WO2021089010A1 - 基于共振振镜进行焦点调制的光学显微装置及方法 - Google Patents

基于共振振镜进行焦点调制的光学显微装置及方法 Download PDF

Info

Publication number
WO2021089010A1
WO2021089010A1 PCT/CN2020/127251 CN2020127251W WO2021089010A1 WO 2021089010 A1 WO2021089010 A1 WO 2021089010A1 CN 2020127251 W CN2020127251 W CN 2020127251W WO 2021089010 A1 WO2021089010 A1 WO 2021089010A1
Authority
WO
WIPO (PCT)
Prior art keywords
signal
scanning
resonant galvanometer
sample
lock
Prior art date
Application number
PCT/CN2020/127251
Other languages
English (en)
French (fr)
Inventor
廖九零
吴婷
李慧
郑炜
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2021089010A1 publication Critical patent/WO2021089010A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens

Definitions

  • the present invention relates to the field of optical technology, in particular to an optical microscopy device and a microscopy method capable of improving imaging signal-to-noise ratio and resolution.
  • Optical microscope can observe cell and subcellular structure and has become an important tool in biological tissue imaging technology.
  • Traditional wide-field microscopes can only observe the microstructure of the surface.
  • Confocal or multiphoton imaging techniques can work with fluorescent dyes, have excellent optical tomography capabilities, and can observe biological tissues with a certain thickness.
  • “Fluorescence microscopy system and method based on focus modulation” (CN104568884A) divides the beam into modulated light and non-modulated light, so that the two beams interfere at the focal point, and the longitudinal focus modulation enhances the longitudinal resolution and imaging of the microscope system depth.
  • "Microscope based on lock-in amplification” (CN106932374A) adds optical switches and lock-in amplifiers to conventional microscopes for signal amplification and noise filtering. However, although the use of optical switches or similar modulation methods changes the intensity of the illuminating light, it also causes the background noise to change at the same time, that is to say, the background noise and the target signal are still mixed together, which is not conducive to the change signal obtained from the detection. Restore the signal of the target point.
  • the present invention scans the focus and its vicinity through a high-speed resonant galvanometer, correlates the clock signal of the resonant galvanometer with the reference signal of the lock-in amplifier, and can detect submerged
  • the focus signal in the noise realizes the enhancement of the resolution and the improvement of the imaging depth, and has the advantages of simple implementation method and fast acquisition speed.
  • the present invention provides an optical microscopy device, including: a light source assembly for generating incident light incident on a target area of a sample; a focusing assembly for focusing incident light on the target area of the sample; a resonance galvanometer , For changing the position of the focus formed by the focusing component on the sample target area by scanning; the photoelectric detection component receives the optical signal generated by the sample target area being irradiated by the focused incident beam, and combines the The optical signal is converted into an electric signal; the lock-in amplifier collects the electric signal of the photoelectric detection component, and outputs the electric signal at the focal point based on the correlation between the resonant galvanometer clock signal and the lock-in amplifier reference signal.
  • the correlation between the clock signal of the resonant galvanometer and the reference signal of the lock-in amplifier is specifically: using the clock signal of the resonant galvanometer as the reference signal of the lock-in amplifier; or, setting the lock
  • the reference signal of the phase amplifier is used as the trigger signal of the resonance galvanometer, and the frequency of the trigger signal provided by the lock-in amplifier is not higher than the scanning frequency of the resonance galvanometer.
  • the scanning range of the resonant galvanometer is preferably 3-5 times the resolution in the fast scan direction.
  • the scanning direction of the resonant galvanometer is perpendicular to the fast scanning direction.
  • the scanning speed of the resonant galvanometer is preferably 4kHz, 8kHz or 12kHz, so as to ensure the imaging speed.
  • the resonant galvanometer adopts an external clock for triggering or an internal clock for triggering.
  • the focusing assembly includes a stage that can move in one direction, two directions, or three directions.
  • the focusing assembly further includes a scanning galvanometer, which cooperates with the stage to focus incident light on different target areas of the sample.
  • the present invention also provides a method for using the above-mentioned optical microscopy device, which includes the following steps:
  • the correlation between the clock signal of the resonant galvanometer and the reference signal of the lock-in amplifier is specifically: using the clock signal of the resonant galvanometer as the reference signal of the lock-in amplifier; or The reference signal of the lock-in amplifier is used as the trigger signal of the resonant galvanometer, and the frequency of the trigger signal provided by the lock-in amplifier is not higher than the scanning frequency of the resonant galvanometer.
  • the method further includes judging the position of the target area in the sample, and when the target area is located on the surface of the sample, the resonant galvanometer does not focus on the focus in step S2.
  • the present invention uses a resonant galvanometer to quickly scan the focus and cooperates with a lock-in amplifier to obtain signals, so that the optical microscopic device and microscopic imaging
  • the method can detect the focus signal submerged in noise more simply and quickly, and obtain deeper imaging depth and higher imaging resolution.
  • the background noise changes together with the target signal.
  • the focus modulation process using a resonant galvanometer the background noise remains unchanged, which is more conducive to recovering the target point signal.
  • the technical solution of the present invention will be able to adapt to a wider range of applications such as neural activity imaging in a deeper part of the cortex.
  • FIG. 1 is a schematic diagram of the structure of an optical microscopy device according to an embodiment of the present invention
  • Figure 2 is a schematic diagram of the scanning method
  • Figure 3 is a schematic diagram of focus modulation
  • Figure 4 is a schematic diagram of the signal relationship between the resonant galvanometer and the lock-in amplifier
  • Fig. 5 is a schematic diagram of the optical path of the optical microscopy device according to an embodiment of the present invention.
  • the optical microscopy device based on resonant galvanometer scanning to perform focus modulation of the present invention can be used in a variety of different imaging instruments or imaging systems, such as confocal microscopes, single-photon or multi-photon microscopes, and the like.
  • a resonant galvanometer is used to achieve high-speed scanning of the focus position in a small range to achieve the purpose of focus modulation.
  • a lock-in amplifier is used to detect the optical signal at the focus to improve the signal-to-noise ratio, resolution and resolution of the optical microscopy device. Imaging depth.
  • FIG. 1 is a schematic structural diagram of an optical microscopy device based on resonant galvanometer scanning for focus modulation according to an embodiment of the present invention.
  • the optical microscopy device includes a light source assembly 101, a resonant galvanometer 102, a focusing assembly 103, a photodetection assembly 104, and a lock-in amplifier 105.
  • the light source assembly 101 is used to generate an incident light beam irradiated to a target area of the sample.
  • the light source assembly 101 may preferably include a laser, and the incident beam generated is a parallel laser beam with better collimation, or a beam that has undergone beam expansion/or splitting treatment. Therefore, the light source assembly 101 may also include a collimating lens, a beam expander, a beam splitter used with a laser, or other optical devices that achieve beam collimation, beam expansion, and beam splitting effects.
  • the emitted laser can be either continuous laser or pulsed laser according to different application scenarios.
  • the laser beam has high energy and can excite fluorescence or other excitation light in the target area of the sample.
  • the focusing component 103 focuses the incident light generated by the light source component 101 in the target area of the sample.
  • the focusing assembly 103 may include, for example, a scanning element, a scanning lens, a tube lens, an imaging objective lens, and a stage. Each lens is used to shape and focus the incident light.
  • the scanning element can include an x-direction galvanometer and a y-direction galvanometer.
  • the stage can be a displacement stage that can move along the z-axis. Therefore, the scanning element is controlled.
  • the incident light can be focused to any position of the sample.
  • the stage can also be a stage that can be displaced in the three directions of x, y, and z, or it can be a micro-electromechanical system that can realize three-dimensional scanning, or it can be the aforementioned scanning method Multiple combinations.
  • FIG. 2 shows a schematic diagram of achieving focus scanning by the focusing assembly 103.
  • the focus component 103 controls the focus to scan along the x direction. After scanning a line in the x direction point by point, the focus is moved to the next position along the y direction perpendicular to the x direction. For example, the y-axis coordinates of the focus position are as shown in the figure. Y1 moves to y2, and then continues to scan one line in the x direction. Repeat this way until a plane is completely scanned.
  • the x direction is defined as the fast scanning direction
  • the y direction is defined as the slow scanning direction.
  • the optical microscopy device of the present invention is also provided with a resonant galvanometer 102, which is used to change the position of the focal point focused on the target area of the sample.
  • the resonant galvanometer 102 may be arranged upstream of the focusing assembly 103 to change the focal position by changing the way of incident light emitted by the light source assembly 101.
  • the resonant galvanometer 102 can also be arranged downstream of the focusing assembly 103 to change the focus position by changing the way of focusing the beam of the focusing assembly 103.
  • the resonant galvanometer 102 Compared with the prior art MEMS device or galvanometer whose maximum scanning speed can only reach 1kHz, the resonant galvanometer 102 has a higher scanning speed, which will help improve the overall signal acquisition speed of the device. Compared with the piezoelectric ceramic device with a faster scanning speed in the prior art, the resonant galvanometer 102 has a larger amount of deformation, which can meet the modulation requirement of the focus position.
  • the refresh rate of the digital micro-mirror array is relatively fast, the micro-mirror generally has only three states, which cannot achieve continuous scanning in a small area at the focal point. Therefore, it is impossible to guarantee a certain scan to the target point, and it is difficult to restore the vicinity of the target point.
  • the modulation intensity curve which has an impact on imaging.
  • high-speed scanning can be performed at a fixed frequency, for example, the scanning frequency is 4 kHz, 8 kHz, or 12 kHz, which has a scanning speed much higher than that of the focusing assembly 103. Therefore, after the focusing component 103 forms a focus on the target area of the sample, the position of the focus in the sample is changed at a high speed within a small range of the focus position and its vicinity, so as to realize the modulation of the focus point. In addition, during the modulation process of the focus position, the scanning range of the resonant galvanometer 102 is also much smaller than the scanning range of the focusing assembly 103. In fact, only when the scanning speed is fast enough and matched with an appropriate scanning range will it not affect the high-speed imaging, which is difficult to achieve with the existing common spatial light modulators.
  • the resonant galvanometer 102 is arranged upstream of the focusing assembly 103 as an example for description. Specifically, due to the addition of the resonant galvanometer 102, the focal position of the objective lens can quickly change within a small range of the target area.
  • Figure 3 illustrates this process, and Figure 3a shows that the incident light is focused into the sample after passing through the focusing assembly 103.
  • the resonant galvanometer 102 can be triggered by an external clock, or can be triggered by an internal clock. When the resonant galvanometer 102 is triggered by an internal clock, continuous scanning can be achieved, and its clock frequency can be output and detected as an electrical signal.
  • the resonant galvanometer 102 can also use the drive signal output by the lock-in amplifier 105 mentioned later as a trigger signal. At this time, the repetition period of the resonant galvanometer 102 is limited by the drive signal of the lock-in amplifier 105. No matter which method is adopted, it is important that the clock signal of the resonant galvanometer 102 is correlated with the lock-in amplifier 105 described below.
  • the photodetection component 104 receives the optical signal, such as a fluorescent signal, generated by the incident light beam irradiated by the focused and scanned target area of the sample by the resonant galvanometer 102, and converts the optical signal into an electrical signal.
  • the optical signal such as a fluorescent signal
  • An example is the use of a photomultiplier tube
  • avalanche diodes or sCMOS cameras can also be used.
  • the photodetection component 104 may also include components such as filters and focusing lenses.
  • the lock-in amplifier 105 collects the electrical signal of the photoelectric detection component 104, and the reference signal of the lock-in amplifier 105 is correlated with the clock signal of the resonant galvanometer 102, thereby outputting the electrical signal at the focal point.
  • the signal output by the lock-in amplifier is the signal at the focal point from which the background noise is filtered out.
  • the operation of the lock-in amplifier is mainly based on the weak signal detection technology of the cross-correlation method.
  • the lock-in amplifier uses a reference signal with the same frequency and a fixed phase relationship with the signal to be measured as a reference to filter out noises different from the reference frequency, thereby extracting a useful signal group Minute. Since the lock-in amplifier needs to provide a reference signal when measuring the input signal, the reference signal of the lock-in amplifier can be provided externally or internally. Therefore, the lock-in amplifier is divided into internal reference and external reference modes.
  • the internal reference mode mainly uses the signal generated by the signal generator inside the lock-in amplifier as the reference signal, and at the same time, the generated signal is also the driving signal provided to the external device.
  • the external reference signal is a periodic signal provided externally. For the resonant galvanometer 102, it can only scan at a specific frequency and cannot perform speed control. Its external trigger signal can only trigger when it starts to scan, but cannot control the period of its single scan.
  • the lock-in amplifier 105 can work in two ways:
  • the lock-in amplifier 105 needs to provide a periodic signal as the trigger signal of the resonant galvanometer 102. Since the scanning frequency of the resonant galvanometer 102 is fixed, use a higher frequency than its frequency. A high trigger signal cannot achieve a higher acquisition speed and cannot guarantee the stability of the phase difference. Therefore, the frequency of the trigger signal provided at this time should not be higher than the scanning frequency of the resonant galvanometer 102. Generally, the rising or falling edge of the trigger signal triggers the resonant galvanometer to start scanning.
  • the rising edge is selected as the trigger signal, and the phase difference is the corresponding signal S2 and lock when the resonant galvanometer 102 scans to the target point.
  • the phase difference ⁇ 2 between the internal reference signals R2 of the phase amplifier. Since the frequency of the trigger signal is lower than the natural frequency of the resonant galvanometer, another trigger signal has not been waited for after the resonant galvanometer finishes scanning, so there is an interruption in the target signal, which is different from the continuous signal in Fig. 4a.
  • the background signal N2 is basically unchanged, and the frequency and phase difference of the reference signal R2 and the signal to be measured S2 are known, according to the signal output by the lock-in amplifier 102, those skilled in the art can restore the target signal according to conventional means.
  • Fig. 5 is a schematic diagram of the optical path of the optical microscopy device based on galvanometer scanning modulation of the present invention.
  • the light beam emitted by the light source assembly 101 for example, a parallel laser beam
  • the resonant galvanometer 102 which can scan at a high speed in a small range.
  • the scanning range is preferably between 3 times and 5 times the resolution of the fast scan direction.
  • the fast-scan resolution is the resolution in the fast-scan direction corresponding to the finally collected image.
  • the scan range should not be too large, otherwise the signal intensity of the target point will be 0 during most of the modulation process, which will result in the amount of collected data The increase and invalid modulation. Further, considering that scanning is performed between two adjacent target points, target points that are too close may cause interference. Therefore, it is preferable that the scanning direction of the resonant galvanometer 102 is perpendicular to the fast scanning direction.
  • the light beam passing through the resonant galvanometer 102 irradiates the focusing assembly 103.
  • the focusing assembly 103 includes, for example, a scanning galvanometer 1031, a scanning lens 1032, a tube lens 1033, a dichroic mirror 1034, an imaging objective lens 1035, and a stage 1036.
  • the incident light beam passing through the resonant galvanometer 102 passes through the scanning galvanometer 1031, and is reflected into the scanning lens 1032 and the tube lens 1033 for beam expansion.
  • the imaging objective lens 1035 is finally focused on the target area of the sample, and the sample is placed on the stage 1036.
  • the photoelectric detection component 104 may include a filter 1041, which can filter out ambient background light and excitation light to allow signal light, such as a fluorescent signal, to pass.
  • the optical signal is condensed into the photomultiplier tube 1043 via the focusing lens 1042, and the photomultiplier tube 1043 converts the collected optical signal into an electrical signal, which can also be transmitted to the lock-in amplifier 105 to be received.
  • the lock-in amplifier 105 also collects the clock signal of the resonant galvanometer 102.
  • the phase lock The signal output by the amplifier 105 is the signal at the focal point where the background noise is filtered out.
  • the focus image with high resolution and high signal-to-noise ratio can be obtained after being collected by a capture card and transferred to the computer 106 for storage and display.
  • the resonant galvanometer 102 vibrates at a specific frequency, such as 4 kHz, 8 kHz or 12 kHz.
  • a specific frequency such as 4 kHz, 8 kHz or 12 kHz.
  • the resonant galvanometer 102 is not used for scanning modulation.
  • the resonant galvanometer 102 is used to modulate the focus position only when the noise is greater at the deeper position of the sample.
  • the electrical signal and the clock signal of the resonant galvanometer are connected to the lock-in amplifier, the clock signal of the resonant galvanometer is used as the reference signal of the lock-in amplifier, and the lock-in amplifier outputs the optical signal at the focal point.
  • the present invention has the advantage that no complicated modulation signal is required, and the signal at the focal point can be obtained without calculation.
  • Common modulation devices such as digital micro-mirror arrays and spatial light modulators need to load complex modulation patterns, and the patterns need to be changed many times. For the obtained data, time-consuming calculations are required to restore the focus position signal. Both are very unfavorable for rapid imaging.
  • the resonant galvanometer clock signal can be used as the reference signal, or the lock-in amplifier can be used to provide the trigger signal and the reference signal. Only simple scanning can realize the modulation at the focal point, and the lock-in amplifier can directly output the focal point.
  • the signal value can easily detect the focus signal submerged in noise, realize the enhancement of the resolution and the improvement of the imaging depth, and has the advantages of simple implementation method and fast acquisition speed.

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Optics & Photonics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

一种光学显微装置及相应的显微成像方法，装置包括产生入射光的光源组件(101)，对入射光聚焦的聚焦组件(103)，能够通过扫描的方式改变聚焦组件(103)于样品目标区域形成的焦点位置的共振振镜(102)，接受光信号并将光信号转换为电信号的光电检测组件(104)，以及能够收集共振振镜(102)的时钟信号和光电检测组件(104)的电信号的锁相放大器(105)。光学显微装置及显微成像方法能够更加简便快速地探测出淹没在噪声中的焦点信号，获得更深的成像深度和更高的成像分辨率，由于实现方法简单、采集速度更快，因此能够适应更多的应用场景。

Description

基于共振振镜进行焦点调制的光学显微装置及方法 技术领域
本发明涉及光学技术领域，尤其涉及一种能够提高成像信噪比及分辨率的光学显微装置及显微方法。
 背景技术
光学显微镜能够观察细胞和亚细胞结构，已成为生物组织成像技术中的重要工具。传统的宽场显微镜仅能观察表面的显微结构，共聚焦或者多光子成像技术能够结合荧光染料工作，具有优秀的光学层析能力，能够观察具有一定厚度的生物组织。
但此类荧光显微成像技术成像深度还有提高的空间。由于生物组织是非均匀的，入射光会被组织散射和吸收。随着探测深度的增加，激发光在焦点处的功率会发生指数衰减，使得信号减弱，同时由于在非焦点处的噪声增强，从而使得成像信噪比变差，特别是到达一定深度后，噪声超过信号强度，此时无法分辨信号和噪声。为了抑制在生物组织内散射光激发的背景荧光所带来的影响，已有相关文献和专利进行了相关技术的报导。文献《Two-photon focal modulation microscopy in turbid media》（ Applied Physics Letters, 2011, 99(23):2151.）已经理论分析证明了，当对样品较深处进行成像时，从样品表面到焦点之间区域激发出的荧光可以认为是背景噪声。通过对焦点附近进行调制，然后解调可以抑制背景噪声，从而获得较大成像深度。专利《荧光焦点调制显微系统和方法》(CN101802675A)采用空间光调制器以预定频率周期性改变焦点周围相干激发光的空间分布，然后进行解调获得焦点处的荧光。《基于焦点调制的荧光显微系统及方法》(CN104568884A)将光束分为调制光和非调制光，使得两束光在焦点处发生干涉，通过纵向焦点调制增强显微系统的纵向分辨率和成像深度。《基于锁相放大的显微镜》（CN106932374A）在常规显微镜中加入光开关和锁相放大器进行信号放大和噪声滤除。然而，采用光开关或者类似的调制方法虽然改变了照射光强，但是也使得背景噪声同时随之发生变化，也就是说背景噪声和目标信号仍然混合在一起，不利于从探测得到的变化信号中还原出目标点的信号。
尽管现有技术已经做出了一些尝试，但是，以上这些技术都需要预加载设计好的调制信号，一般都采用调制器对聚焦光束进行调制，并通过计算解调得出焦点处信号。如此，现有技术中的系统运行相对复杂，这也直接导致其无法做到很快的采集速度，比如在对小鼠大脑皮层较深处神经元活动进行监测时，神经放电过程在毫秒级别，在较低的成像速度下难以捕捉到这个过程。因而，现有技术中相对复杂且慢速的信号采集直接影响了显微镜的适用范围。
 技术解决方案
有鉴于此，为了克服上述现有技术的缺陷，本发明通过高速的共振振镜对焦点及其附近进行扫描，将共振振镜时钟信号与锁相放大器的参考信号相关联，能够探测出淹没在噪声中的焦点信号，实现分辨率的增强和成像深度的提高，具有实现方法简单，采集速度快的优点。
具体地，本发明提出了一种光学显微装置，包括：光源组件，用于产生入射至样品目标区域的入射光；聚焦组件，用于将入射光聚焦至所述样品目标区域；共振振镜，用于通过扫描的方式改变所述聚焦组件于所述样品目标区域形成的焦点的位置；光电检测组件，接收所述样品目标区域受聚焦后的入射光束照射而产生的光信号，并将该光信号转换为电信号；锁相放大器，收集光电检测组件的电信号，基于所述共振振镜时钟信号与所述锁相放大器参考信号之间的关联输出焦点处的电信号。
优选地，所述共振振镜时钟信号与所述锁相放大器参考信号之间的关联具体为：将所述共振振镜的时钟信号作为所述锁相放大器的参考信号；或者，将所述锁相放大器的参考信号作为所述共振振镜的触发信号，并且所述锁相放大器提供的触发信号频率不高于所述共振振镜的扫描频率。
为了避免采集数据量过大以及无效的调制，所述共振振镜的扫描范围优选为快扫方向分辨率的3-5倍。
进一步，为减少目标点之间的干扰，所述共振振镜的扫描方向与快扫描方向垂直。
进一步，所述共振振镜的扫描速度优选为4kHz，8kHz或者12kHz，从而保证成像速度。
进一步，所述共振振镜采用外部时钟进行触发或者采用内部时钟进行触发。
为了灵活调整目标区域在样品中的位置，所述聚焦组件包括载物台，所述载物台能够在一个方向、两个方向或者三个方向上移动。当所述载物台不能三个方向移动时，所述聚焦组件还包括扫描振镜，所述扫描振镜和所述载物台配合以实现在样品不同的目标区域聚焦入射光。
同时，本发明还提出一种使用上述光学显微装置的方法，其包括如下步骤：
S1、产生用于照射样品目标区域的入射光；
S2、采用聚焦组件实现对样品目标区域聚焦扫描；
S3、通过共振振镜的扫描，对样品目标区域的焦点产生调制；
S4、接收样品目标区域产生的光信号，并将光信号转换为电信号；
S5、将所述电信号接入锁相放大器（105），基于所述共振振镜（102）时钟信号与所述锁相放大器（105）参考信号之间的关联输出焦点处的电信号。
其中优选地，所述共振振镜时钟信号与所述锁相放大器参考信号之间的关联具体为：将所述共振振镜的时钟信号作为所述锁相放大器的参考信号；或者，将所述锁相放大器的参考信号作为所述共振振镜的触发信号，并且所述锁相放大器提供的触发信号频率不高于所述共振振镜的扫描频率。
进一步，所述方法还包括判断目标区域在所述样品中的位置，当目标区域位于所述样品表面处时，所述步骤S2中共振振镜不对焦点产生调制。
为了应对现有技术中显微装置运行复杂、采集速度慢的技术问题，本发明通过使用共振振镜对焦点进行快速扫描，配合锁相放大器对信号进行获取，使得光学显微装置及显微成像方法能够更加简便快速地探测出淹没在噪声中的焦点信号，获得更深的成像深度和更高的成像分辨率。相对于使用锁相放大器配合光开光的这类背景噪声与目标信号一起变化的技术方案而言，采用共振振镜进行焦点调制过程中，背景噪声不变，更利于还原出目标点信号。整体上，由于实现方法简单、采集速度更快，因此，本发明的技术方案将能够适应比如皮层较深处的神经活动成像等更多的应用范围。
 附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例的光学显微装置的结构示意图；
图2为扫描方式示意图；
图3为焦点调制示意图；
图4为共振振镜及锁相放大器信号关系示意图；
图5为本发明实施例的光学显微装置的光路示意图。
附图标记：
101光源组件
102共振振镜
103聚焦组件
104光电检测组件
105锁相放大器
1031扫描振镜
1032扫描透镜
1033套筒透镜
1034二向色镜
1035成像物镜
1036载物台
1041滤光片
1042聚焦透镜
1043光电倍增管
106电脑
 本发明的实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明基于共振振镜扫描进行焦点调制的光学显微装置可以用于多种不同的成像仪器或成像系统中，例如共聚焦显微镜、单光子或多光子显微镜等。具体通过共振振镜来实现焦点位置在小范围内的高速扫描以达到对焦点调制的目的，同时采用锁相放大器探测出焦点处的光信号，提高光学显微装置的信噪比、分辨率及成像深度。
图1为本发明实施例的基于共振振镜扫描进行焦点调制的光学显微装置的结构示意图。光学显微装置包括光源组件101、共振振镜102、聚焦组件103、光电检测组件104及锁相放大器105。
本发明实施例中，光源组件101用来产生照射至样品目标区域的入射光束。例如光源组件101可以优选包括激光器，产生的入射光束是准直性较佳的平行激光光束，还可以是经过扩束/或者分光处理的光束。因而光源组件101还可以包括配合激光器使用的准直透镜、扩束镜、分光镜，或者实现光束准直、扩束、分光等效果的其它光学器件。所发出的激光根据不同的应用场景既可以是连续的激光，也可以是脉冲激光。激光光束具有较高的能量，能在样品目标区域激发荧光或其他激发光。
聚焦组件103将光源组件101产生的入射光聚焦在样品的目标区域中。聚焦组件103例如可以包括扫描元件、扫描透镜、套筒透镜、成像物镜及载物台。其中各个透镜用于对入射光进行整形、聚焦，扫描元件可以包含x方向振镜、y方向振镜，相应地，载物台可以为能够沿z轴方向移动的位移台，因此，控制扫描元件和载物台的配合，能够实现将入射光聚焦到样品的任意位置。可替换地，载物台也可以是自身能够在x、y、z三个方向进行位移的载物台，或者可以是能实现三维扫描的微机电系统，也可以是前面提到的扫描方式的多种组合。
图2表示通过聚焦组件103实现焦点扫描的示意图。首先，聚焦组件103控制焦点沿着x方向扫描，当x方向逐点扫描完一行之后，将焦点沿与x方向相垂直的y方向移动到下一个位置，例如焦点位置的y轴坐标从图中的y1移动到y2，然后在x方向又继续进行一行扫描。如此循环，直到将一个平面完全扫描出来。在此，根据扫描的进度，将x方向定义为快扫方向，y方向定义为慢扫方向。
与现有技术不同，本发明的光学显微装置中还设置有共振振镜102，其用来改变聚焦到样品目标区域的焦点的位置。具体地，共振振镜102可以设置在聚焦组件103的上游以通过改变光源组件101发出的入射光的方式来改变焦点位置。可选地，共振振镜102也可以设置在聚焦组件103的下游，以通过改变聚焦组件103聚焦后的光束的方式来改变焦点位置。相对于现有技术中最高扫描速度仅能达到1kHz的MEMS器件或者检流计振镜，共振振镜102具有更高的扫描速度，这将有助于提升装置整体的信号采集速度。相对于现有技术中的扫描速度较快的压电陶瓷器件，共振振镜102具有更大的形变量，能够满足对焦点位置的调制要求。此外，虽然数字微镜阵列刷新的速度较快，但是其微镜一般只有三个状态，无法实现在焦点处的小范围连续扫描，因而无法保证一定扫描到目标点，也难以还原出目标点附近的调制强度曲线，从而对成像产生影响。
对于本实施例的共振振镜102来说，可以以固定的频率进行高速扫描，例如扫描频率为4kHz、8kHz或者12kHz，其具有远高于聚焦组件103的扫描速度。从而在聚焦组件103于样品的目标区域形成焦点后，高速地在该焦点位置及其附近的小范围内改变焦点在样品中的位置，实现对焦点的调制。此外，在对焦点位置的调制过程中，共振振镜102的扫描范围也远小于聚焦组件103的扫描范围。实际上，只有当扫描速度足够快速，且配合有适当的扫描范围时才不会对高速成像产生影响，这一点现有常见的空间光调制器是难以达到的。
在本实施例中，以共振振镜102设置在聚焦组件103上游为例进行说明。具体地，由于共振振镜102的加入，导致物镜的焦点位置能够在目标区域小范围内快速发生改变，图3示意了这一过程，图3a为入射光经过聚焦组件103后聚焦到样品中较深处的特定位置，也就是焦点本来的位置，由于样品的散射，在较深位置处，理想焦点附近都被照明，有用的信号被淹没在噪声中，因而以方形示出；图3b为通过共振振镜102的扫描，使得该焦点位置在一个小范围内变化，即本来焦点位置处的以方形示意的特定形式的光场会在所述的小范围里不停高速地变化位置，图中仅示例了额外的两个位置，但扫描可以是在多个位置之间连续的。位置高速变化的焦点处光场在短时间内的叠加形成一个小体积范围内光场的变化，这个变化即体现了对入射光焦点的调制，或者说利用焦点位置的变化形成一个焦点周围特定体积范围内光场的调制。共振振镜102可以采用外部时钟进行触发，也可以采用内部时钟进行触发。当共振振镜102使用内部时钟触发时可以实现持续扫描，其时钟频率可以作为电信号输出并被探测到。共振振镜102也可以使用后文提及的锁相放大器105输出的驱动信号作为触发信号，此时共振振镜102的重复周期受锁相放大器105的驱动信号限制。无论采用哪种方式，重要的是共振振镜102的时钟信号与下文所述的锁相放大器105进行关联。
光电检测组件104接收样品目标区域受聚焦且经共振振镜102扫描后的入射光束照射而产生的光信号，例如荧光信号等，并将该光信号转换为电信号，一个例子是使用光电倍增管，此外还可以选用雪崩二极管或者sCMOS相机等。为了更好地收集光信号，光电检测组件104还可以包括滤光片、聚焦透镜等元器件。
锁相放大器105收集光电检测组件104的电信号，并且锁相放大器105的参考信号与共振振镜102的时钟信号相关联，从而输出焦点处的电信号。通过将光电检测组件104得到的电信号与共振振镜102的时钟信号进行运算，锁相放大器输出的信号即为滤除了背景噪声的焦点处信号。锁相放大器的运行主要基于互相关方法的微弱信号检测技术，利用与待测信号具有相同频率和固定相位关系的参考信号为基准，滤除与参考频率不同的噪声，从而提取出有用的信号组分。由于锁相放大器在测量输入信号时，需要提供参考信号，因此，锁相放大器的参考信号可由外部提供，也可由内部提供，故锁相放大器分为内部参考和外部参考方式。内部参考方式主要是由锁相放大器内部的信号发生器产生的信号作为参考信号，同时，该产生的信号也是提供给外部器件的驱动信号。外部参考信号是外部提供的周期性信号。对于共振振镜102来说，其只能以特定频率进行扫描，无法进行速度控制，其外部触发信号只能触发其何时开始扫描，而不能控制其单次扫描的周期。
基于共振振镜的这个特性，锁相放大器105可以有两种工作方式：
（1）将共振振镜102的时钟信号作为锁相放大器105的外部参考信号R1，参考图4a所示，上方为目标信号S1和背景噪声N1，真实的目标信号与共振振镜102的时钟信号同步，此时待测目标信号与锁相放大器105的参考信号相位差为0；
（2）当共振振镜102的时钟信号不方便输出时，需要将锁相放大器105提供周期性信号作为共振振镜102的触发信号，由于共振振镜102的扫描频率固定，使用比其频率更高的触发信号无法实现更高的采集速度，而且无法保证相位差的稳定性，因而此时提供的触发信号频率应不高于共振振镜102的扫描频率。一般以触发信号的上升沿或下降沿触发共振振镜开始扫描，如图4b所示，选取上升沿作为触发信号，相位差为共振振镜102扫到目标点时对应的待测信号S2和锁相放大器内部参考信号R2之间的相位差Δφ2。由于触发信号的频率低于共振振镜的固有频率，共振振镜结束扫描后还没等到另一个触发信号，因此目标信号存在中断，与图4a中连续信号不同。在背景信号N2基本不变，已知参考信号R2、待测信号S2的频率与相位差的情况下，根据锁相放大器102输出的信号，本领域技术人员按照惯用手段能够还原出目标信号。
图5为本发明的基于振镜扫描调制的光学显微装置的光路示意图。如图5所示，光源组件101出射的光束，例如平行激光光束，入射到共振振镜102上，共振振镜102能够以高速小范围扫描。在本实施例中，为了产生目标点明暗对比的调制变化，扫描的范围优选为快扫方向分辨率的3倍-5倍之间。快扫分辨率为最终采集到的图像对应的快扫方向分辨率，该扫描范围不宜过大，否则在调制过程中大部分时间内目标点的信号强度都为0，如此则会导致采集数据量的增加以及无效的调制。进一步，考虑到在相邻两个目标点间进行扫描，靠的太近的目标点可能会产生干扰，因此优选地，共振振镜102的扫描方向与快扫方向垂直。
经过共振振镜102的光束照射到聚焦组件103中。具体地，聚焦组件103例如包括扫描振镜1031、扫描透镜1032、套筒透镜1033、二向色镜1034、成像物镜1035和载物台1036。经过共振振镜102的入射光束经过扫描振镜1031，反射进入扫描透镜1032和套筒透镜1033扩束。之后穿过二向色镜1034由成像物镜1035最终聚焦至样品目标区域上，样品放置于载物台1036上。
样品被入射光照射的目标区域产生的光信号，例如荧光，经由成像物镜1035收集后，经过二向色镜1034反射至光电检测组件104。在本实施例中，光电检测组件104可以包括滤光片1041，其能够将环境背景光和激发光滤除而使信号光，例如荧光信号通过。随后，光信号经由聚焦透镜1042会聚到光电倍增管1043中，光电倍增管1043将收集到的光信号转换为电信号，该电信号也能够传输到锁相放大器105中从而被接收。
以锁相放大器105使用外部参考信号为例，锁相放大器105同时还采集共振振镜102的时钟信号，通过将光电检测组件104得到的电信号与共振振镜102的时钟信号进行运算，锁相放大器105输出的信号即为滤除了背景噪声的焦点处信号。通过采集卡采集，传输至电脑106储存显示后即可得到高分辨率高信噪比的焦点处图像。
共振振镜102以特定的频率进行振动，例如4 kHz，8 kHz或者12 kHz，当通过共振振镜102对目标点附近进行快速扫描时，目标点的光强度随着焦点位置的变化产生强弱变化，尽管仍然淹没在噪声中，但是这样的变化与共振振镜102的时钟信号同步，根据共振振镜102时钟信号与锁相放大器105参考信号的相位差，锁相放大器105可以直接读出目标点的荧光强度。
在具体使用本发明的光学显微装置时，对于样品表面处，其信号强度没有淹没在噪声中时，不用使用共振振镜102进行扫描调制。当对样品较深位置处噪声较大时才开始使用共振振镜102对焦点位置进行调制。进一步利用上述光学显微系统进行焦点调制的方法还包括如下步骤：
S1、产生用于照射样品目标区域的光束；
S2、采用聚焦组件实现对样品目标区域聚焦扫描；
S3、通过共振振镜高速且小范围扫描，对样品目标区域的焦点产生调制；
S4、接收样品目标区域产生的光信号，并将光信号转换为电信号；
S5、将电信号和共振振镜的时钟信号接入锁相放大器，共振振镜的时钟信号作为锁相放大器的参考信号，锁相放大器输出焦点处的光信号。
本发明相比于现有技术中的其他调制方案的优势在于不需要复杂的调制信号，无需计算即可获得焦点处的信号。常见的调制器件如数字微镜阵列和空间光调制器，都需要加载复杂的调制图案，而且需要多次改变图案，对于得到的数据还需要经过耗时的计算才能还原出焦点位置的信号，这都非常不利于快速成像。而本发明可以采用共振振镜时钟信号作为参考信号，也可以使用锁相放大器提供触发信号和参考信号，仅需简单的扫描即可实现焦点处的调制，并且锁相放大器能直接输出焦点处的信号值，能够方便地探测出淹没在噪声中的焦点信号，实现分辨率的增强和成像深度的提高，具有实现方法简单，采集速度快的优点。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本申请。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本申请的精神或范围的情况下，在其它实施例中实现。因此，本申请将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种光学显微装置，包括：
    光源组件（101），用于产生入射至样品目标区域的入射光；
    聚焦组件（103），用于将入射光聚焦至所述样品目标区域；
    共振振镜（102），用于通过扫描的方式改变所述聚焦组件（103）于所述样品目标区域形成的焦点的位置；
    光电检测组件（104），接收所述样品目标区域受聚焦后的入射光束照射而产生的光信号，并将该光信号转换为电信号；
    锁相放大器（105），收集所述光电检测组件（104）的电信号，基于所述共振振镜（102）时钟信号与所述锁相放大器（105）参考信号之间的关联输出焦点处的电信号。
  2. 根据权利要求1所述的光学显微装置，其特征在于，
    所述共振振镜（102）时钟信号与所述锁相放大器（105）参考信号之间的关联具体为：
    将所述共振振镜（102）的时钟信号作为所述锁相放大器（105）的参考信号；
    或者，将所述锁相放大器（105）的参考信号作为所述共振振镜（102）的触发信号，并且所述锁相放大器（105）提供的触发信号频率不高于所述共振振镜（102）的扫描频率。
  3. 根据权利要求1所述的光学显微装置，其特征在于，
    所述共振振镜（102）的扫描范围为快扫方向分辨率的3-5倍。
  4. 根据权利要求3所述的光学显微装置，其特征在于，
    所述共振振镜（102）的扫描方向与快扫方向垂直。
  5. 根据权利要求1或3所述的光学显微装置，其特征在于，
    所述共振振镜（102）的扫描速度为4 kHz或8 kHz或12 kHz。
  6. 据权利要求1所述的光学显微装置，其特征在于，
    所述共振振镜（102）采用外部时钟进行触发或者采用内部时钟进行触发。
  7. 根据权利要求1所述的光学显微装置，其特征在于，
    所述聚焦组件（103）包括载物台（1036），所述载物台能够在一个方向、两个方向或者三个方向上移动。
  8. 根据权利要求7所述的光学显微装置，其特征在于，
    所述聚焦组件（103）还包括扫描振镜（1031），所述扫描振镜（1031）和所述载物台（1036）配合以实现在样品不同的目标区域聚焦入射光。
  9. 一种使用权利要求1-8任一项所述光学显微装置的方法，其包括如下步骤：
    S1、产生用于照射样品目标区域的入射光；
    S2、采用聚焦组件（103）实现对样品目标区域聚焦扫描；
    S3、通过共振振镜（102）的扫描，对样品目标区域的焦点产生调制；
    S4、接收样品目标区域产生的光信号，并将光信号转换为电信号；
    S5、将所述电信号接入锁相放大器（105），基于所述共振振镜（102）时钟信号与所述锁相放大器（105）参考信号之间的关联输出焦点处的电信号。
  10. 根据权利要求9所述的方法，其特征在于，
    还包括判断目标区域在所述样品中的位置，当目标区域位于所述样品表面处时，所述步骤S2中共振振镜（102）不对焦点产生调制。
PCT/CN2020/127251 2019-11-07 2020-11-06 基于共振振镜进行焦点调制的光学显微装置及方法 WO2021089010A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911083385.1A CN110702605B (zh) 2019-11-07 2019-11-07 基于共振振镜进行焦点调制的光学显微装置及方法
CN201911083385.1 2019-11-07

Publications (1)

Publication Number Publication Date
WO2021089010A1 true WO2021089010A1 (zh) 2021-05-14

Family

ID=69204558

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/127251 WO2021089010A1 (zh) 2019-11-07 2020-11-06 基于共振振镜进行焦点调制的光学显微装置及方法

Country Status (2)

Country Link
CN (1) CN110702605B (zh)
WO (1) WO2021089010A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110702605B (zh) * 2019-11-07 2021-08-17 中国科学院深圳先进技术研究院 基于共振振镜进行焦点调制的光学显微装置及方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007278768A (ja) * 2006-04-04 2007-10-25 Toshiba Corp 顕微鏡装置
CN102313594A (zh) * 2011-05-18 2012-01-11 福建师范大学 不同衍射角光强分布的探测装置
CN104614318A (zh) * 2015-01-28 2015-05-13 浙江大学 一种快速的超分辨显微成像方法和装置
CN206684042U (zh) * 2017-04-17 2017-11-28 大连理工大学 基于锁相放大的显微镜
CN110702605A (zh) * 2019-11-07 2020-01-17 中国科学院深圳先进技术研究院 基于共振振镜进行焦点调制的光学显微装置及方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002332802A1 (en) * 2001-08-29 2003-03-10 Musc Foundation For Research Development Line scanning confocal microscope
CN104568884B (zh) * 2014-12-31 2017-03-29 深圳先进技术研究院 基于焦点调制的荧光显微系统及方法
JP2016033676A (ja) * 2015-11-02 2016-03-10 株式会社ニコン 顕微鏡装置
CN106932374A (zh) * 2017-04-17 2017-07-07 大连理工大学 基于锁相放大的显微镜
CN109211855B (zh) * 2018-08-10 2021-11-16 国家纳米科学中心 多光束多光子显微成像装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007278768A (ja) * 2006-04-04 2007-10-25 Toshiba Corp 顕微鏡装置
CN102313594A (zh) * 2011-05-18 2012-01-11 福建师范大学 不同衍射角光强分布的探测装置
CN104614318A (zh) * 2015-01-28 2015-05-13 浙江大学 一种快速的超分辨显微成像方法和装置
CN206684042U (zh) * 2017-04-17 2017-11-28 大连理工大学 基于锁相放大的显微镜
CN110702605A (zh) * 2019-11-07 2020-01-17 中国科学院深圳先进技术研究院 基于共振振镜进行焦点调制的光学显微装置及方法

Also Published As

Publication number Publication date
CN110702605A (zh) 2020-01-17
CN110702605B (zh) 2021-08-17

Similar Documents

Publication Publication Date Title
CN106290284B (zh) 结构光照明的双光子荧光显微系统与方法
CN108680544B (zh) 一种结构化照明的光切片荧光显微成像方法和装置
CN102004307B (zh) 使用同心双锥面镜实现全内反射荧光显微的系统与方法
CN103543135B (zh) 一种基于荧光寿命分布的纳米精度光斑对准方法和装置
CN110146473B (zh) 一种轴向超分辨的双光子荧光显微装置及方法
CN108303421B (zh) 三维高速宽视场层析成像方法及装置
CN111971606B (zh) 具有高空间分辨率的时间分辨成像方法
CN111202499A (zh) 快速高效自适应光学补偿的受激拉曼散射成像系统和方法
TWI452335B (zh) 應用共聚焦顯微鏡結構的被測物圖像獲取方法及系統
JP2009109788A (ja) レーザー走査型顕微鏡
CN202102170U (zh) 使用同心双锥面镜实现全内反射荧光显微的系统
CN110220875B (zh) 一种基于荧光差分法的晶格光切片荧光显微成像装备及方法
JP6253395B2 (ja) 画像生成システム
CN114895450B (zh) 基于二次谐波的超分辨显微成像系统及成像方法
CN108982455B (zh) 一种多焦点光切片荧光显微成像方法和装置
WO2021089010A1 (zh) 基于共振振镜进行焦点调制的光学显微装置及方法
KR20190000056A (ko) 레이저 스캐닝 공초점 현미경
WO2024087614A1 (zh) 一种荧光发射比率超分辨成像方法
CN107490566A (zh) 基于二元光学元件的艾里光束光片照明显微成像装置
CN109557653B (zh) 一种基于算法恢复的差分共聚焦显微成像方法和装置
KR101080382B1 (ko) 공초점 레이저 주사 현미경
JP6379810B2 (ja) 観察装置
CN110261320B (zh) 一种荧光交错差分显微成像的方法和装置
CN112326609B (zh) 基于偏振复用的实时三维荧光差分超分辨成像方法和装置
CN111580260B (zh) 一种高速光学三维光切片显微系统及方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20884267

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20884267

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20884267

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 20/02/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 20884267

Country of ref document: EP

Kind code of ref document: A1