodulation processing on the received signal light corresponding to the different preset frequencies described in step 4; finally, obtain a super-resolution image with low background noise.

[0030] Furthermore, the emitted laser can be continuous light or pulsed light, and the size of the cross-sectional size of its light spot needs to be smaller than the aperture of the subsequent frequency modulation device to ensure the modulation effect.

[0031] Furthermore, the beam splitter and beam combiner must have polarization characteristics to ensure the correct separation and combination of polarized light.

[0032] Furthermore, the phase modulation device may be a vortex phase plate or a spatial light modulator; the frequency modulation device may be an acousto-optic modulator or an electro-optic modulator to achieve precise frequency control.

[0033] Furthermore, the light beam incident on the sample surface needs to be adjusted into left-handed circularly polarized light and right-handed circularly polarized light by a polarization device to optimize the interaction with the sample.

[0034] Furthermore, the vertically polarized light incident on the sample surface converges into a hollow light spot, and the parallel polarized light converges into a solid light spot, so as to achieve a specific imaging effect.

[0035] Furthermore, the signal is demodulated by a modem device, which can be a hardware means such as a phase-locked amplifier or a software means such as a Fourier transform; during the demodulation process, the background light signal with the same frequency as the vertically polarized light will be removed in the modem device, thereby effectively reducing the Poisson background noise.

[0036] In order to implement the above method, Figure 1As shown, the embodiment of the present application also provides a super-resolution microscopic imaging device for removing Poisson background noise, including: a laser 1, a single-mode optical fiber 2, a collimating lens 3, a first 1 / 2 wave plate 4, a polarization beam splitter 5, a delay device 6, a first 1 / 4 wave plate 7, a second 1 / 2 wave plate 8, a first reflector 9, a third 1 / 2 wave plate 10, a second reflector 11, a spatial light modulator 12, a polarization beam combiner 13, a third reflector 14, a dichroic mirror 15, a scanning galvanometer 16, a second 1 / 4 wave plate 17, a fourth reflector 18, a scanning lens 19, a field lens 20, a microscope objective 21, a sample stage 22, a bandpass filter 23, a collecting lens 24, a pinhole 25, a multimode optical fiber 26, a detector 27, a first acousto-optic modulator 28, a second acousto-optic modulator 29, a signal generator 30, a radio frequency driver 31, a phase-locked amplifier 32, an acquisition card 33 and a controller 34.

[0037] Among them, the single-mode optical fiber 2, the collimating lens 3, the first 1 / 2 wave plate 4, the polarization beam splitter 5, the delay device 6, the first acousto-optic modulator 28, the first 1 / 4 wave plate 7, the second 1 / 2 wave plate 8, and the first reflector 9 are sequentially located on the optical axis of the outgoing light beam of the laser 1; the direction of the transmission axis of the first 1 / 2 wave plate 4 is adjusted so that the intensity of the S polarization component of the light beam after passing through is much greater than the intensity of the P polarization component, with a difference of two orders of magnitude.

[0038] The first acousto-optic modulator 28 is located on the optical axis of the light beam modulated by the polarization beam splitter 5 .

[0039] The time delay device 6 is used to adjust the optical path lengths of the P light and the S light so that the arrival times of the P light and the S light on the sample surface are equal.

[0040] The polarization beam combiner 13 is located on the optical axis of the light beam modulated by the spatial light modulator 12 .

[0041] The third half wave plate 10 is used to compensate the phase of the S light.

[0042] Among them, the third reflector 14 and the dichroic mirror 15 are successively located on the optical axis of the outgoing light of the polarization beam combiner 13 after being deflected by the reflector; the scanning galvanometer 16 is located on the optical axis of the light beam after being reflected by the dichroic mirror 15, and the scanning galvanometer 16 deflects the optical path of the light beam passing through the second 1 / 4 wave plate 17.

[0043] Among them, the scanning lens 19, the field lens 20, the microscope objective lens 21, and the sample stage 22 are sequentially located on the optical axis of the light beam emitted by the scanning galvanometer 16. The scanning lens 19 and the field lens 20 are respectively used to focus and collimate the light beam emitted by the scanning galvanometer.

[0044] Among them, the bandpass filter 23, the collecting lens 24, the pinhole 25, the multimode optical fiber 26, and the detector 27 are successively located on the optical axis of the light beam reflected by the reflector after passing through the dichroic mirror 15; the pinhole 25 is located at the focal plane of the collecting lens 24; the bandpass filter is used to filter out the stray light in the signal light emitted by the dichroic mirror, and the bandpass filter can be removed when the sample to be tested is a non-fluorescent sample, such as by using an electric device of a flip mirror; the detector is used to detect the light intensity signal of the signal light beam, and the detector uses a photomultiplier tube (PMT); the collecting lens is used to focus the filtered signal light beam onto the detector; the pinhole is used to perform spatial filtering on the signal light beam, and it is located at the focal plane of the focusing lens, and the diameter of the pinhole used should be smaller than the diameter of an Airy disk.

[0045] The controller 34 is connected to the spatial light modulator 12 (not shown in the figure) to load the modulation phase of the spatial light modulator 12 ; the controller 34 is connected to the scanning galvanometer 16 to realize the scanning of the scanning galvanometer 16 .

[0046] The signal generator 30 provides different preset frequencies for the RF driver 31, so that the first AOM 28 and the second AOM 29 can generate modulation frequencies of different frequencies; at the same time, the signal generator 30 provides a reference signal to the phase-locked amplifier 32, which can be used to unlock the preset frequency.

[0047] In the above device, the numerical aperture NA of the microscope objective lens 21 is 1.45; the diameter of the pinhole 25 used is 0.65 Airy disk diameter, and the detector 27 is a photomultiplier tube (PMT).

[0048] The phase-locked amplifier 32 compares the frequency of the signal collected by the photomultiplier tube 27 with the preset frequency loaded by the signal generator 30 to achieve signal separation and filtering and gating of specific frequency signals.

[0049] The acquisition card 33 controls the input and output of two-dimensional data to the controller 34 , and reads the signal separated by the lock-in amplifier 32 .

[0050] The RF driver 31 is used to generate ultrasonic waves, so that the refractive index in the first AOM 28 and the second AOM 29 can change periodically. A high-speed lock-in amplifier (LIA) is used to generate a specific signal to modulate the RF driver and output it from the two signal generation channels built into the lock-in amplifier, with an amplitude of 5V and frequencies of f1 and f2 respectively. The fluorescence signal (F hexc ) is considered as background signal and is removed accordingly, e.g. Figure 2 shown.

[0051] The principle of this application is as follows: When the output frequency of the signal generator is f1 and f 2 When f is applied to the solid light spot and the hollow light spot focused on the sample surface, 1 and f 2 The fluorescence signal generated after excitation is also modulated by time, and the formula is as follows:

[0052]

[0053]

[0054] Among them, F exc (x,y,t) and F hexc (x, y, t) are the fluorescence signals generated by the solid spots and hollow spots respectively, which change with time; F exc (x,y) and F hexc (x, y) are the fluorescence signals generated by the solid spot and the hollow spot without time modulation, respectively; f exc (f 1 ,t) and f hexc (f 2 , t) are the modulation functions of solid and hollow spots with respect to frequency and time, respectively. The background signal is related to F hexc Proportional, as shown in the formula:

[0055]

[0056] Among them, c, q, σ are coefficient, quantum efficiency, absorption cross section, and BKGD is background signal. Then we can get low background saturation competition imaging, as shown in the formula:

[0057]

[0058] Among them, dmSAC is the imaging of this application, and SAC is the traditional imaging.

[0059] further, Figure 3 The experimental results of conventional saturation absorption competition microscopy (SAC) and dual modulation SAC (dmSAC) were compared. 40 nm fluorescent microspheres were used for characterization. In SAC, the fluorescent part excited by the hollow beam should be filtered out. However, in the actual phase lock detection, speckle-like noise appeared, such as Figure 3 As shown in a in the figure. The hollow beam power is 300 μW. The above noise becomes the main factor limiting the improvement of SAC resolution and signal-to-noise ratio. Figure 3 Figure b shows the confocal imaging of solid beam scanning. The parameters are as follows: solid beam power, 10 μW; hollow beam power, 300 μW; time constant, 500 μs; modulation frequency f 1 : 200 kHz. When the hollow beam is transmitted at a frequency f 2When modulated at 50 kHz, it is found that the peripheral noise is eliminated, such as Figure 3 As shown in d. When the hollow beam laser power is further increased to 900 μW, a higher resolution can be achieved ( Figure 3 In e), the background noise is effectively suppressed.

[0060] Those skilled in the art can understand that the above are only preferred examples of the invention and are not intended to limit the invention. Although the invention is described in detail with reference to the above examples, those skilled in the art can still modify the technical solutions recorded in the above examples or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A super-resolution microscopy imaging method for removing Poisson background noise, characterized in that: The following steps are involved: Step 1). Collimating and adjusting the laser output by the laser; Step 2). Split the collimated laser beam into two linearly polarized beams through a beam splitter, namely parallel polarized light and vertical polarized light; Step 3). Phase modulating the vertically polarized light; Step 4). The split parallel polarized light and vertical polarized light are modulated into two sequential lights with different preset frequencies by a frequency modulation device; Step 5). Combining the sequential light into a beam of light through a beam combining device; Step 6). Project the combined beam onto the sample surface and perform point scanning on the sample; Step 7). Receive the signal light emitted by the sample during the scanning process, and demodulate the received signal light; Obtain a super-resolution image after removing Poisson background noise.

2. The super-resolution microscopy method according to claim 1, characterized in that: The laser output is continuous light or pulsed light.

3. The super-resolution microscopy method according to claim 1 or 2, characterized in that: The phase modulation device is a vortex phase plate or a spatial light modulator.

4. The super-resolution microscopy method according to claim 1 or 2, characterized in that: The frequency modulation device is an acousto-optic modulator or an electro-optic modulator.

5. The super-resolution microscopic imaging method according to claim 1, characterized in that: The demodulation process is performed by a modem device, which is a phase-locked amplifier; during the demodulation process, the background light signal with the same frequency as the vertically polarized light will be removed in the modem device at the same time.

6. The super-resolution microscopy method according to claim 1, characterized in that: The light beam incident on the sample surface is circularly polarized light; the vertically polarized light in the circularly polarized light converges into a hollow light spot, and the parallel polarized light in the circularly polarized light converges into a solid light spot.

7. The super-resolution microscopic imaging method according to claim 4, characterized in that: The signal generator provides different preset frequencies for the radio frequency driver, so that the acousto-optic modulator can generate modulation frequencies of different frequencies.

8. The super-resolution microscopic imaging method according to claim 7, characterized in that: The demodulation process is performed by a modem device, which is a phase-locked amplifier. The signal generator provides a reference signal to the phase-locked amplifier to unlock a preset frequency.

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