An ultralong focal depth vector polarized light imaging system and method for minimum light flux depth imaging

Abstract

The application discloses a super-long focal depth vector polarized light imaging system and method for minimum light flux depth imaging, and relates to the field of optical imaging. The system comprises a laser for emitting excitation light; a vector polarized light generator for converting the excitation light into vector polarized light; a phase modulation element for phase modulating the vector polarized light; an imaging module comprising a high numerical aperture objective lens and a silicon oil immersion objective lens for high-precision positioning of fluorescent molecules in biological tissues; and a detection module for receiving and detecting fluorescent signals emitted by the fluorescent molecules to realize positioning and imaging analysis of the fluorescent molecules. The application significantly expands the imaging focal depth through vector polarized light and phase modulation, and can realize high-resolution imaging in tissues with a depth of more than 1 micrometer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical imaging, and more particularly to an ultra-long focal depth vector polarized light imaging system and method for minimum light flux depth imaging. BACKGROUND

[0002] Minimum light flux super-resolution nanoscale imaging technology is a high-resolution fluorescence microscope technology that can locate single fluorescent molecules at the nanoscale. However, traditional minimum light flux super-resolution imaging is affected by optical distortion, absorption and scattering when imaging in depth, limiting its application in biological tissues. In the prior art, methods for extending the focal depth usually sacrifice lateral resolution, and the present application aims to solve this problem. SUMMARY

[0003] Therefore, the present application provides an ultra-long focal depth vector polarized light imaging system and method for minimum light flux depth imaging.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0005] An ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging, comprising:

[0006] a laser for emitting excitation light; a vector polarized light generator for converting the excitation light into vector polarized light; a phase modulation element for phase modulating the vector polarized light; an imaging module comprising a high numerical aperture objective lens and a silicon oil immersion objective lens for high-precision positioning of fluorescent molecules in biological tissues; and a detection module for receiving and detecting fluorescent signals emitted by the fluorescent molecules to achieve positioning and imaging analysis of the fluorescent molecules.

[0007] Optionally, it further comprises a real-time anti-drifting module, which comprises: an infrared laser for providing an infrared laser beam; a third polarization beam splitter for reflecting the infrared laser beam; a λ / 4 wave plate for changing the polarization direction of the infrared laser beam; a second dichroic mirror for reflecting the infrared laser beam so that it passes through the objective lens to achieve wide-field illumination; and a high-frame-rate camera for receiving and detecting scattering signals generated by the interaction of the infrared laser beam with gold nanoparticles on the fluorescent sample to perform real-time position estimation on the fluorescent sample and real-time position compensation by the sample stage.

[0008] Optionally, the imaging module further comprises a first dichroic mirror and a first filter, the first dichroic mirror transmits the excitation light and reflects the fluorescent signal, and the first filter is used to transmit the fluorescent signal so that the detection module receives the fluorescent signal.

[0009] Optionally, the detection module includes a single-photon detector for high-sensitivity detection of the fluorescence signal.

[0010] Optionally, the laser, vector polarization light generator, phase modulation element, imaging module, and detection module are sequentially arranged in the optical path, and the real-time anti-drift module is arranged in parallel with the imaging module in the optical path.

[0011] Optionally, the detection module includes a single-photon detector for high-sensitivity detection of the fluorescence signal.

[0012] A method for ultra-long depth-of-focus vector polarized light imaging for minimum luminous flux depth imaging, utilizing any one of the ultra-long depth-of-focus vector polarized light imaging systems for minimum luminous flux depth imaging, is characterized by comprising the following steps:

[0013] Use a laser to provide the excitation light;

[0014] The excitation light is converted into vector-polarized light using a vector-polarized light generator;

[0015] A phase modulation element is introduced into the optical path to modulate the phase of the vector polarized light, thereby forming a needle-like or tunnel-like light intensity distribution at the focal point and thus extending the depth of focus.

[0016] The vector-polarized light is focused onto fluorescent molecules in biological tissue, exciting the fluorescent molecules to emit fluorescent signals.

[0017] The fluorescence signal is received by the imaging module and detected by the detection module to achieve high-precision positioning and imaging analysis of the fluorescent molecules.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention provides an ultra-long focal depth vector polarized light imaging system and method for minimum light flux depth imaging, which has the following beneficial effects:

[0019] 1. Ultra-long depth of focus: Through vector polarization and phase modulation, the imaging depth of focus is significantly extended, enabling high-resolution imaging of tissues with a depth of more than 1 micrometer.

[0020] 2. Maintain lateral resolution: While expanding the depth of focus, the lateral resolution is not sacrificed, thus maintaining the high-precision positioning capability of the minimum light flux super-resolution technology.

[0021] 3. Adaptability to complex environments: This method is more robust to optical distortion and scattering, and is suitable for complex imaging environments such as biological tissues.

[0022] 4. Compatibility: This technology can be seamlessly integrated into existing minimum light throughput super-resolution imaging systems without complex hardware modifications. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 This is an optical schematic diagram of an ultra-long depth-of-focus imaging system.

[0025] Figure 2 This is a diagram showing the intensity distribution and phase modulation effect of vector-polarized light.

[0026] Figure 3 Figure showing the experimental results of performance improvement of ultra-long focal depth imaging methods in depth imaging;

[0027] In this array, 1 is a laser, 2 is a first polarization beam splitter, 3 is a vector polarization light generator, 4 is a 0 / π phase plate, 5 is a second polarization beam splitter, 6 is a first reflecting mirror, 7 is a 0-2π phase plate, 8 is a second reflecting mirror, 9 is a third reflecting mirror, 10 is an electro-optic deflector, 11 is a first dichroic mirror, 12 is a second dichroic mirror, 13 is a silicone oil objective lens, 14 is a sample stage, 15 is a first filter, 16 is a single-photon detector, 17 is an infrared laser, 18 is a third polarization beam splitter, 19 is a λ / 4 waveplate, 20 is a second filter, and 21 is a high frame rate camera. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention discloses an ultra-long focal depth vector polarization imaging system for minimum luminous flux depth imaging, comprising:

[0030] A laser is used to emit excitation light; a vector polarization light generator is used to convert the excitation light into vector polarization light; a phase modulation element is used to modulate the phase of the vector polarization light; an imaging module includes a high numerical aperture objective lens and a silicone oil immersion objective lens for high-precision localization of fluorescent molecules in biological tissues; and a detection module is used to receive and detect the fluorescence signals emitted by the fluorescent molecules to achieve localization and imaging analysis of the fluorescent molecules.

[0031] With the above system, the embodiment also discloses a super-long focal depth vector polarized light imaging method for minimum light flux depth imaging, which is characterized by comprising the following steps:

[0032] Providing excitation light by using a laser;

[0033] Converting the excitation light into vector polarized light by a vector polarized light generator;

[0034] Introducing a phase modulation element in an optical path to perform phase modulation on the vector polarized light to form a needle-shaped or tunnel-shaped light intensity distribution at a focal point, thereby extending the focal depth;

[0035] Focusing the vector polarized light on fluorescent molecules in biological tissues to excite the fluorescent molecules to emit fluorescent signals;

[0036] Receiving the fluorescent signals by an imaging module and detecting the fluorescent signals by a detection module to realize high-precision positioning and imaging analysis of the fluorescent molecules.

[0037] Specifically, the vector polarized light is generated as follows:

[0038] The embodiment uses cylindrical vector polarized light (for example, azimuthal polarized light) as excitation light, and the polarization direction thereof depends on the azimuthal angle. Phase modulation is performed on the excitation light by a phase plate or a spatial light modulator (SLM) to optimize the focal depth and the light intensity distribution. A circularly symmetric 0 / π phase plate is introduced in the optical path to divide the excitation light into two parts: unmodulated light and modulated light. By interference effect, the two parts of light form a needle-shaped or tunnel-shaped light intensity distribution at the focal point, thereby significantly extending the focal depth. The above vector polarized light and phase modulation technology are integrated into a minimum light flux super-resolution imaging system. By using the excitation light mode with extended focal depth, high-precision positioning of fluorescent molecules in biological tissues is realized, and single-nanometer-level resolution can be maintained even at a depth of more than 1 micrometer.

[0039] Further, the cylindrical vector polarized light is azimuthal polarized light; the phase modulation element is a circularly symmetric 0 / π phase plate or a spatial light modulator (SLM); the imaging system comprises a high numerical aperture objective lens and a silicon oil immersion objective lens to improve refractive index matching; the phase modulation element is a circularly symmetric 0 / π phase plate or a spatial light modulator (SLM); and the imaging module comprises a high numerical aperture objective lens and a silicon oil immersion objective lens. The device of the embodiment can realize sub-ten nanometer positioning accuracy of a single fluorophore in a fluorescent molecule solution with a depth of more than 1 micrometer.

[0040] As Figure 1The diagram shows the optical principle of the ultra-long depth-of-focus imaging system in this embodiment. The device includes: a laser 1, a first polarization beam splitter 2, a vector polarization light generator 3, a 0 / π phase plate 4, a second polarization beam splitter 5, a first mirror 6, a 0-2π phase plate 7, a second mirror 8, a third mirror 9, an electro-optic deflector 10, a first dichroic mirror 11, a second dichroic mirror 12, a silicone oil objective lens 13, a sample stage 14, a first filter 15, a single-photon detector 16, an infrared laser 17, a third polarization beam splitter 18, a λ / 4 waveplate 19, a second filter 20, and a high frame rate camera 21.

[0041] Figure 2 This is a diagram showing the intensity distribution of the light beam along the XZ direction. Figure 2 The left side shows the results of azimuth polarization and 0 / π phase modulation. Figure 2 The middle section shows the 0-2π phase modulation result. Figure 2 The right side shows the result of superimposing the first two modulation methods.

[0042] In this process, the beam emitted from laser 1 is split into two beams by the first polarization beam splitter 2. The vector polarization light generator 3 and the 0 / π phase plate 4 are aligned on the transmitted optical axis, producing beams such as... Figure 2 The light intensity distribution shown on the left; the 0-2π phase plate 7, the first reflecting mirror 6, and the second reflecting mirror 8 are on the reflected optical axis, producing a light intensity distribution as shown on the left. Figure 2 The light intensity distribution shown in the middle section. The two beams are combined into one beam by the second polarizing beam splitter 5, producing a result as shown in the image. Figure 2 The light intensity distribution is shown on the right. The light beam is deflected by the third reflecting mirror 9 and enters the electro-optic deflector 10, causing a small-range, high-speed deflection of the beam at a fixed position in the XY direction. Subsequently, after passing through the first dichroic mirror 11, the second dichroic mirror 12, and the silicone oil objective lens 13, the beam is focused onto the fluorescent sample on the sample stage 14, exciting a fluorescence signal. This fluorescence signal is reflected by the first dichroic mirror 11, transmitted through the first filter 15, and enters the single-photon detector 16. The obtained fluorescence signal is used for localization and imaging analysis of the fluorescent sample.

[0043] In addition, the device is equipped with a real-time anti-drift module. The infrared laser 17 emits an infrared laser beam, which is reflected by the third polarization beam splitter 18, transmitted through the λ / 4 waveplate 19, and reflected by the second dichroic mirror 12. The beam is then used to achieve wide-field illumination through the objective lens, causing scattering of the gold nanoparticles on the fluorescent sample. This scattered signal is reflected by the second dichroic mirror 12, passes through the λ / 4 waveplate 19 a second time, and is transmitted through the third polarization beam splitter 18. After passing through the second filter 20, it enters the high frame rate camera 21. The obtained scattered signal is used to estimate the position of the fluorescent sample in real time, and the sample stage 14 performs real-time position compensation, thus achieving the effect of real-time anti-drift.

[0044] In this embodiment, the vector polarized light generator used is azimuth-polarized light.

[0045] Among them, the first dichroic mirror 11 transmits the laser beam but reflects the fluorescence signal.

[0046] Among them, the second dichroic mirror 12 transmits both laser beams and fluorescence signals, while reflecting infrared laser signals.

[0047] The infrared laser beam passes through the λ / 4 waveplate 19 twice, and its polarization direction is rotated by 90 degrees, which is beneficial for the transmission of its scattered signal to the third polarization beam splitter 18.

[0048] In this embodiment, a silicone oil immersion objective with a high numerical aperture (NA) (e.g., NA = 1.35) is used to improve refractive index matching and eliminate the spherical aberration effect during depth imaging.

[0049] In this case, adding a circular symmetrical 0 / π phase plate 4 to the optical path can also enable phase modulation via SLM.

[0050] The 0-2π phase plate 7 added to the optical path can also be phase modulated by SLM.

[0051] The experimental steps in this embodiment are as follows:

[0052] Place fluorescently labeled biological samples into an imaging system;

[0053] Adjust the parameters of the phase plate and SLM to optimize the depth of focus and intensity distribution of the excitation light;

[0054] The sample was scanned using a minimum light flux super-resolution imaging system to record the localization information of fluorescent molecules;

[0055] Data analysis was used to verify the improvement in depth and resolution achieved by the ultra-long depth-of-focus imaging method.

[0056] like Figure 3 As shown, this method achieves a localization effect in a single-molecule solution. Fluorescent molecules are randomly distributed at focal planes of -600nm, -300nm, 0nm, +300nm, and +600nm. The leftmost column shows the actual distribution of fluorescent molecules; the rightmost column shows the localization results of traditional minimum flux super-resolution microscopy, which cannot resolve most fluorescent molecules at large focal depths; conversely, the middle column shows the implementation effect of this method, which can resolve most fluorescent molecules at large focal depths. Therefore, this method achieves sub-10 nanometer localization accuracy for single fluorophores in fluorescent molecule solutions with depths exceeding 1 micrometer.

[0057] Compared with the traditional minimum light flux super-resolution imaging, the super-long focal depth imaging method significantly improves the performance of depth imaging while maintaining high resolution.

[0058] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts between the various embodiments can be referred to each other. For the device disclosed by the embodiments, since it corresponds to the method disclosed by the embodiments, the description is relatively simple, and the related parts can be referred to the method part.

[0059] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging, characterized in that, Comprise: laser, first polarization beam splitter, vector polarized light generator, 0 / π phase plate, second polarization beam splitter, first mirror, 0-2π phase plate, second mirror, third mirror, electro-optical deflector, first dichroic mirror, second dichroic mirror, silicon oil objective, sample stage, first filter, single photon detector, infrared laser, third polarization beam splitter, λ / 4 wave plate, second filter, high frame rate camera; laser, for emitting excitation light; vector polarized light generator, for converting the excitation light into vector polarized light; phase modulation element, for phase modulating the vector polarized light; imaging module, comprising high numerical aperture objective and silicon oil immersion objective, for high precision positioning of fluorescent molecules in biological tissue; detection module, for receiving and detecting the fluorescent signal emitted by the fluorescent molecules, to realize positioning and imaging analysis of the fluorescent molecules; further comprising real-time anti-drifting module, the real-time anti-drifting module comprises: infrared laser, for providing infrared laser beam; third polarization beam splitter, for reflecting the infrared laser beam; λ / 4 wave plate, for changing the polarization direction of the infrared laser beam; second dichroic mirror, for reflecting the infrared laser beam, so that it passes through the objective to realize wide field illumination; high frame rate camera, for receiving and detecting the scattering signal generated by the interaction of the infrared laser beam and the gold nanoparticles on the fluorescent sample, to realize real-time position estimation of the fluorescent sample, and real-time position compensation by the sample stage; the laser, vector polarized light generator, phase modulation element, imaging module and detection module are sequentially arranged on the optical path, and the real-time anti-drifting module is arranged in parallel with the imaging module on the optical path; wherein the light beam emitted by the laser is divided into two beams by the first polarization beam splitter, the vector polarized light generator and the 0 / π phase plate are on the transmission optical axis to generate light intensity distribution; the 0-2π phase plate, the first mirror and the second mirror are on the reflection optical axis to generate light intensity distribution; the two beams of light are combined into one beam by the second polarization beam splitter.

2. The ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging according to claim 1, characterized in that, The imaging module further comprises a first dichroic mirror and a first filter, the first dichroic mirror transmits the excitation light and reflects the fluorescent signal, and the first filter is used to transmit the fluorescent signal so that the detection module receives the fluorescent signal.

3. The ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging according to claim 1, characterized in that, The detection module comprises a single photon detector for high sensitivity detection of the fluorescent signal.

4. The ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging according to claim 1, characterized in that, The detection module comprises a single photon detector for high sensitivity detection of the fluorescent signal.

5. An ultralong focal depth vector polarized light imaging method for minimum light flux depth imaging, using the ultralong focal depth vector polarized light imaging system for minimum light flux depth imaging according to any one of claims 1-4, characterized in that, Comprise the following steps: using a laser to provide excitation light; converting the excitation light into vector polarized light by a vector polarized light generator; introducing a phase modulation element in the optical path to phase modulate the vector polarized light to form a needle-shaped or tunnel-shaped light intensity distribution at the focal point, thereby expanding the focal depth; focusing the vector polarized light on the fluorescent molecules in biological tissue to excite the fluorescent molecules to emit fluorescent signals; receiving the fluorescent signals by an imaging module and detecting the fluorescent signals by a detection module to realize high precision positioning and imaging analysis of the fluorescent molecules.

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