Ultra-long focal depth vector polarized light imaging system and method for minimum luminous flux depth imaging

Abstract

The invention discloses an ultra-long focal depth vector polarized light imaging system and method for minimum luminous flux depth imaging, and relates to the field of optical imaging. Comprising a laser used for emitting exciting light; the vector polarized light generator is used for converting the exciting light into vector polarized light; the phase modulation element is used for performing phase modulation on the vector polarized light; the imaging module comprises a high numerical aperture objective lens and a silicone oil immersion objective lens and is used for performing high-precision positioning on fluorescent molecules in the biological tissue; and the detection module is used for receiving and detecting fluorescence signals emitted by the fluorescence molecules so as to realize positioning and imaging analysis of the fluorescence molecules. Through vector polarized light and phase modulation, the imaging focal depth is obviously expanded, and high-resolution imaging can be carried out in tissues with the depth exceeding 1 micron.

Description

Technical Field

[0001] The present invention 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 Art

[0002] Minimum-flux super-resolution nanoimaging is a high-resolution fluorescence microscopy technique capable of localizing single fluorescent molecules at the nanometer scale. However, conventional minimum-flux super-resolution imaging suffers from optical distortion, absorption, and scattering during deep imaging, limiting its application in biological tissues. Existing methods for extending the depth of focus often sacrifice lateral resolution, a problem addressed by the present invention. Summary of the Invention

[0003] In view of this, the present invention provides an ultra-long focal depth vector polarized light imaging system and method for minimum light flux depth imaging.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

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

[0006] A laser is used to emit excitation light; a vector polarized light generator is used to convert the excitation light into vector polarized light; a phase modulation element is used to phase modulate the vector polarized light; an imaging module, including a high numerical aperture objective lens and a silicone oil immersion objective lens, is used to perform high-precision positioning of fluorescent molecules in biological tissues; and a detection module is used to receive and detect the fluorescent signals emitted by the fluorescent molecules to achieve positioning and imaging analysis of the fluorescent molecules.

[0007] Optionally, a real-time anti-drift module is also included, which includes: 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 can achieve wide-field illumination through the objective lens; and a high-frame rate camera for receiving and detecting the scattered signal generated by the interaction between the infrared laser beam and the gold nanoparticles on the fluorescent sample, so as to perform real-time position estimation of the fluorescent sample and perform real-time position compensation by the sample stage.

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

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

[0010] Optionally, 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-drift module and the imaging module are arranged in parallel on the optical path.

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

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

[0013] Using a laser to provide excitation light;

[0014] Converting the excitation light into vector polarized light by a vector polarized light generator;

[0015] Introducing a phase modulation element into the optical path to phase modulate the vector polarized light to form a needle-shaped or tunnel-shaped light intensity distribution at the focus, thereby extending the focal depth;

[0016] focusing the vector polarized light onto fluorescent molecules in biological tissue to excite the fluorescent molecules to emit fluorescent signals;

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

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

[0019] 1. Ultra-long focal depth: Through vector polarization and phase modulation, the imaging focal depth is significantly extended, enabling high-resolution imaging in tissues with a depth exceeding 1 micron.

[0020] 2. Maintaining lateral resolution: While extending the focal depth, the lateral resolution is not sacrificed, 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 flux super-resolution imaging systems without the need for complex hardware modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 This is the optical principle diagram of the ultra-long focal depth imaging system;

[0025] Figure 2 This is the intensity distribution and phase modulation effect diagram of vector polarized light;

[0026] Figure 3 This figure shows the experimental results of the performance improvement of the ultra-long focal depth imaging method in depth imaging;

[0027] Among them, 1 is a laser, 2 is the 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 reflector, 7 is a 0-2π phase plate, 8 is a second reflector, 9 is a third reflector, 10 is an electro-optical 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 wave plate, 20 is a second filter, and 21 is a high frame rate camera. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] An embodiment of the present invention discloses an ultra-long focal depth vector polarized light imaging system for minimum light flux depth imaging, comprising:

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

[0031] Utilizing the above system, this embodiment further discloses a method for ultra-long focal depth vector polarization imaging for minimum light flux depth imaging, which is characterized by comprising the following steps:

[0032] Using a laser to provide excitation light;

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

[0034] Introducing a phase modulation element into the optical path to phase modulate the vector polarized light to form a needle-shaped or tunnel-shaped light intensity distribution at the focus, thereby extending the focal depth;

[0035] focusing the vector polarized light onto fluorescent molecules in biological tissue to excite the fluorescent molecules to emit fluorescent signals;

[0036] The fluorescence signal is received by an imaging module and detected by a detection module to achieve high-precision positioning and imaging analysis of the fluorescent molecules.

[0037] Specifically, the generation of vector polarized light:

[0038] This embodiment uses cylindrical vector polarized light (such as azimuthally polarized light) as excitation light, and its polarization direction depends on the azimuth angle. The excitation light is phase modulated by a phase plate or a spatial light modulator (SLM) to optimize the depth of focus and light intensity distribution. A circularly symmetrical 0 / π phase plate is introduced into the optical path to divide the excitation light into two parts: unmodulated light and modulated light. Through the interference effect, the two parts of light form a needle-shaped or tunnel-shaped light intensity distribution at the focus, thereby significantly extending the depth of focus. The above-mentioned vector polarized light and phase modulation technology are integrated into a minimum flux super-resolution imaging system. By utilizing the excitation light mode with extended focal depth, high-precision positioning of fluorescent molecules in biological tissues can be achieved, and single-nanometer resolution can be maintained even at positions with a depth exceeding 1 micron.

[0039] Furthermore, the cylindrical vector polarized light is azimuthally polarized light; the phase modulation element is a circularly symmetric 0 / π phase plate or a spatial light modulator (SLM); the imaging system includes a high numerical aperture objective lens and a silicone 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 includes a high numerical aperture objective lens and a silicone oil immersion objective lens. The device of the present invention can achieve sub-ten-nanometer positioning accuracy of a single fluorophore in a fluorescent molecular solution at a depth exceeding 1 micron.

[0040] like Figure 1As shown, the optical principle diagram of the ultra-long focal depth imaging system of 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 reflector 6, a 0-2π phase plate 7, a second reflector 8, a third reflector 9, an electro-optical 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 wave plate 19, a second filter 20, and a high frame rate camera 21.

[0041] Figure 2 is the light intensity distribution diagram of the beam in the XZ direction, Figure 2 The left side shows the azimuth polarization and 0 / π phase modulation results. Figure 2 The middle part is the 0-2π phase modulation result. Figure 2 The right side shows the result of superposition of the first two modulations.

[0042] The light beam emitted by the 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 on the optical axis of the transmission to generate the following Figure 2 The light intensity distribution shown on the left; 0-2π phase plate 7, second reflector 8 and third reflector 9 are on the reflected optical axis, producing Figure 2 The light intensity distribution shown in the middle. The two beams are combined into one beam by the second polarization beam splitter 5, producing Figure 2 The light intensity distribution is shown on the right. The light beam is deflected by the third reflector 9 and enters the electro-optical deflector 10, which deflects the beam within a fixed position within a small range in the X and Y directions at high speed. The beam then passes through the first dichroic mirror 11, the second dichroic mirror 12, and the silicone oil objective lens 13, focusing the beam onto the fluorescent sample on the sample stage 14, stimulating a fluorescent signal. This fluorescent signal is reflected by the first dichroic mirror 11, transmitted through the first filter 15, and enters the single-photon detector 16. The resulting fluorescence signal is used for localization and imaging analysis of the fluorescent sample.

[0043] The device is also equipped with a real-time drift prevention module. An infrared laser beam emitted by an infrared laser 17 is reflected by a third polarization beam splitter 18, transmitted through a λ / 4 wave plate 19, and reflected by a second dichroic mirror 12. Wide-field illumination is achieved through the objective lens, which scatters the gold nanoparticles on the fluorescent sample. This scattered signal is reflected by the second dichroic mirror 12, passes through the λ / 4 wave plate 19 a second time, and transmits through the third polarization beam splitter 18. It then passes through a second filter 20 and enters a high-frame-rate camera 21. The resulting scattered signal is used to estimate the position of the fluorescent sample in real time and is compensated for by the sample stage 14 in real time, achieving real-time drift prevention.

[0044] The vector polarized light generator used in this embodiment generates azimuthally polarized light.

[0045] The first dichroic mirror 11 transmits the laser beam and reflects the fluorescent signal.

[0046] The second dichroic mirror 12 transmits the laser beam and the fluorescent signal, but reflects the infrared laser.

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

[0048] In this embodiment, a silicone oil immersion objective lens with a high numerical aperture (NA) (eg, NA=1.35) is used to improve refractive index matching and eliminate the influence of spherical aberration caused by deep imaging.

[0049] In this case, a circularly symmetrical 0 / π phase plate 4 is added to the optical path, and phase modulation can also be performed through the SLM.

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

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

[0052] placing a fluorescently labeled biological sample in an imaging system;

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

[0054] Use a minimum light flux super-resolution imaging system to scan the sample and record the positioning information of fluorescent molecules;

[0055] Through data analysis, the improvement in depth and resolution of the ultra-long focal depth imaging method was verified.

[0056] like Figure 3 As shown, in a single-molecule solution, the positioning effect that can be achieved by this method. Some fluorescent molecules are randomly distributed at the focal planes of -600nm, -300nm, 0nm, +300nm, and +600nm. The leftmost column shows the actual distribution of fluorescent molecules; the rightmost column shows the positioning of traditional minimum flux super-resolution microscopy. It can be seen that it cannot distinguish most fluorescent molecules under large focal depths; in contrast, the middle column shows the implementation effect adopted in this method, which can distinguish most fluorescent molecules under large focal depths. It can be seen that this method achieves sub-ten-nanometer positioning accuracy of single fluorophores in fluorescent molecule solutions with a depth of more than 1 micron.

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

[0058] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method description.

[0059] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging, characterized in that: include: A laser, used for emitting excitation light; A vector polarized light generator, used for converting the excitation light into vector polarized light; A phase modulation element is used to phase modulate the vector polarized light; an imaging module includes a high numerical aperture objective lens and a silicone oil immersion objective lens, which are used to perform high-precision positioning of fluorescent molecules in biological tissues; and a detection module is used to receive and detect the fluorescent signals emitted by the fluorescent molecules to achieve positioning and imaging analysis of the fluorescent molecules.

2. The ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging according to claim 1, characterized in that: It also includes a real-time anti-drift module, which includes: 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 can achieve wide-field illumination through the objective lens; and a high-frame rate camera for receiving and detecting the scattered signal generated by the interaction between the infrared laser beam and the gold nanoparticles on the fluorescent sample, so as to perform real-time position estimation of the fluorescent sample and perform real-time position compensation by the sample stage.

3. The ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging according to claim 1, characterized in that: The imaging module further includes a first dichroic mirror and a first filter. The first dichroic mirror transmits the excitation light and reflects the fluorescence signal. The first filter is used to transmit the fluorescence signal so that the detection module can receive the fluorescence signal.

4. The ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging according to claim 1, characterized in that: The detection module includes a single-photon detector for detecting the fluorescence signal with high sensitivity.

5. The ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging according to claim 2, characterized in that: The laser, vector polarized light generator, phase modulation element, imaging module and detection module are sequentially arranged on an optical path, and the real-time anti-drift module and the imaging module are arranged in parallel on the optical path.

6. The ultra-long focal depth vector polarization light imaging system for minimum light flux depth imaging according to claim 1, characterized in that: The detection module includes a single-photon detector for detecting the fluorescence signal with high sensitivity.

7. A method for ultra-long focal depth vector polarization imaging for minimum light flux depth imaging, utilizing the ultra-long focal depth vector polarization imaging system for minimum light flux depth imaging according to any one of claims 1 to 6, characterized in that: The following steps are involved: 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 into the optical path to phase modulate the vector polarized light to form a needle-shaped or tunnel-shaped light intensity distribution at the focus, thereby extending the focal depth; focusing the vector polarized light onto fluorescent molecules in biological tissue to excite the fluorescent molecules to emit fluorescent signals; The fluorescence signal is received by an imaging module and detected by a detection module to achieve high-precision positioning and imaging analysis of the fluorescent molecules.

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