WO2020107140A1

WO2020107140A1 - Super-resolution stimulated emission depletion fluorescence lifetime imaging device

Info

Publication number: WO2020107140A1
Application number: PCT/CN2018/117378
Authority: WO
Inventors: 严伟; 屈军乐; 王璐玮; 叶彤
Original assignee: 深圳大学
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2020-06-04

Abstract

A super-resolution stimulated emission depletion fluorescence lifetime imaging device, used for live cell imaging and resolving the problem of low accuracy of live cell imaging in the prior art, and comprising: a depletion laser provider (Laser1); a depletion laser adjustment component provided at the output end of the depletion laser provider (Laser1); an excitation laser provider (Laser2) provided at one side of the depletion laser provider (Laser1); a synchronization controller provided between the excitation laser provider (Laser2) and the depletion laser provider (Laser1); an excitation laser adjustment component provided at the output end of the excitation laser provider (Laser2); a vortex phase plate (VPP) provided on one side of the depletion laser adjustment component; a quarter waveplate (QWP) provided on one side of the vortex phase plate (VPP); a second dichroic mirror (DM2) provided on one side of the quarter waveplate (QWP); a first dichroic mirror (DM1) provided on one side of the second dichroic mirror (DM2); a sample fluorescence acquisition component provided on one side of the first dichroic mirror (DM1); a fluorescence imaging component provided on one side of the second dichroic mirror (DM2); and a reference fluorescence signal collection component provided on one side of the excitation laser adjustment component, so that the accuracy of live cell imaging can be improved.

Description

Excited emission loss fluorescence lifetime super-resolution imaging device

Technical field

The invention relates to the technical field of optical microscopic imaging, in particular to a super-resolution imaging device for stimulated emission loss fluorescence lifetime.

Background technique

Light has a wave-particle duality, and the wave nature of light is reflected in the fact that it is an electromagnetic wave, with the properties of electric field, magnetic field and propagation direction perpendicular to each other; and interference phenomenon will occur during the propagation process, forming interference fringes between light and dark. Therefore, an ideal point light source is imaged by the optical system. Due to the existence of the diffraction phenomenon, the ideal image point cannot be obtained, but a Fraunhofer diffraction image with a certain size. The particle nature of light is reflected in its discrete energies, which are photons. As an actual object, photons exchange energy with other substances during propagation, and this change in energy becomes the basis of spectroscopy.

At the end of the 20th century, German scientist Stefan W. Hell proposed Stimulated Emission Depletion (STED) technology, which improved the resolution of the optical microscope by an order of magnitude. A typical STED super-resolution system requires two beams of light: One beam is excitation light and the other is loss light. The core idea of STED super-resolution technology is to use stimulated emission to selectively deplete the excited state fluorescent molecules in the edge area of the spot, thereby reducing the luminous range of effective fluorescence and compressing the effective PSF scale to improve the system resolution.

technical problem

In the process of using the STED microscope, in order to improve the imaging resolution, it is necessary to apply extremely high laser power (laser power>100mW) to the sample, and excessive loss of laser power will damage the sample and cause the photobleaching effect of the fluorescent molecules. Therefore, the imaging accuracy of living cells using STED microscopy is low.

Technical solution

The invention provides a stimulated emission loss fluorescence lifetime super-resolution imaging device, comprising: a loss laser providing part for providing a loss laser; a loss laser adjusting component provided at an output end of the loss laser providing part for adjusting the loss The linear polarization direction of the laser and the intensity of the laser, and widening the depletion laser; the excitation laser provider provided on the side of the depletion laser provider for providing excitation laser; the excitation laser provider and the laser A synchronous controller between the loss laser provider; an excitation laser adjustment component provided at the output end of the excitation laser provider for adjusting the polarization characteristic and intensity of the excitation laser, and controlling the excitation laser and the loss laser Pulse interval, and separate the excitation laser; a spiral phase plate provided on the side of the lossy laser adjustment component away from the lossy laser supply; a glass plate provided on the side of the spiral phase plate away from the lossy laser adjustment component Film; a second dichroic mirror disposed on the side of the slide away from the lossy laser adjustment assembly, the first light exit port of the second dichroic mirror is used to transmit lossy laser light, and the second light exit of the second dichroic mirror The port is used to reflect fluorescence; the first dichroic mirror disposed on the side of the second dichromatic mirror away from the slide and located on the optical path of the depletion laser and the excitation laser at the same time is used to transmit the depletion laser and reflect the excitation laser, Fine-tune the transmission direction of the excitation laser; the sample fluorescence collection component provided on the side of the first dichromatic mirror; the fluorescence imaging component provided on the side of the second light exit port; the reference provided on the side of the excitation laser adjustment component Fluorescent signal collection assembly.

Beneficial effect

By using excitation laser and depletion laser, and adjusting the excitation laser and depletion laser, it is possible to use stimulated emission to selectively deplete the excited state fluorescent molecules in the edge area of the laser spot. After the depletion laser is superimposed on the excitation laser spot, the laser spot is excited The surrounding superimposed area is subjected to strong stimulated radiation, and the excited state fluorescent molecules will quickly radiate energy back to the ground state, so it can be seen that the lifetime of the fluorescent molecules in the superimposed area around the excitation laser spot changes greatly; while the excited fluorescent molecules in the center area The effect of the received radiation is small, or it is not affected by the radiation, and it still emits fluorescence in the form of spontaneous radiation. Therefore, it can be seen that the life span of the fluorescent molecules in the central area changes little, so that when extracting the fluorescent signal generated by the luminescence information of the fluorescent molecules, it can be obtained At the same time as the fluorescence intensity of the fluorescent molecule, the fluorescence lifetime information of the fluorescent molecule is obtained, thereby realizing fluorescence lifetime imaging with super-resolution information and improving the accuracy of live cell imaging.

BRIEF DESCRIPTION

FIG. 1 is a schematic structural diagram of a stimulated emission loss fluorescence lifetime super-resolution imaging device according to an embodiment of the present invention.

Best Mode of the Invention

A stimulated emission loss fluorescence lifetime super-resolution imaging device, comprising: a loss laser provider for supplying loss laser; a loss laser adjustment component provided at the output end of the loss laser provider for adjusting the line of the loss laser Polarization direction and laser intensity, and the loss laser is broadened; the excitation laser provider provided on one side of the loss laser provider is used to provide excitation laser; the excitation laser provider and the loss laser are provided A synchronous controller between the supply parts; an excitation laser adjustment component provided at the output end of the excitation laser supply part for adjusting the polarization characteristics and intensity of the excitation laser, and controlling the pulse interval between the excitation laser and the depletion laser, And separate the excitation laser; a spiral phase plate provided on the side of the lossy laser adjustment component away from the lossy laser providing member; a glass slide provided on the side of the spiral phase plate away from the lossy laser adjustment component; A second dichroic mirror on the side of the slide away from the lossy laser adjustment assembly, the first light exit port of the second dichroic mirror is used to transmit loss laser, and the second light exit port of the second dichroic mirror is used for Reflective fluorescence; a first dichroic mirror disposed on the side of the second dichroic mirror away from the slide and on the optical path of the depletion laser and excitation laser at the same time, used to transmit the depletion laser, reflect the excitation laser, and fine-tune the excitation laser The transmission direction of the sample; the sample fluorescence collection component provided on the side of the first dichromatic mirror; the fluorescence imaging component provided on the side of the second light exit port; the reference fluorescence signal collection provided on the side of the excitation laser adjustment component Components.

Further, the lossy laser adjustment component includes: a second half-wave plate disposed at the output end of the lossy laser provider; a second gland disposed on a side of the second half-waveplate away from the lossy laser provider A laser prism; a widened glass rod provided on the side of the second Glan laser prism away from the second half-wave plate.

Further, the lossy laser adjustment assembly further includes: a third lens disposed on a side of the widened glass rod away from the second Glan laser prism; a third lens disposed on a side of the third lens remote from the widened glass rod Single-mode polarization-maintaining fiber; a fourth lens disposed on the side of the single-mode polarization-maintaining fiber away from the third lens; a third half disposed on the side of the fourth lens away from the single-mode polarization-maintaining fiber A wave plate; a third Glan laser prism disposed on a side of the third half wave plate away from the fourth lens.

Further, the excitation laser adjustment assembly includes: a single-mode optical fiber disposed at the output end of the excitation laser supply; a first lens disposed at the output end side of the single-mode optical fiber; the first lens disposed away from the single A first half-wave plate on the side of the mode fiber; a corner reflector provided on the side of the first half-wave plate away from the first lens, the first half-wave plate is located on the incident light of the corner reflector On the road; a first Granite laser prism disposed on the optical path of the corner reflector to reflect light is used to divide the excitation laser into two beams.

Further, the sample fluorescence collection component includes: a galvanometer scanner disposed on the transmission light path and the reflection light path of the first dichromatic mirror, and used for synchronous scanning of the overlapping excitation laser and loss laser to realize the sample Area array imaging; a fifth lens disposed on the light exit side of the galvanometer scanner; a sixth lens disposed on the side of the fifth lens away from the galvanometer scanner; disposed on the sixth lens A high-power numerical aperture objective lens on a side far from the fifth lens; a stage provided on the high-power numerical aperture objective lens on a side away from the sixth lens.

Further, the reference fluorescence signal collection component includes: a laser detector disposed on one side of the excitation laser adjustment component; a time-dependent single photon counter disposed at the output end of the laser detector, the time-dependent single photon counter output The end is coupled to the fluorescence imaging assembly.

Further, the fluorescent imaging assembly includes: a seventh lens disposed on the reflected optical path of the second dichromatic mirror; a filter disposed on the side of the seventh lens away from the second dichromatic mirror; The filter is away from the multimode fiber on the side of the seventh lens; a photomultiplier tube is provided at the output end of the multimode fiber, and the output end of the photomultiplier tube is coupled to the fluorescent imaging component.

Further, reflection mirrors are provided in the loss laser adjustment assembly, in the excitation laser adjustment assembly, and between the spiral phase plate and the glass slide.

Further, an electronically controlled baffle is provided between the spiral phase plate and the glass slide. Further, the slide is a quarter slide.

Embodiments of the invention

In order to make the purpose, features, and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the drawings in the embodiments of the present invention. Obviously, the description The embodiments are only a part of the embodiments of the present invention, but not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present invention.

Please refer to FIG. 1, which is a stimulated emission loss fluorescence lifetime super-resolution imaging device, including: loss laser provider, loss laser adjustment component, excitation laser provider, synchronization controller, excitation laser adjustment component, spiral phase plate VPP, Slide QWP, second dichromatic mirror DM2, first dichromatic mirror DM1, sample fluorescence collection component, imaging component and reference fluorescence signal collection component.

The lossy laser provider is used to provide lossy lasers. In this embodiment, the lossy laser provider is a femtosecond laser Laster1 and the output loss laser wavelength is 760 nanometers; the lossy laser adjustment component is set at the output of the lossy laser provider output laser The side of the end is used to adjust the linear polarization direction and laser intensity of the loss laser and broaden the loss laser; the excitation laser provider is provided on either side of the loss laser provider, but does not overlap with the loss laser adjustment component , Used to generate excitation laser, in this embodiment, the excitation laser provider is picosecond laser Laster2, the output excitation laser wavelength is 635 nanometers; the synchronization controller is set between the loss laser provider and the excitation laser provider, loss The laser provider can emit the depletion laser, and at the same time trigger the excitation laser provider to emit the excitation laser through the synchronous controller, so that the depletion laser provider and the excitation laser provider can emit the laser with the same pulse frequency; the excitation laser adjustment component is set in the excitation laser Provide the output end of the output excitation laser to adjust the polarization characteristics and intensity of the excitation laser, and control the pulse interval between the excitation laser and the loss laser, and separate the excitation laser; the spiral phase plate VPP is set in the loss laser adjustment component Far away from the side of the lossy laser provider, the lossy laser can convert the wavefront from Gaussian to circular through the spiral phase plate VPP; the slide QWP is set on the side of the spiral phase plate VPP away from the lossy laser adjustment component, in this embodiment , The slide QWP is a quarter slide, used to convert the loss laser from linear polarization to circular polarization; the second dichroic mirror DM2 is placed on the side of the quarter slide away from the loss laser adjustment component, and the second The dichroic mirror DM2 has two light exit ports, the first light exit port is used for transmission loss laser and the second light exit port is used for reflecting fluorescence; On the optical path of the loss laser and the excitation laser, the first dichroic mirror DM1 is used to transmit the loss laser and reflect the excitation laser, and fine-tune the transmission direction of the excitation laser, so that the excitation laser and the loss laser can be better overlapped; One side of a dichromatic mirror DM1 is used to collect the fluorescence signal reflected by the sample; the fluorescence imaging component is provided on the side of the second light outlet for imaging the collected fluorescence signal; the reference fluorescence signal collection component is provided on the excitation laser adjustment component One side is used to detect the excitation laser as a reference fluorescence for measuring the life of the sample.

Loss laser adjustment components include: second half-wave plate H2, second Glan laser prism G2, and widened glass rod GR; the second half-wave plate H2 is provided at the output end of the loss laser provider to ensure that the loss laser is linearly polarized light , And adjust the polarization direction of the lossy laser if necessary; the second Glan laser prism G2 is provided on the side of the second half-wave plate H2 away from the lossy laser provider, for cooperation with the second half-wave plate H2, The intensity of the lost laser is controlled; the widened glass rod GR is disposed on the side of the second Glan laser prism G2 away from the second half-wave plate H2, and is used to widen the pulse of the lost laser to 1 picosecond.

The lossy laser adjustment assembly also includes: a third lens L3, a single-mode polarization-maintaining fiber PMF, a fourth lens L4, a third half-wave plate H3, and a third Glan laser prism G3; the third lens L3 is disposed away from the widened glass rod GR One side of the second Glan laser prism G2; the single-mode polarization-maintaining fiber PMF is disposed on the side of the third lens L3 away from the broadened glass rod GR. In this embodiment, the length of the single-mode polarization-maintaining fiber PMF is 100 meters. Yu further broadens the lossy laser so that the pulse width of the lossy laser reaches 200 picoseconds; the fourth lens L4 is disposed on the side of the single-mode polarization-maintaining fiber PMF away from the third lens L3; the third half-wave plate H3 is disposed on the fourth lens L4 The side away from the single-mode polarization-maintaining fiber PMF; the third Glan laser prism G3 is disposed on the side of the third half-wave plate H3 away from the fourth lens L4.

The excitation laser adjustment component includes: a single-mode fiber SMF, a first lens L1, a first half-wave plate H1, a corner reflector RR, and a first Glan laser prism G1; a single-mode fiber SMF is provided at the output end of the excitation laser supply, Used to adjust the mode of the excitation laser emitted from the excitation laser supply; the first lens L1 is set on the output side of the single-mode fiber SMF output excitation laser; the first half-wave plate H1 is set on the first lens L1 away from the single mode The side of the fiber SMF; the corner reflector RR is provided on the side of the first half-wave plate H1 away from the first lens L1, used to extend or shorten the optical path of the excitation laser, thereby controlling the time between the excitation laser and the loss laser Pulse interval.

Sample fluorescence collection components include: galvanometer scanner Scanner, fifth lens L5, sixth lens L6, high-power numerical aperture objective lens OL and stage 3Dstage; galvanometer scanner Scanner is set in the first dichromatic mirror DM1 transmission light path and reflected light On the road, it is used to scan the overlapping excitation laser and loss laser synchronously to realize the area array imaging of the sample; the fifth lens L5 is provided on the light exit side of the galvanometer scanner Scanner; the sixth lens L6 is provided on the fifth The lens L5 is away from the side of the galvanometer scanner Scanner; the high-power numerical aperture objective lens OL is disposed on the side of the sixth lens L6 away from the fifth lens L5. Specifically, the high-power numerical aperture objective lens OL has a magnification of 100 times and a numerical aperture of 1.4, used to focus the overlapping excitation laser and depletion laser, and collect the fluorescent signal emitted from the sample; the stage 3Dstage is set on the side of the high-power numerical aperture objective lens OL away from the sixth lens L6.

The reference fluorescence signal collection component includes: a laser detector Detector and a time-dependent single photon counter TCSPC; the laser detector Detector is disposed on one side of the excitation laser adjustment component, and is used to detect the excitation laser pulse and use the excitation laser pulse as the fluorescence lifetime Reference signal; the time-dependent single photon counter TCSPC is set on the output side of the laser detector Detector, and the output end of the time-correlated single photon counter TCSPC is coupled with the fluorescence imaging component for measuring the laser detector Detector collected The time that each fluorescent molecule is in an excited state after being excited by an excitation laser.

A second lens L2 is also provided between the first Glan laser prism G1 and the laser detector Detector.

The fluorescence imaging component includes: a seventh lens L7, a filter Filter, a multimode fiber MMF and a photomultiplier tube PMT; a seventh lens L7 is disposed on the reflected optical path of the second dichroic mirror DM2; a filter Filter is disposed away from the seventh lens L7 The side of the second dichroic mirror DM2 is used to transmit the fluorescent signal that needs to be collected in the wavelength band and filter the stray light that needs to be collected in the wavelength band outside; the multimode fiber MMF is disposed on the side of the filter Filter away from the seventh lens L7, with In order to transmit the collected fluorescent signal to the photomultiplier tube PMT, the core of the multimode fiber MMF is used as a small hole to receive the fluorescent signal focused by the seventh lens L7 and exclude the effects of stray light; the photomultiplier tube PMT is set in the multimode The output end of the optical fiber MMF, and the photomultiplier tube PMT is coupled with the fluorescence imaging component. In this embodiment, the fluorescence imaging component is a computer computer, and the photomultiplier tube PMT is used to amplify the collected fluorescence signal.

Reflectors are provided in the loss laser adjustment module, the excitation laser adjustment module, and the spiral phase plate VPP and the quarter glass slide. The mirror is used to reflect incident light and change the transmission direction of the beam. Specifically, The reflection mirror in the lossy laser adjustment assembly is the first reflection mirror M1, and the first reflection mirror M1 is disposed between the third half-wave plate H3 and the fourth lens L4; the reflection mirror in the excitation laser adjustment assembly is the second reflection The mirror M2 and the second mirror M2 are arranged between the first dichroic mirror DM1 and the first Glan laser prism G1; the mirror arranged between the spiral phase plate VPP and the quarter glass plate is the third mirror M3.

An electronically controlled baffle E1 is provided between the spiral phase plate VPP and the quarter glass slide. When the electronically controlled baffle E1 is closed, it can block the lost laser, and only the excitation laser can illuminate the sample, which is confocal. For imaging, when the baffle is opened, the depletion laser and the excitation laser overlap on the sample to form STED super-resolution imaging.

The working process or principle of the stimulated emission loss fluorescence lifetime super-resolution imaging device provided by the present invention is as follows: the imaging device has two laser light sources, which are: a loss laser providing part for emitting loss laser, and an excitation laser emitting excitation laser Provided parts; the loss laser firstly passes through the second half-wave plate H2 and the second Glan laser prism G2, so that the linear polarization direction and laser intensity of the loss laser are adjusted to ensure that the loss laser entering the optical path system is linearly polarized light. The widened glass rod GR is used to widen the femtosecond loss laser. The pulse width after broadening is about 1 picosecond. The beam of the loss laser is focused by the third lens L3 and coupled into a 100-meter single-mode polarization-maintaining fiber PMF. The pulse width of the depletion laser is further extended to about 200 picoseconds; after the depletion laser passes through the single-mode polarization-maintaining ray, the beam is expanded by the fourth lens L4 and enters the third half-wave plate H3 and the third Glan laser prism G3, again Adjust the polarization characteristics and intensity of the lost laser. After the loss laser beam passes through the spiral phase plate VPP, the wavefront changes from Gaussian to ring-shaped; after passing through a quarter glass plate, it changes from linear polarization to circularly polarized light. The lost light is sequentially transmitted through the second dichroic mirror DM2 and the first dichroic mirror DM1 and enters the scanning system. The spot diameter is enlarged to be equal to or slightly larger than the aperture of the high-power numerical aperture objective lens OL through the fifth lens L5 and the sixth lens L6, and the incident parallel light is focused on the sample.

The excitation laser is generated by the excitation laser provider, and is triggered by the depletion laser signal to emit the excitation laser of the same repetition frequency. Through single-mode fiber SMF coupling, the first lens L1 magnifies and sequentially enters the first half-wave plate H1 and the first Glan laser prism G1, so that the polarization characteristics and intensity of the excitation laser are adjusted to ensure that the excitation laser entering the optical path system is Linearly polarized light. The excitation light changes the optical path of the optical path through the corner reflector RR, thereby controlling the pulse interval between the excitation laser and the depletion laser. The first Glan laser prism G1 divides the excitation laser light into two beams, and the reflected light is detected by the laser detector Detector as a reference signal for measuring the fluorescence lifetime of the sample. After the transmitted light is reflected by the mirror, it meets the depleted laser at the first dichroic mirror DM1. The transmitted light of the excitation laser light and the depletion laser light are spatially overlapped and enter the fluorescence collection component together.

In the stimulated emission loss fluorescence lifetime super-resolution imaging device, the excitation laser is provided by the excitation laser provider, the excitation laser provider is connected to the loss laser provider through the external wiring, and the excitation laser is triggered by the control system of the loss laser provider Provide the output pulse type excitation laser. The pulse interval of the depletion laser and the excitation laser has a very obvious effect on the effect of super resolution. In order to obtain the ideal STED super resolution image, the pulse interval between the two laser pulses is controlled by adjusting the angle reflector RR, so that the excitation laser pulse In the front, the depletion laser pulse is behind, and the pulse interval between the excitation laser and the depletion laser is kept at about 200 ps. The specific value can be determined by the actual super-resolution imaging effect. The excitation laser and the depletion laser coincide on the galvanometer scanner Scanner. The fifth lens L5 and the sixth lens L6 magnify the light spot to the aperture size of the high-power numerical aperture objective OL. After the superposed laser is focused by the high-power numerical aperture objective OL, Excite the fluorescent sample. After the fluorescence signal is collected by the high-power numerical aperture objective lens OL, it passes through the sixth lens L6, the fifth lens L5, the galvanometer scanner Scanner, the first dichromatic mirror DM1, the second dichromatic mirror DM2, and the seventh lens L7, and then couples into the multimode Fiber MMF. The end surface of the core of the multimode fiber MMF is used as a small hole, and the collected fluorescent signal is transmitted to the photomultiplier tube PMT through the optical fiber for signal amplification, and finally imaged on the computer computer.

Because the wavefront of the STED laser is distributed in a ring shape, and the center intensity is almost zero, the intensity of the ring area is relatively large. When the depletion laser is superimposed on the spot of the excitation laser, the superimposed area around the spot of the excitation laser is subjected to strong stimulated radiation. Fluorescent molecules in the state rapidly radiate energy back to the ground state, so the lifespan changes greatly; while the excited fluorescent molecules in the central region are not affected or are weakly affected, and still emit fluorescence in the form of spontaneous radiation, so the lifespan change is small.

The phase graph analysis method is used to convert the data collected by the time-dependent single photon counter TCSPC from the time domain to the frequency domain, which can avoid the errors caused by the exponential analysis, and can also display the fluorescence information of each pixel in the form of visualization Out, so it is a very useful method of fluorescence lifetime data analysis. According to the theory of phase diagram analysis, the fluorescence signal is modulated by the excitation laser at the same frequency (ω), and its amplitude and phase will change. Therefore, the frequency domain information in the phase diagram includes the phase delay (φ) and the amplitude modulation rate (m ). Under the determined modulation frequency, all the data collected by the time-dependent single photon counter TCSPC are converted to the phase space using the formulas g=m×cos(φ) and s=m×sin(φ); where g and s represent The coordinate values (g, s) of the phase space. In the above data, the decay trend of the fluorescent molecules at each pixel position is completely recorded. For single-component samples, the coordinate values g and s vary with fluorescence lifetime and modulation frequency, ie: g = 1/(1+ω ² τ ² ) and s = ωτ/(1+ω ² τ ² ) . According to the above formula, a semi-circular curve centered on the coordinate (0.5, 0) and having a radius of 0.5 can be established in the phase diagram. In the coordinate axis of the phase diagram, the origin of the coordinate, that is, the coordinate (0,0) represents the life is infinite, the coordinate (1,0) represents the life is zero, the fluorescence lifetime value of the single-component sample must fall on this semicircular curve , And different positions on the semicircle represent different fluorescence lifetimes.

After converting the fluorescence lifetime data to the phase space, due to the different phase delay and amplitude modulation information of the collected fluorescent molecules, the fluorescent molecules will be distributed in the phase space with different coordinates. According to the principle of phase map analysis, in single-component confocal imaging, the life data is converted into a phase map, and the phase center coordinates are calculated by fuzzy c-mean clustering algorithm (Fuzzy-mean clustering algorithm), namely The average fluorescence lifetime of all fluorescent molecules, the phase center coordinate lies on the semicircular curve. The closer to the phase center coordinate, the more concentrated the fluorescent molecules and the more the number of fluorescent molecules. In the STED imaging mode, the excited fluorescent molecules at different positions radiate energy in two forms: spontaneous emission and stimulated emission. If the intensity of the center region of the spot of the depletion laser is zero, the excited molecules there will not be affected by the depletion light, and the lifetime value should be very close to the average lifetime in the confocal mode. However, due to aberrations and other factors, the central area of the ring-loss light will produce a non-zero intensity, which will increase as the laser energy increases. At this time, part of the fluorescent molecules in the center of the spot of the excitation laser will also be stimulated to radiate, thereby reducing their lifetime, so almost all fluorescent molecules in the phase diagram are far away from the phase center coordinates of the confocal image. Nevertheless, the intensity of the center of the lost light is still less than the intensity of the ring region. The result is that the excited fluorescent molecules in the area that is less affected by the loss of light have a phase coordinate closer to the coordinate of the confocal center; The excited state fluorescent molecules in the region have a phase coordinate farther away from the confocal center coordinate. Fluorescent molecules with super-resolution information are less affected by the depleted light, and their phase coordinates are closer to the confocal center coordinates; while the remaining fluorescent molecules have a longer life span, so they are farther away from the confocal center coordinates.

When processing STED-FLIM data, the double exponential curve can not only fit the fluorescence lifetime well, but also simplify the data processing process. Therefore, the fluorescence lifetime in the STED mode is regarded as a combination of long lifetime and short lifetime, which respectively represent the central region fluorescent molecule with weak stimulated radiation effect and the circular region fluorescent molecule with strong stimulated radiation effect. The phase center coordinate in STED mode represents the average life of the sample, so it can be regarded as a criterion. The line segment connecting the phase center coordinate and the coordinate (0,0) point of the STED image divides the phase space into two parts. The fluorescent molecules located in the upper left area of the line segment are dominated by long life, basically from the center of the spot of the excitation laser; The fluorescent molecules in the lower right area are predominantly short-lived, mostly from the ring area of the laser spot and the background noise. The areas located on both sides of the line segment are regarded as "selected areas" and "abandoned areas", and the fluorescent molecules in the "abandoned areas" can be removed to improve the resolution. By using this method, not only can the "useless" fluorescent molecules be removed to achieve improved resolution, but also enough fluorescent molecules can be retained to form a better super-resolution image. Therefore, in this device, the use of a phase diagram analysis method to select fluorescent molecules that are less affected by stimulated radiation can achieve further improvement in imaging resolution at low power.

In the several embodiments provided in this application, it should be understood that the disclosed device may be implemented in other ways. For example, the device embodiments described above are only schematic. In addition, the displayed or discussed mutual coupling or direct coupling may be indirect coupling or communication connection through some interfaces, devices or modules, and may be in electrical, mechanical, or other forms.

It should be noted that, for the foregoing embodiments, those skilled in the art should know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

The above is a description of a stimulated emission loss fluorescence lifetime super-resolution imaging device provided by the present invention. For those skilled in the art, according to the ideas of the embodiments of the present invention, the specific implementation and application scope may change. In summary, the content of this specification should not be construed as limiting the invention.

Industrial applicability

It solves the technical problem of low imaging accuracy in live cells in the prior art.

Claims

A stimulated emission loss fluorescence lifetime super-resolution imaging device, which is characterized by comprising:

Loss laser supplier, used to provide loss laser;

A loss laser adjustment component provided at the output end of the loss laser provider, for adjusting the linear polarization direction and laser intensity of the loss laser, and broadening the loss laser;

An excitation laser provider provided on one side of the depletion laser provider, for providing excitation laser;

A synchronous controller provided between the excitation laser provider and the loss laser provider;

An excitation laser adjustment component provided at the output end of the excitation laser provider, for adjusting the polarization characteristics and intensity of the excitation laser, and controlling the pulse interval between the excitation laser and the depletion laser, and separating the excitation laser;

A spiral phase plate provided on a side of the lossy laser adjusting component away from the lossy laser providing member;

A glass slide arranged on a side of the spiral phase plate away from the lossy laser adjustment component;

A second dichroic mirror disposed on the side of the slide away from the lossy laser adjustment assembly, the first light exit port of the second dichroic mirror is used to transmit loss laser, and the second light exit port of the second dichroic mirror For reflected fluorescence;

A first dichromatic mirror disposed on the side of the second dichromatic mirror away from the slide and on the optical path of the loss laser and the excitation laser at the same time, is used to transmit the loss laser, reflect the excitation laser, and fine-tune the transmission direction of the excitation laser ;

A sample fluorescence collection component provided on one side of the first dichromatic mirror;

A fluorescence imaging component provided on the side of the second light outlet;

A reference fluorescence signal collection component provided on one side of the excitation laser adjustment component.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the loss laser adjustment component comprises:

A second half-wave plate provided at the output end of the lossy laser providing part;

A second Glan laser prism disposed on a side of the second half-wave plate away from the lossy laser providing member;

A widened glass rod disposed on a side of the second Granger laser prism away from the second half-wave plate.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 2, wherein the loss laser adjustment component further comprises:

A third lens disposed on the side of the widened glass rod away from the second Gran laser prism;

A single-mode polarization-maintaining fiber disposed on the side of the third lens away from the widened glass rod;

A fourth lens disposed on a side of the single-mode polarization-maintaining fiber away from the third lens;

A third half-wave plate disposed on the side of the fourth lens away from the single-mode polarization-maintaining fiber;

A third Glan laser prism disposed on a side of the third half-wave plate away from the fourth lens.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the excitation laser adjustment component comprises:

Single-mode optical fiber set at the output end of the excitation laser supply;

A first lens disposed on the side of the output end of the single-mode optical fiber;

A first half-wave plate disposed on a side of the first lens away from the single-mode optical fiber;

An angle reflector disposed on a side of the first half-wave plate away from the first lens, the first half-wave plate is located on an incident light path of the angle reflector;

A first Glan laser prism disposed on the optical path of the reflected light of the corner reflector is used to divide the excitation laser into two beams.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the sample fluorescence collection component comprises:

A galvanometer scanner disposed on the transmission light path and the reflection light path of the first dichromatic mirror is used to simultaneously scan the superimposed excitation laser and the depletion laser to realize the imaging of the area array of the sample;

A fifth lens provided on the light exit side of the galvanometer scanner;

A sixth lens disposed on the side of the fifth lens away from the galvanometer scanner;

A high-power numerical aperture objective lens disposed on the side of the sixth lens away from the fifth lens;

A stage provided on the side of the high-power numerical aperture objective lens away from the sixth lens.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the reference fluorescence signal collection component comprises:

A laser detector provided on one side of the excitation laser adjustment assembly;

A time-dependent single photon counter provided at the output end of the laser detector, the output end of the time-dependent single photon counter is coupled to the fluorescence imaging component.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the fluorescence imaging component comprises:

A seventh lens disposed on the reflected optical path of the second dichromatic mirror;

A filter disposed on a side of the seventh lens away from the second dichromatic mirror;

A multimode optical fiber disposed on the side of the filter away from the seventh lens;

A photomultiplier tube disposed at the output end of the multimode optical fiber, the output end of the photomultiplier tube is coupled to the fluorescent imaging component.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the loss laser adjustment component, the excitation laser adjustment component, and the spiral phase plate and the slide All rooms are equipped with reflectors.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein an electronically controlled baffle is provided between the spiral phase plate and the glass slide.
The stimulated emission loss fluorescence lifetime super-resolution imaging device according to claim 1, wherein the slide is a quarter slide.