WO2022151469A1 - 纳米颗粒散射光共聚焦成像的装置和方法 - Google Patents

纳米颗粒散射光共聚焦成像的装置和方法 Download PDF

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WO2022151469A1
WO2022151469A1 PCT/CN2021/072443 CN2021072443W WO2022151469A1 WO 2022151469 A1 WO2022151469 A1 WO 2022151469A1 CN 2021072443 W CN2021072443 W CN 2021072443W WO 2022151469 A1 WO2022151469 A1 WO 2022151469A1
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laser
imaging
nanoparticles
scattered light
unit
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French (fr)
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宋茂勇
王丰邦
麻春艳
毕磊
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中国科学院生态环境研究中心
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    • 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
    • G01N21/65—Raman scattering

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  • the present disclosure relates to the technical fields of nanomaterial observation and biomolecule detection, in particular to a device and method for confocal imaging of nanoparticle scattering light.
  • optical microscopy imaging techniques have been developed for label-free nanoparticles, such as dark-field microscopy, confocal Raman microscopy, orthogonal polarization microscopy, etc. These optical microscopy imaging techniques can collect the scattered light signal of label-free nanoparticles, and the intracellular label-free nanoparticles can be observed in situ, which provides great convenience for in-depth study of nanoparticle cellular biological effects and processes.
  • the disadvantage of these imaging modes is that in order to improve the scattered light signal of nanoparticles and reduce the interference of the cell's own transmitted light, reflected light, fluorescence and other backgrounds, it cannot synchronize the intracellular microstructures, subcellular organelles, proteins and other biomolecules. imaging, and it is difficult to accurately locate the intracellular distribution and interaction process of nanoparticles.
  • nanoparticles In order to overcome the limitations of label-free detection of nanoparticles, many studies have modified the surface of nanoparticles to add easily detectable labels, and detected the labeling signal to track the distribution and content of nanoparticles in living cells.
  • fluorescent probes are modified on the surface of nanoparticles, and the labeled fluorescent probes in living cells are detected by high-resolution fluorescence microscopy imaging, so as to observe the distribution position and content of nanoparticles in cells in real time.
  • confocal laser scanning microscope uses laser light to detect fluorescent probes that label cells, combined with cell computer image processing technology, to observe intracellular microstructure and specific biomolecules, and to detect pH and Ca ions in subcellular areas. It can carry out quantitative analysis and real-time dynamic imaging, which is the most widely used molecular biology analysis instrument. It is especially worth noting that there is a pinhole in front of the light source and the detector of the confocal scanning microscope. Only the fluorescence generated by the laser on the focal plane of the sample can be detected by the reflection of the dichroic mirror, so that the formed focal plane can be detected. The images have high spatial resolution.
  • the scattered light of nanoparticles is generally several orders of magnitude higher than the fluorescence of labeled probes.
  • Using laser scanning confocal fluorescence microscopy to collect scattered light of unlabeled nanoparticles will accurately locate the intracellular distribution of nanoparticles.
  • no laser scanning confocal microscope is capable of simultaneous fluorescence imaging of light scattered by unlabeled nanoparticles and labeled biomolecules, and hyperspectral imaging of light scattered by nanoparticles.
  • the scattered light of nanoparticles has the same detection wavelength as the incident laser light, so it cannot pass through the filter of the fluorescence microscope, resulting in serious interference to the detection of single-particle scattering.
  • One of the main purposes of the present disclosure is to provide a device and method for confocal imaging of scattered light of nanoparticles.
  • a device for confocal imaging of nanoparticle scattered light comprising a laser unit, a first pinhole, a beam splitter turntable, a scanning focusing unit, a motorized stage, and a first detection an imaging unit, a second pinhole, a spectroscopic unit, and a second detection imaging unit; wherein,
  • the laser light emitted by the laser unit reaches the beam splitter turntable through the first pinhole, and the light reflected by the beam splitter turntable is irradiated on the sample through the focusing scanning unit;
  • Part of the laser light transmitted from the sample enters the first detection imaging unit for imaging
  • the mixed light emitted from the sample returns through the scanning and focusing unit to the beam splitter turntable.
  • the mixed light passing through the beam splitter turntable passes through the second pinhole and enters the spectroscopic unit for light splitting, and then enters the second detection and imaging unit for detection and imaging.
  • the excitation unit and the beam splitter turntable are selected so that the scattered light signal emitted by the unlabeled nanoparticles and the fluorescent signal emitted by the labeled biomolecule in the sample are simultaneously detected and imaged by the second detection and imaging unit.
  • the supercontinuum laser of the excitation unit and the flat beam splitter of the beam splitter turntable are selected so that the scattered light signal emitted by the unlabeled nanoparticles in the sample is detected and imaged by the second detection and imaging unit.
  • FIG. 1 is a schematic structural diagram of a device for confocal imaging of nanoparticle scattering light in an embodiment of the disclosure
  • FIG. 2 is a schematic top view of a beam splitter turntable in an embodiment of the disclosure
  • FIG. 3 is a synchronous imaging diagram of scattered light and fluorescence in step (5) in Example 1 of the present disclosure.
  • 100-laser unit 101-405nm monochromatic laser, 102-488nm monochromatic laser, 103-543nm monochromatic laser, 104-supercontinuum laser, 200-first pinhole, 201-second pinhole; 300-th 1 lens; 301-second lens; 400-beam splitter turntable; 401-first dichroic mirror; 402-second dichroic mirror; 403-third dichroic mirror; 404-first plate beam splitter Mirror; 405-Second Plate Beamsplitter; 500-Focus Scanning Unit; 501-x-y-axis Scanning Galvo; 502-Objective Lens; 600-Object Stage; 701-Fiber; 702-Split Prism; 703-Graster; The first detector; 801 - the second detector.
  • the imaging of nanoparticles on the confocal laser microscope is basically a method of modification with fluorescent probes.
  • This indirect imaging method not only changes the original surface modification of the nanoparticles, but also produces phototoxicity and false positive results.
  • it is difficult to perform simultaneous imaging with fluorescent probes of other wavelengths.
  • the present disclosure utilizes a laser to perform confocal imaging on the scattered light of nanoparticles, and can perform spatially precise positioning and high-resolution imaging on non-labeled nanoparticles.
  • the present disclosure specifically relates to a device and method for confocal imaging based on the scattered light of nanoparticles generated by lasers. Monochromatic lasers and related long-pass dichroic mirrors are selected according to the excitation wavelength of fluorescent probes.
  • the narrow transition range between the wavelength and the starting wavelength)” can half reflect the wavelength laser, and then use different PMTs to synchronously collect the scattered light of nanoparticles reflected from the sample and the fluorescence of labeled biomolecules to achieve synchronous confocal of nanoparticles and biomolecules
  • the technology can also realize confocal hyperspectral analysis of the scattered light of nanoparticles by using a supercontinuum laser and a flat beam splitter according to the difference in the scattering efficiency of nanoparticles to different wavelengths of laser light.
  • the biomolecule can be DNA, protein, and other biological signals that can be labeled with fluorescent probes.
  • the present disclosure fills the gap in the simultaneous confocal imaging of unlabeled nanoparticles and fluorescently labeled biomolecules in the sample and the confocal hyperspectral imaging analysis of the scattered light of nanoparticles, and can realize the scattered light of unlabeled nanoparticles.
  • Simultaneous fluorescence imaging of probe-labeled organisms and hyperspectral imaging of scattered light from nanoparticles with in situ real-time imaging, label-free imaging of nanoparticles, high resolution of particle size, single particle imaging, high fluorescence compatibility, accurate spatial positioning, Long dynamic tracking time, co-localization analysis, interaction analysis and qualitative analysis, three-dimensional stereo imaging and scattered light hyperspectral analysis, etc.
  • the present disclosure discloses a nanoparticle scattering light confocal imaging device, comprising a laser unit, a first pinhole, a beam splitter turntable, a scanning focusing unit, a motorized stage, a first detection and imaging unit, a second pinhole, a spectroscopic unit and a second detection imaging unit; wherein,
  • the laser light emitted by the laser unit reaches the beam splitter turntable through the first pinhole, and the light reflected by the beam splitter turntable is irradiated on the sample through the focusing scanning unit;
  • Part of the laser light transmitted from the sample enters the first detection imaging unit for imaging
  • the mixed light emitted from the sample returns through the scanning and focusing unit to the beam splitter turntable.
  • the mixed light passing through the beam splitter turntable passes through the second pinhole and enters the spectroscopic unit for light splitting, and then enters the second detection and imaging unit for detection and imaging.
  • the laser unit includes an adjustable laser unit composed of a plurality of monochromatic lasers and supercontinuum lasers;
  • the beam splitter turntable is configured according to the laser wavelengths of different lasers in the laser system, and includes an adjustable beam splitter turntable composed of a plurality of dichroic mirrors and a plurality of flat beam splitters , where the wavelength of the monochromatic laser lies in the narrow transition interval between the cutoff wavelength and the onset wavelength of the dichroic mirror.
  • the beamsplitter dial selects a dichroic mirror; the dichroic mirror can transmit 40 to 60% of the scattered light from nanoparticles and ⁇ 90% biomolecular fluorescence;
  • the beamsplitter dial selects a flat-panel beamsplitter; the flat-panel beamsplitter can transmit 50 to 70% of the scattered light from nanoparticles emitted by the sample .
  • the mixed light includes the scattered light of nanoparticles emitted from the sample and the fluorescence of the excited probe label; the mixed light is split by the spectroscopic unit to form a monochromatic spectrum, and the second detection imaging unit is used
  • the two detectors in the device detect the light signal in the scattered light band and the light signal in the fluorescence band at the same time, respectively, so as to realize the simultaneous imaging of the scattered light and the fluorescence.
  • the angle between the laser beam passing through the first pinhole and the turntable of the beam splitter is 45 degrees.
  • the scanning focusing unit includes a scanning galvanometer and an objective lens
  • the scanning galvanometer includes an x-y axis scanning galvanometer, and the scanning frequency ranges from 200 to 8000 Hz.
  • the nanoparticle has no imaging label, and the scattered light signal generated by the nanoparticle to the laser is detected;
  • the light splitting unit includes a light splitting prism and a grating.
  • the first detection imaging unit includes a photomultiplier tube
  • the second detection imaging unit includes a photomultiplier tube and a spectral charge coupled element.
  • the present disclosure also discloses a method for simultaneous imaging of the scattered light of the label-free nanoparticles and the fluorescence of the labelled biomolecules, using the above-mentioned device, including:
  • the excitation unit and the beam splitter turntable are selected so that the scattered light signal emitted by the unlabeled nanoparticles and the fluorescent signal emitted by the labeled biomolecule in the sample are simultaneously detected and imaged by the second detection and imaging unit.
  • the present disclosure also discloses a method for hyperspectral imaging of scattered light of nanoparticles, using the above-mentioned device, including:
  • the supercontinuum laser of the excitation unit and the flat beam splitter of the beam splitter turntable are selected so that the scattered light signal emitted by the unlabeled nanoparticles in the sample is detected and imaged by the second detection and imaging unit.
  • a novel device for confocal imaging of nanoparticle scattered light in the present disclosure includes a laser unit 100 , a first pinhole 200 , a first lens 300 , and a beam splitter
  • PMT photomultiplier tube
  • the laser light emitted by the laser unit 100 after passing through the first pinhole 200 and the first lens 300, is incident on the beam splitter turntable 400 at an angle of 45°, and the dichroic mirror or The flat beam splitter is reflected to the x-y axis scanning galvanometer 501, and then enters the condenser objective 502 to focus on the sample on the motorized stage 600, and the scattered light from the nanoparticles emitted from the sample and the fluorescence of the probe label return the same way And through the dichroic mirror or plate beam splitter, through the second pinhole 201 and the second lens 301, and then through the fiber 701 into the dispersive prism 702, through the grating 703, and finally to the second detector 801 to achieve non-marking Confocal imaging of light scattered by nanoparticles.
  • the scattered light and fluorescence separated by the prism enter different PMTs, and the light transmitted from the sample enters the PMT of the first detector 800 .
  • the laser unit 100 has a plurality of monochromatic lasers, these laser generators can emit lasers of different wavelengths, and the monochromatic lasers of each wavelength have a corresponding long-wavelength pass dichroic mirror of laser semi-reflection,
  • the wavelength is in the narrow "cut on" wavelength range of the long-pass dichroic mirror, and the monochromatic laser passes through the first pinhole 200 at an incident angle of 45° to the long-pass dichroic mirror on the beam splitter turntable 400, about 50%
  • the laser light is reflected onto the sample and the remaining 50% of the laser light is transmitted through the dichroic mirror.
  • the laser generated by the laser generator has a good monochromatic color, and the unlabeled nanoparticles in the sample irradiated by the laser produce good scattered light with the same wavelength.
  • the range of the scattered light signal collected by the PMT is the laser wavelength ⁇ 5 nm.
  • the laser adopts a monochromatic laser, and select a long-pass dichroic mirror in the "cut on" interval close to the laser wavelength on the beam splitter turntable, so that the nanoparticles emitted by the sample scatter 40-60% of the light, preferably 50% and more than 90% of the biomolecule fluorescence is transmitted through the dichroic mirror, enabling simultaneous imaging of the scattered light of the unlabeled nanoparticles and the fluorescence of the probe-labeled biomolecules.
  • the supercontinuum laser 104 is used as the laser, and the flat beam splitter on the beam splitter turntable is selected, 50-70% of the scattered light spectrum emitted by the sample can pass through the beam splitter to achieve nanoparticle scattered light hyperspectral imaging.
  • the laser unit 100 in this embodiment has multiple monochromatic lasers and one supercontinuum laser, and the monochromatic lasers are mainly: 405nm monochromatic laser 101, 488nm monochromatic laser 102, 543nm monochromatic laser 103, supercontinuum laser 104
  • the first three monochromatic lasers are mainly used for simultaneous imaging of unlabeled nanoparticles scattered light and probe-labeled biomolecules, and supercontinuum lasers are used for nanoparticle scattered light hyperspectral imaging.
  • the scattered light of nanoparticles and the fluorescence of fluorescent probe-labeled biomolecules generated by monochromatic laser irradiation on the sample return through the original optical path, and then enter the long-pass dichroic mirror at an angle of 45°, of which about 50% scattering
  • the light and more than 90% of the fluorescence pass through the dichroic mirror and reach the beam splitting prism 702 through the second pinhole 201.
  • the light signals in the scattered light and fluorescence bands are simultaneously detected by different PMTs to realize the scattered light and probes of the nanoparticles. Simultaneous fluorescence imaging of labeled biomolecules, etc.
  • the spectrum emitted by the supercontinuum laser 104 is a continuous laser, and the spectrum covers 320-2400 nm.
  • the scattered light from nanoparticles generated by continuum laser irradiation on the sample returns through the original optical path, and then reaches the flat beam splitter at an angle of 45°, where the scattered light in the range of 380-1100nm passes through with a transmittance of about 50% or 70%.
  • the light signals in the scattered light and fluorescence wavelength bands are simultaneously detected by different PMTs to realize the scattered light hyperspectral imaging of nanoparticles.
  • the beam splitter turntable 400 has three long-pass dichroic mirrors with different "cut on" wavelengths (namely the first dichroic mirror 401, the second dichroic mirror 402 and the third dichroic mirror 402). 403) and two flat beam splitters (ie the first flat beam splitter 404 and the second flat beam splitter 405), the three long-pass dichroic mirrors are mainly used to semi-reflect the 405, 488 and 543 nm laser light respectively. to the sample, and semi-transmits the scattered light from the nanoparticles and fully transmits the fluorescence; the functions of the two flat beamsplitters are 50% reflected laser light and 50% transmitted through nanoparticles scattered light, 30% reflected laser light and 70% transmitted Light is scattered by nanoparticles.
  • the scanning and focusing unit 500 includes an x-y axis scanning galvanometer 501 and a condenser objective lens 502.
  • the frequency of the x-y axis scanning galvanometer 501 is adjustable, and the adjustable range is 200-8000 Hz.
  • the scanning focusing unit 500 can focus the laser on the focal plane of the sample in a scanning manner, and irradiate the unlabeled nanoparticles and fluorescently labeled biomolecules in the focal plane, and the generated scattered light and fluorescence can pass through the second pinhole 201 Reach the detector, which excludes the background scattered light signal of the sample that is not in the longitudinal focal plane and the transverse viewing area, and does not reduce the scattered light signal of the label-free nanoparticles at the focal point.
  • the long-pass dichroic mirror of the beam splitter turntable 400 can fully transmit the fluorescent signal greater than the laser wavelength generated by the labeled biomolecules in the sample, and can semi-transmit the fluorescent signal equal to the laser wavelength generated by the unlabeled nanoparticles in the sample. Scatter the light signal so that both wavelengths can pass through the dichroic mirror.
  • the signal for detecting nanoparticles is scattered light, and there is no need to image and label the nanoparticles, and the laser energy can make the nanoparticles in the sample generate scattered light signals.
  • the laser unit 100 can generate laser light according to the excitation wavelength of the fluorescent probe of the labeled biomolecules, which can not only make the unlabeled nanoparticles emit scattered light, but also can excite the fluorescence of the labeled biomolecules, so that the nanoparticles in the cells can emit light.
  • the scattered light spectrum and the fluorescence spectrum of the labeled biomolecule do not overlap.
  • the laser can make the nanoparticles emitted in the sample scatter light and label the biomolecules to fluoresce.
  • the composite beam of these two optical signals returns to the original path and passes through the dichroic mirror, then enters the second pinhole 201, and is then split into light.
  • the prism 702 is decomposed into dispersed spectra, and different PMTs are used to receive scattered light spectral signals and fluorescence spectral signals respectively.
  • the laser signal passing through the sample is also PMTed to achieve simultaneous imaging of label-free nanoparticles and fluorescently labeled biomolecules.
  • the laser generator can generate monochromatic laser light, and the unlabeled nanoparticles in the sample irradiated by the laser generate scattered light that is also monochromatic and has the same wavelength. 5nm.
  • the laser semi-reflective long-wavelength dichroic mirror has a narrow "cut on" wavelength range that can transmit 50% of the scattered light of unlabeled nanoparticles, so that the light signal collected by PMT detection of unlabeled nanoparticles is Scattered light.
  • the laser is focused on the cell sample through the pinhole, the unlabeled nanoparticles scattered light emitted by the sample and the fluorescence of the labeled biomolecules return the same way, and reach the PMT through the second pinhole 201, where the scattered light signal of the nanoparticles and The fluorescent signals of labeled biomolecules are all confocal, enabling single-particle imaging of nanoparticles.
  • the scattered light of unlabeled nanoparticles and the fluorescence synchronous imaging of probe-labeled biomolecules are confocal, so the scattered light and fluorescence synchronous imaging can not only scan and image in the XY plane, but also in the Z-axis direction. Three-dimensional stereo imaging can be achieved.
  • the present embodiment also discloses a method for synchronous imaging of unlabeled nanoparticles scattered light and labeled biomolecule fluorescence in a sample by using the above-mentioned device, including the following steps:
  • the unlabeled nanoparticles are exposed, and incubated in an incubator for a certain period of time to obtain ingested nanoparticles and fluorescently labeled organisms.
  • the live cell sample is obtained, and the sample is placed on the sample stage; or the unlabeled nanoparticles are exposed to the cells, and after a certain period of incubation in the incubator, the cell biomolecules are labeled with specific fluorescent probes to obtain the ingested nanoparticles and fluorescence. Label the living cell sample of the organism and place the sample on the sample stage. Or fix the fluorescently modified nanoparticles in the gel to obtain a gel sample with embedded nanoparticles, and place the sample on the motorized stage;
  • the optimal laser and the semi-reflecting dichroic mirror of the laser are selected, so that the laser is reflected on the scanning focusing unit 500 .
  • the nanoparticles do not need to be labeled, and can simultaneously observe the distribution position and relative content of unlabeled nanoparticles and fluorescently labeled organisms in the sample in situ, observe the morphology change of the nanoparticles at the single particle level, and dynamically observe the unlabeled nanoparticles for a long time. trajectories in living cells.
  • the present embodiment also discloses a method for hyperspectral imaging of nanoparticle scattered light in a sample using the above-mentioned device, characterized in that the method comprises the following steps:
  • the imaging parameters such as the intensity of the light source, the type of the flat beam splitter, and the scanning speed of the galvanometer are adjusted.
  • the nanoparticle scattering light spectrum imaging is performed.
  • step (6) Analyzing the nanoparticle scattering light hyperspectral signal collected in step (5) to obtain the characteristic scattering light spectrum of the nanoparticle.
  • the nanoparticles do not need to be labeled, and can perform hyperspectral imaging of the scattered light of the nanoparticles in the sample in situ, and analyze and compare the characteristics of the morphology changes, surface modification and species of the nanoparticles at the single particle level.
  • FIG. 1-2 The structure of the device for synchronous imaging of the scattered light of non-labeled nanoparticles in living cells and the fluorescence of labeled biomolecules in this embodiment is shown in Figures 1-2, including a laser unit 100, a laser semi-reflecting long-wavelength dichroic mirror, a scanning focus The unit 500, the sample stage 600 on which the living cell culture system can be mounted, the beam splitting prism 702, and the photomultiplier tube (ie, the second detector 801) for detecting the light signal.
  • the laser light emitted by the laser unit is semi-reflected by the long-wavelength dichroic mirror to the scanning focusing unit 500 and focused on the cell sample, and the scattered light reflected from the unlabeled nanoparticles on the focal plane of the sample and the fluorescence of the labeled biomolecules
  • the scattered light and fluorescent light are separated by the beam splitter prism 702 and enter different avalanche diodes.
  • the device scans the cells by focusing the laser on the focal plane of the cell sample and scanning the cells in the x-y direction, and detects the scattered light of the nanoparticles in the local cell on the focal plane, without reducing the scattered light of the nanoparticles, and at the same time reducing the longitudinal direction from the cell sample.
  • the interference of lateral scattered light combined with PMT and computer noise reduction processing, can improve the sensitivity and signal-to-noise ratio.
  • the above-mentioned device is used to simultaneously image the scattered light of unlabeled nanoparticles and the fluorescence of labeled nuclei in living cells, including the following steps:
  • step (6) Analyze the intensity and location of the unlabeled AgNPs scattered light signal and the labeled cell nucleus fluorescence signal in step (5), co-localize and analyze the spatial relationship between the two, and accurately locate the intracellular distribution of AgNPs at the single particle level.
  • Fig. 3 is the imaging picture of step 5. It can be seen from Fig. 3 that unlabeled AgNPs and fluorescently labeled nuclei can be simultaneously observed in AgNPs exposed cells, as well as the cell morphology of living cells, while the blank control group only Fluorescently labeled cell nuclei can be observed without the presence of AgNPs, which proves that the detected scattered light signal is indeed from unlabeled AgNPs; from the superimposed pictures, the co-localization analysis of the light signal can see that the AgNPs are located outside the cell, indicating that the device of the present disclosure can effectively The method can precisely locate the intracellular distribution of AgNPs at the single particle level.
  • the device and method for confocal imaging of nanoparticle scattering light of the present disclosure have at least one of the following advantages over the prior art:
  • the device and method for confocal imaging of scattered light of nanoparticles of the present disclosure have at least one or a part of the following advantages over the prior art:

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Abstract

一种纳米颗粒散射光共聚焦成像的装置和方法，装置包括激光单元（100）、第一针孔（200）、第一透镜（300）、分束镜转盘（400）、扫描单元（500）、电动载物台（600）、第一检测器（800）、第二针孔（201）、分光色散棱镜（702）和第二检测器（801）。装置无需事先对纳米颗粒进行标记，能够同步收集无标记纳米颗粒散射光和荧光标记生物分子的荧光，也能够原位成像，直接观察活细胞中纳米颗粒分布位置和含量。

Description

纳米颗粒散射光共聚焦成像的装置和方法 技术领域
本公开涉及纳米材料观察和生物分子检测等技术领域，具体涉及一种纳米颗粒散射光共聚焦成像的装置和方法。
背景技术
随着越来越多的纳米颗粒应用到生物医学等领域，动态追踪其在生物体内的分布位置和过程已成为其生物安全性评价热点研究内容。由于纳米材料的尺寸非常小，电子显微镜作为一种高分辨显微成像技术，一直是观察无标记纳米颗粒在细胞内分布位置和形貌状态主要方法。然而，尽管有很高的分辨率，电子显微镜不能进行原位追踪活细胞内纳米颗粒动态分布过程，而且其生物样品制备过程需要复杂步骤和较长的时间，可能会使胞内纳米颗粒发生形貌变化和转化。电子显微成像技术的这些不足之处限制了人类深入研究胞内纳米颗粒分布过程和相互作用机制。因此，开发一种简单方便显微成像技术来动态追踪细胞内纳米颗粒是十分必要的。随着光学显微成像技术的发展，为解决直接原位动态追踪纳米颗粒在活细胞中分布和相互作用过程问题提供了机遇。
由于大多数纳米颗粒都具有良好的散射光性质，目前针对无标记纳米颗粒已经开发了许多光学显微成像技术，如暗场显微镜，共聚焦拉曼显微镜，正交偏振显微镜等。这些光学显微成像技术能够收集无标记纳米颗粒的散射光信号，而原位观察到胞内无标记纳米颗粒，为深入研究纳米颗粒细胞生物效应和过程提供了很大的便利性。但不足的是，这些成像模式会为了提高纳米颗粒散射光信号，去降低细胞自身透射光、反射光、荧光等背景的干扰，而不能同步对细胞内细微结构、亚细胞器、蛋白质等生物分子进行成像，而难以准确定位纳米颗粒胞内分布位置和相互作用过程。
为了克服检测无标记纳米颗粒手段的限制，众多研究通过对纳米颗粒表面进行修饰以添加上易于检测的标记，通过检测标记信号以追踪纳 米颗粒在活细胞内的分布和含量。例如，将荧光探针修饰在纳米颗粒表面，通过高分辨荧光显微镜成像检测活细胞中标记的荧光探针，以实时观察纳米颗粒在细胞中的分布位置和含量。
作为一种高分辨荧光显微镜，激光共聚焦扫描显微镜利用激光检测标记细胞的荧光探针，再结合细胞计算机图像处理技术，可以观察细胞内微结构和特定生物分子，在亚细胞检测pH、Ca离子、膜电位等生理状态及细胞形态的变化，并能够进行定量分析和实时动态成像，是目前应用最广泛的分子生物学分析仪器。尤其值得注意得是，激光共聚焦扫描显微镜的光源和探测器前方都各有一个针孔，只有通过二向色镜反射在样品焦平面上激光产生的荧光才能被检测到，使得形成的焦平面图像具有很高的空间分辨率。因此，在纳米生物学领域，众多研究者利用激光共聚焦扫描显微镜对标记在纳米颗粒上的荧光探针进行高分辨成像，再结合细胞生物分子特异性荧光探针，实现精准定位和实时追踪纳米颗粒在活细胞中分布位置和形貌状态的目的。然而，这种荧光标记会改变纳米颗粒表面性质而影响纳米颗粒的生物效应，并且长时间成像会有荧光漂白和光毒性等问题。加之，很多纳米材料表面难以形成稳定的荧光标记修饰，使荧光标记在细胞中容易从纳米颗粒上脱落，导致观察到加阳性结果。故这种方法只适用少数纳米材料。
根据文献报道，纳米颗粒的散射光一般要比标记探针的荧光高出几个数量级，利用激光扫描共聚焦荧光显微镜收集无标记纳米颗粒的散射光，将会准确定位纳米颗粒在胞内分布位置。但迄今为止，没有激光扫描共聚焦显微镜能够对非标记纳米颗粒散射光和标记生物分子的荧光同步成像以及纳米颗粒散射光高光谱成像。这主要是因为，不同于标记生物分子的荧光信号，纳米颗粒的散射光与入射激光具备相同的检测波长，因此无法在通过荧光显微镜的滤光片，导致对单粒子散射检测的严重干扰。
公开内容
本公开的主要目的之一在于提出一种纳米颗粒散射光共聚焦成像的装置和方法。
具体地，作为本公开的一个方面，提供了一种纳米颗粒散射光共聚焦成像的装置，包括激光单元、第一针孔、分束镜转盘、扫描聚焦单元、电动载物台、第一检测成像单元、第二针孔、分光单元和第二检测成像单元；其中，
激光单元发出的激光，经过第一针孔到达分束镜转盘，经分束镜转盘反射的光通过聚焦扫描单元照射在样品上；
从样品上透射出的部分激光进入第一检测成像单元成像；
从样品上发射出的混合光原路返回经过扫描聚焦单元到达分束镜转盘，透过分束镜转盘的混合光经过第二针孔后进入分光单元分光，之后进入第二检测成像单元检测成像。
作为本公开的另一个方面，还提供了一种无标记纳米颗粒散射光与标记生物分子荧光同步成像方法，采用如上所述的装置，包括：
将含有无标记纳米颗粒和荧光标记的生物分子的样品放置在电动载物台上；
选择激发单元和分束镜转盘使样品中无标记纳米颗粒发射的散射光信号和标记生物分子发射的荧光信号分别同时被第二检测成像单元检测成像。
作为本公开的又一个方面，还提供了一种纳米颗粒散射光高光谱成像方法，采用如上所述的装置，包括：
将含有无标记纳米颗粒的样品放置在电动载物台上；
选择激发单元的超连续谱激光器和分束镜转盘的平板分束镜使样品中无标记纳米颗粒发射的散射光信号被第二检测成像单元检测成像。
附图说明
图1为本公开实施例中纳米颗粒散射光共聚焦成像的装置结构示意图；
图2为本公开实施例中分束镜转盘的俯视方向示意图；
图3为本公开实施例1中步骤(5)散射光和荧光同步成像图。
上图中，附图标记含义如下：
100-激光单元，101-405nm单色激光器，102-488nm单色激光器， 103-543nm单色激光器，104-超连续谱激光器，200-第一针孔，201-第二针孔；300-第一透镜；301-第二透镜；400-分束镜转盘；401-第一二向色镜；402-第二二向色镜；403-第三二向色镜；404-第一平板分束镜；405-第二平板分束镜；500-聚焦扫描单元；501-x-y轴扫描振镜；502-物镜；600-载物台；701-光纤；702-分光棱镜；703-光栅；800-第一检测器；801-第二检测器。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本公开作进一步的详细说明。
目前，在激光共聚焦显微镜上对纳米颗粒进行成像基本上都是通过荧光探针修饰的方法，这种间接成像的方法不仅会改变纳米颗粒原有的表面修饰、产生光毒性和假阳性结果，而且难以和其他波长的荧光探针进行同步成像。这些缺点限制了激光共聚焦显微镜在动态示踪活细胞内纳米颗粒方面的应用，亟需一种非标记成像的方式。大多数纳米颗粒都对光具有良好的散色性，因此，暗场显微镜常被用来收集散射光，达到对样品中非标记纳米颗粒原位成像。但暗场显微镜基本上都是宽敞显微镜并难以同步对样品中荧光成像，而不能实现纳米颗粒在空间上的精准定位和探针标记生物分子等的荧光同步成像。本公开是利用激光对纳米颗粒散射光进行共聚焦成像，对非标记纳米颗粒可以在空间上进行精准定位和高分辨成像。本公开具体是一种基于激光产生的纳米颗粒散射光进行共聚焦成像的装置和方法，根据荧光探针的激发波长选择单色激光以及相关的长波通二向色镜，其其“cut on(截至波长与起始波长之间)”的狭窄过渡区间可以半反射波长激光，然后利用不同PMT同步采集样品反射出来的纳米颗粒散射光和标记生物分子荧光，实现对纳米颗粒和生物分子同步共聚焦成像，同时，该技术还可以根据纳米颗粒对不同波长激光散射效率的差异性，利用超连续谱激光器和平板分束镜实现纳米颗粒散射光共聚焦高光谱分析。该生物分子可以是能被荧光探针标记的DNA、蛋白以及其他生物信号等。与现有技术相比，本公开填补了对样品中无标记纳米颗粒和荧光标记生物分子同步共聚焦成像以及纳米颗 粒散射光共聚焦高光谱成像分析方面的空白，能够实现非标记纳米颗粒散射光和探针标记生物的荧光同步成像以及纳米颗粒散射光的高光谱成像，具有原位实时成像、纳米颗粒非标记成像、颗粒尺寸高分辨率、单颗粒成像、荧光兼容性高、空间定位准确、动态追踪时间长、共定位分析、相互作用分析和定性分析、三维立体成像和散射光高光谱分析等特点。
本公开公开了一种纳米颗粒散射光共聚焦成像的装置，包括激光单元、第一针孔、分束镜转盘、扫描聚焦单元、电动载物台、第一检测成像单元、第二针孔、分光单元和第二检测成像单元；其中，
激光单元发出的激光，经过第一针孔到达分束镜转盘，经分束镜转盘反射的光通过聚焦扫描单元照射在样品上；
从样品上透射出的部分激光进入第一检测成像单元成像；
从样品上发射出的混合光原路返回经过扫描聚焦单元到达分束镜转盘，透过分束镜转盘的混合光经过第二针孔后进入分光单元分光，之后进入第二检测成像单元检测成像。
在本公开的一些实施例中，所述激光单元包括多个单色激光器和超连续谱激光器构成的可调节激光单元；
在本公开的一些实施例中，所述分束镜转盘是根据激光***中不同激光器的激光波长配置的，包括多个二向色镜和多个平板分束镜构成的可调节分束镜转盘，其中单色激光器的波长位于二向色镜截止波长和起始波长之间狭窄的过渡区间。
在本公开的一些实施例中，当激光单元选择单色激光器时，分束镜转盘选择二向色镜；所述二向色镜能透过样品发射出40至60％的纳米颗粒散射光和≥90％的生物分子荧光；
在本公开的一些实施例中，当激光单元选择超连续谱激光器时，分束镜转盘选择平板分束镜；所述平板分束镜能透过样品发射出的50至70％纳米颗粒散射光。
在本公开的一些实施例中，所述混合光包括从样品上发射出来的纳米颗粒散射光和激发探针标记的荧光；混合光经分光单元分光，形成单 色光谱，使用第二检测成像单元中的两个检测器分别同时检测散射光波段的光信号和荧光波段的光信号，实现散射光和荧光同步成像。
在本公开的一些实施例中，经过所述第一针孔的激光与分束镜转盘之间的夹角为45度。
在本公开的一些实施例中，所述扫描聚焦单元包括扫描振镜和物镜；
在本公开的一些实施例中，所述扫描振镜包括x-y轴扫描振镜，扫描频率范围为200至8000Hz。
在本公开的一些实施例中，所述纳米颗粒无成像标记，检测的是纳米颗粒对激光产生的散射光信号；
在本公开的一些实施例中，所述分光单元包括分光棱镜和光栅。
在本公开的一些实施例中，所述第一检测成像单元包括光电倍增管；
在本公开的一些实施例中，所述第二检测成像单元包括光电倍增管和光谱电荷耦合元件。
本公开还公开了一种无标记纳米颗粒散射光与标记生物分子荧光同步成像方法，采用如上所述的装置，包括：
将含有无标记纳米颗粒和荧光标记的生物分子的样品放置在电动载物台上；
选择激发单元和分束镜转盘使样品中无标记纳米颗粒发射的散射光信号和标记生物分子发射的荧光信号分别同时被第二检测成像单元检测成像。
本公开还公开了一种纳米颗粒散射光高光谱成像方法，采用如上所述的装置，包括：
将含有无标记纳米颗粒的样品放置在电动载物台上；
选择激发单元的超连续谱激光器和分束镜转盘的平板分束镜使样品中无标记纳米颗粒发射的散射光信号被第二检测成像单元检测成像。
在一个优选实施例中，如图1-2所示，本公开的一种新型纳米颗粒散射光共聚焦成像的装置，包括激光单元100、第一针孔200、第一透镜300、分束镜转盘400、扫描单元500、电动载物台600、第二针孔201、第二透镜301、光纤701、分光色散棱镜702及用于检测光信号的光电 倍增管(PMT)。所述的激光单元100发射的激光，穿过第一针孔200和第一透镜300后，以45°角入射到经分束镜转盘400上，经分束镜转盘400上二向色镜或平板分束镜反射到x-y轴扫描振镜501，再进入聚光物镜502聚焦到电动载物台600上的样品上，从样品中发射出来的纳米颗粒散射光和探针标记的荧光原路返回并透过二向色镜或平板分束镜，经第二针孔201和第二透镜301后通过光纤701进入分光色散棱镜702，穿过光栅703，最后达到第二检测器801，实现非标记纳米颗粒散射光的共聚焦成像。其中，棱镜分开的散射光和荧光进入不同的PMT，从样品中透射出的光进入第一检测器800的PMT。
其中，所述的激光单元100具有多个单色激光器，这些激光发生器能够发射出不同波长的激光，每个波长的单色激光器有一个相对应的激光半反射的长波通二向色镜，波长处在长波通二向色镜狭窄的“cut on”波长区间，单色激光经第一针孔200以45°入射角到分束镜转盘400上的长波通二向色镜，约50％的激光被反射到样品上，剩余50％激光透过二向色镜。
其中，激光发生器产生的激光单色好，激光照射样品中的无标记纳米颗粒产生散射光单色也良好，波长相同，PMT收集无标记纳米颗粒散射光信号范围为激光波长±5nm。
其中，当激光器采用单色激光器时，并选择分束镜转盘上与激光波长接近的“cut on”区间的长波通二向色镜，使样品发射出的纳米颗粒散射光40-60％、优选50％和生物分子荧光超过90％透过二向色镜，实现非标记纳米颗粒的散射光和探针标记生物分子的荧光同步成像。当激光器采用超连续谱激光器104时，并选择分束镜转盘上的平板分束镜，使样品发出的散射光光谱50-70％透过分束镜，实现纳米颗粒散射光高光谱成像。本实施例中的激光单元100具有多个单色激光器和一个超连续谱激光器，单色激光器主要为：405nm单色激光器101、488nm单色激光器102和543nm单色激光器103、超连续谱激光器104；前三个单色激光器主要用于非标记纳米颗粒散射光和探针标记的生物分子同步成像，超连续谱激光器用于纳米颗粒散射光高光谱成像。
其中，单色激光照射样品上产生的纳米颗粒散射光和荧光探针标记生物分子等的荧光，经原光路返回，再以45°角入射到长波通二向色镜，其中约50％的散射光和大于90％的荧光透过二向色镜，经第二针孔201到达分光棱镜702，处于散射光和荧光波段的光信号被不同的PMT同时检测，实现纳米颗粒的散射光和探针标记生物分子等的荧光同步成像。
其中，超连续谱激光器104发出的光谱为连续激光，光谱覆盖320-2400nm。超连续谱激光器104的发出的光经第一小孔200以45°入射到平板分束镜，平板分束镜在380-1100nm光谱范围内将激光以50％或30％(反射∶透过比例＝50∶50或30∶70)反射到样品上。连续谱激光照射样品上产生的纳米颗粒散射光经原光路返回，再以45°角到平板分束镜，其中在380-1100nm范围内的散射光以约50％或70％的透过率穿过平板分束镜，经第二针孔201到达分光棱镜702，处于散射光和荧光波段的光信号被不同的PMT同时检测，实现纳米颗粒的散射光高光谱成像。
其中，所述的分束镜转盘400具三个不同“cut on”波长的长波通二向色镜(即第一二向色镜401、第二二向色镜402和第三二向色镜403)和两个平板分束镜(即第一平板分束镜404和第二平板分束镜405)，三个长波通二向色镜主要作用是分别将405、488和543nm的激光半反射到样品上，并半透过纳米颗粒散射光和全透过荧光；两个平板分束镜的作用分别是50％反射激光和50％透过纳米颗粒散射光，30％反射激光和70％透过纳米颗粒散射光。
其中，所述的扫描聚焦单元500包括x-y轴扫描振镜501和聚光物镜502，x-y轴扫描振镜501的频率可调，可调范围为200-8000Hz。
其中，扫描聚焦单元500能够将激光以扫描的方式聚焦在样品焦平面上，照射到处于焦平面的无标记纳米颗粒和荧光标记的生物分子，产生的散射光和荧光能够通过第二针孔201到达检测器，这既可以排除不处在纵向焦平面和横向观察区域的样品背景散射光信号，又不降低处在聚焦点的无标记纳米颗粒散射光信号。
其中，分束镜转盘400的长波通二向色镜，可以全透过样品中标记 生物分子产生的大于激光波长的荧光信号，同时能够半透过样品中无标记纳米颗粒产生的等于激光波长的散射光信号，实现两种波长的信号都能透过二向色镜。
其中，检测纳米颗粒的信号是散射光，无需对纳米颗粒成像标记，激光能使使样品中纳米颗粒产生散射光信号。
其中，所述的激光单元100可以根据标记生物分子的荧光探针激发波长，产生的激光既能够使无标记纳米颗粒发射出散射光，又能够激发标记生物分子的荧光，使细胞中纳米颗粒的散射光光谱和标记生物分子的荧光光谱不重叠。
其中，所述的激光能够使样品中发射的纳米颗粒散射光和标记生物分子荧光，这两种光信号的复合光束原路返回经过二向色镜后，进入第二针孔201，再经分光棱镜702分解为分散的光谱，分别用不同的PMT接受散射光光谱信号和荧光光谱信号，同时，透过样品的激光信号也被PMT，达到同步对无标记纳米颗粒、荧光标记生物分子成像。
其中，所述激光发生器能够产生单色性的激光，激光照射样品中的无标记纳米颗粒产生散射光也具有单色性，波长相同，PMT收集无标记纳米颗粒散射光信号范围为激光波长±5nm。
其中，所述的激光半反射的长波通二向色镜，其狭窄的“cut on”波长区间能够透过50％的无标记纳米颗粒散射光，使得PMT检测收集到无标记纳米颗粒光信号是散射光。
其中，所述的激光经过针孔聚焦在细胞样品上，样品发射的无标记纳米颗粒散射光和标记生物分子的荧光原路返回，经过第二针孔201到达PMT，纳米颗粒的散射光信号和标记生物分子荧光信号都具有共聚焦性，能够对纳米颗粒进行单颗粒成像。
其中，非标记纳米颗粒的散射光和探针标记生物分子等的荧光同步成像具有共聚焦性，因此散射光和荧光同步成像不仅可以在XY平面扫描成像，而且可以在Z轴方向扫描成像，进而可以实现三维立体成像。
本实施例还公开了一种采用上述的装置进行样品中无标记纳米颗粒散射光与标记生物分子荧光同步成像方法，包括以下步骤：
(1)将细胞中感兴趣的生物分子通过基因工程技术(如质粒转染)带有荧光蛋白后，暴露无标记的纳米颗粒，培养箱中孵育一定时间，得到摄入纳米颗粒和荧光标记生物的活细胞样品，并将样品放置在样品台上；或将无标记的纳米颗粒暴露细胞，培养箱中孵育一定时间后，用特异性荧光探针标记细胞生物分子，得到摄入纳米颗粒和荧光标记生物的活细胞样品，并将样品放置在样品台上。或将带有荧光修饰纳米颗粒固定在凝胶中，得到包埋纳米颗粒的凝胶样品，将样品放置在电动载物台上；
(2)根据标记生物分子的荧光探针或荧光蛋白的激发光波长，选择最适激光和该激光半反射的二向色镜，使激光被反射到扫描聚焦单元500上。
(3)调节扫描聚焦单元500的扫描速度和焦距，找出最佳扫描速度和最适焦距，使激光聚焦在样品中感兴趣观察区域。
(4)选择合适放大倍数的显微镜物镜，并调节激光的能量，使样品中无标记纳米颗粒发射的散射光信号和标记生物分子发射的荧光信号足够强。
(5)设置扫描的时间和次数后，若进行三维立体成像，则还需要设置Z轴成像高度和步移间距，接着同时打开3个PMT，并设置PMT1采集的无标纳米颗粒散射光范围为激光波长±5nm，PMT2采集的光谱范围为标记荧光探针发射的荧光，PMT3采集透过样品的激光信号，以分别同步对无标记纳米颗粒、荧光标记生物分子和细胞形貌进行成像。其中，在第一检测器800中实现细胞形貌进行成像。
(6)分析步骤(5)中散射光信号和荧光信号的强度和在细胞中的位置，得到无标记纳米颗粒和标记生物分子在活细胞内相对含量和分布位置，共定位分析两者的空间关系。
其中，所述的纳米颗粒无需标记，能够原位同步观察样品中无标记纳米颗粒和荧光标记生物的分布位置和相对含量，在单颗粒水平观察纳米颗粒形貌变化，长时间动态观察无标记纳米在活细胞内运动轨迹。
本实施例还公开了一种采用上述的装置进行的样品中纳米颗粒散 射光高光谱成像方法，其特征在于，所述方法包括以下几个步骤：
(1)将摄入纳米颗粒的细胞或包埋纳米颗粒的凝胶等样品至于电动载物台上，找到样品中的纳米颗粒。
(2)打开超连续谱激光器，选择平板分束镜，将连续光谱的激光反射到样品上，光谱检测范围设置在380-1100nm区间。
(3)根据散射光光谱信号的强度，调整光源的强度、平板分束镜的种类、振镜的扫描速度等成像参数。
(4)调整好成像的参数后，根据样品特征设置成像的分辨率、放大倍数、扫描次数和光谱波长精度(一般2nm)等采集参数，若进行三维立体成像，则还需要设置Z轴成像高度和步移间距。
(5)设置完采集参数后，进行纳米颗粒散射光光谱成像。
(6)分析步骤(5)中采集到的纳米颗粒散射光高光谱信号，到纳米颗粒的特征散射光光谱。
其中，所述的纳米颗粒无需标记，能够原位对样品中纳米颗粒的散射光进行高光谱成像，在单颗粒水平分析和比较纳米颗粒形貌变化、表面修饰和种类等特征。
以下通过具体实施例结合附图对本公开的技术方案做进一步阐述说明。需要注意的是，下述的具体实施例仅是作为举例说明，本公开的保护范围并不限于此。
下述实施例中使用的化学药品和原料均为市售所得或通过公知的制备方法自制得到。
实施例1
本实施例的活细胞内无标记纳米颗粒散射光与标记生物分子荧光同步成像装置，其结构如图1-2所示，包括激光单元100、激光半反射的长波通二向色镜、扫描聚焦单元500、可搭载活细胞培养***的样品载物台600、分光棱镜702和用于检测光信号的光电倍增管(即第二检测器801)。所述的激光单元发射的激光，经长波通二向色镜半反射到扫描聚焦单元500，聚焦到细胞样品上，从样品焦平面上无标记纳米颗粒 反射出来的散射光和标记生物分子的荧光原路返回并透过二向色镜，再经分光棱镜702分开的散射光和荧光，进入不同的雪崩二极管。
本装置通过将激光聚焦在细胞样品焦平面上和x-y方向振镜对细胞进行扫描，检测焦平面上细胞局部的纳米颗粒散射光，不减小纳米颗粒的散射光，同时减少来自细胞样品在纵向和横向的散射光的干扰，结合PMT和计算机对信号进行降噪处理，可以提高灵敏度和信噪比。
采用上述装置对活细胞内无标记纳米颗粒散射光和标记细胞核荧光进行同步成像，包括一下几个步骤：
(1)选取玻璃底的共聚焦小皿，将生长在对数期的巨噬细胞转移到共聚焦小皿中，放入培养箱中过夜至细胞贴壁，将含有2μg/mL 100nm AgNPs(银纳米颗粒)的新鲜培养基替换共聚焦小皿中的旧培养基，放回培养箱中，其中加入不含AgNPs新鲜培养基的共聚焦小皿作为空白对照。
(2)培养2h后，取出共聚焦小皿，移除培养基，用PBS(磷酸缓冲液)洗涤细胞，除去未进入细胞的AgNPs，然后加入含有核酸特异性荧光探针10μM SYTO Green的新鲜培养基到共聚焦小皿中，放回培养箱中孵育10min，使SYTO Green进入细胞并标记细胞核。
(3)待细胞核标记完成后，取出共聚焦小皿，移除含有SYTO Green荧光探针的培养基，用PBS洗涤细胞以除去多余的探针，加入新鲜培养基至共聚焦小皿后，将样品置于装置样品台上。
(4)根据SYTO Green探针的最适激发光，调节激光发生器，选择488nm波长激光，并选择对488nm激光半反射的长波通二向色镜，此时488nm激光处在二向色镜狭窄的“cut on”过渡区域，接着，同时打开PMT1、PMT2和PMT3，设置PMT1接收光谱范围为483-493nm，PMT2接收光谱范围为510-560nm，PMT3接收样品的透射光，从而分别同时检测AgNPs的散射光、标记细胞核探针SYTO Green的荧光和细胞形貌，对无标记AgNPs、荧光标记细胞核和细胞形貌进行同步成像。
(5)调节聚焦平面，聚焦到细胞内无标记AgNPs和标记细胞核后，设置扫描速度和次数，开始用PMT1、PMT2同步收集无标记AgNPs的 散射光和标记细胞核荧光信号，以及PMT3拍摄细胞形貌状态。
(6)分析步骤(5)中无标记AgNPs散射光信号和标记细胞核荧光信号的强度和在细胞中的位置，共定位分析两者的空间关系，在单个颗粒水平准确定位AgNPs在胞内分布。
其中，图3为步骤5的成像图片，从图3中可以看出，AgNPs暴露细胞内可以同步观察到无标记的AgNPs和荧光标记的细胞核，以及活细胞的细胞形貌，而空白对照组只能观察到荧光标记的细胞核，无AgNPs存在，证明检测到散射光信号确实来自无标记AgNPs；从叠加图片中，对光信号共定位分析可以看出AgNPs处在细胞外，表明本公开的装置和方法可以在单颗粒水平精准定位AgNPs在细胞内的分布。
综上，本公开的纳米颗粒散射光共聚焦成像的装置和方法，相对于现有技术至少具有以下优势之一：
基于上述技术方案可知，本公开的纳米颗粒散射光共聚焦成像的装置和方法相对于现有技术至少具有以下优势之一或一部分：
(1)无需对纳米进行标记，能够同步收集无标记纳米颗粒和荧光标记生物分子；
(2)能够原位成像，直接观察活细胞中纳米颗粒分布位置和含量；
(3)成像分辨率高，既能够对纳米颗粒单颗粒成像，也能够在生物分子水平进行荧光成像；
(4)操作步骤简单方便，细胞暴露纳米颗粒后，可直接进行观察；
(5)长时间动态成像，无标记纳米颗粒无光漂白和光毒性，能够长时间实时追踪其在胞内分布和形貌；
(6)背景低，无标记纳米颗粒散射光信号的扫描共聚焦成像方式，能够将光电倍增管(PMT)收集信号聚焦在纳米颗粒上；
(7)能够对非标记纳米颗粒和荧光标记生物分子同步进行3维扫描成像；
(8)纳米颗粒散射光高光谱成像，能够原位直接分析纳米颗粒种类、大小和表面修饰等差异性，对纳米进行定性分析。
以上所述的具体实施例，对本公开的目的、技术方案和有益效果进 行了进一步详细说明，应理解的是，以上所述仅为本公开的具体实施例而已，并不用于限制本公开，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (14)

  1. 一种纳米颗粒散射光共聚焦成像的装置，其中，包括激光单元、第一针孔、分束镜转盘、扫描聚焦单元、电动载物台、第一检测成像单元、第二针孔、分光单元和第二检测成像单元；其中，
    激光单元发出的激光，经过第一针孔到达分束镜转盘，经分束镜转盘反射的光通过聚焦扫描单元照射在样品上；
    从样品上透射出的部分激光进入第一检测成像单元成像；
    从样品上发射出的混合光原路返回经过扫描聚焦单元到达分束镜转盘，透过分束镜转盘的混合光经过第二针孔后进入分光单元分光，之后进入第二检测成像单元检测成像。
  2. 根据权利要求1所述的装置，其中，
    所述激光单元包括多个单色激光器和超连续谱激光器构成的可调节激光单元；
    所述分束镜转盘是根据激光***中不同激光器的激光波长配置的，包括多个二向色镜和多个平板分束镜构成的可调节分束镜转盘，其中单色激光器的波长位于二向色镜截止波长和起始波长之间狭窄的过渡区间。
  3. 根据权利要求2所述的装置，其中，
    当激光单元选择单色激光器时，分束镜转盘选择二向色镜；
    当激光单元选择超连续谱激光器时，分束镜转盘选择平板分束镜。
  4. 根据权利要求2所述的装置，其中，
    所述二向色镜能透过样品发射出40至60％的纳米颗粒散射光和≥90％的生物分子荧光；
    所述平板分束镜能透过样品发射出的50至70％纳米颗粒散射光。
  5. 根据权利要求1所述的装置，其中，
    所述混合光包括从样品上发射出来的纳米颗粒散射光和激发探针标记的荧光；混合光经分光单元分光形成单色光谱，使用第二检测成像单元中的两个检测器分别同时检测散射光波段的光信号和荧光波段的光信号，实现散射光和荧光同步成像。
  6. 根据权利要求1所述的装置，其中，
    经过所述第一针孔的激光与分束镜转盘之间的夹角为45度。
  7. 根据权利要求1所述的装置，其中，
    所述扫描聚焦单元包括扫描振镜和物镜。
  8. 根据权利要求6所述的装置，其中，
    所述扫描振镜包括x-y轴扫描振镜，扫描频率范围为200至8000Hz。
  9. 根据权利要求1所述的装置，其中，
    所述纳米颗粒无成像标记，检测的是纳米颗粒对激光产生的散射光信号。
  10. 根据权利要求1所述的装置，其中，
    所述分光单元包括分光棱镜和光栅。
  11. 根据权利要求1所述的装置，其中，
    所述第一检测成像单元包括光电倍增管。
  12. 根据权利要求1所述的装置，其中，
    所述第二检测成像单元包括光电倍增管和光谱电荷耦合元件。
  13. 一种无标记纳米颗粒散射光与标记生物分子荧光同步成像方法，采用如权利要求1至12任一项所述的装置，包括：
    将含有无标记纳米颗粒和荧光标记的生物分子的样品放置在电动载物台上；
    选择激发单元和分束镜转盘使样品中无标记纳米颗粒发射的散射光信号和标记生物分子发射的荧光信号分别同时被第二检测成像单元检测成像。
  14. 一种纳米颗粒散射光高光谱成像方法，采用如权利要求1至12任一项所述的装置，包括：
    将含有无标记纳米颗粒的样品放置在电动载物台上；
    选择激发单元的超连续谱激光器和分束镜转盘的平板分束镜使样品中无标记纳米颗粒发射的散射光信号被第二检测成像单元检测成像。
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010016267A1 (ja) * 2008-08-08 2010-02-11 独立行政法人科学技術振興機構 粒子プローブ近傍に存在する物質の分布を検出する方法、粒子プローブを用いた画像化方法およびその利用
WO2012000102A1 (en) * 2010-06-30 2012-01-05 The Governors Of The University Of Alberta Apparatus and method for microscope-based label-free microflutdic cytometry
US20130100525A1 (en) * 2011-10-19 2013-04-25 Su Yu CHIANG Optical imaging system using structured illumination
US20160231225A1 (en) * 2013-10-09 2016-08-11 Siemens Aktiengesellschaft In vitro method for the label-free determination of a cell type of a cell
CN106990095A (zh) * 2017-05-23 2017-07-28 北京理工大学 反射式共焦cars显微光谱测试方法及装置
CN107167455A (zh) * 2017-05-23 2017-09-15 北京理工大学 分光瞳激光差动共焦cars显微光谱测试方法及装置
CN107167457A (zh) * 2017-05-23 2017-09-15 北京理工大学 透射式共焦cars显微光谱测试方法及装置
CN107167456A (zh) * 2017-05-23 2017-09-15 北京理工大学 透射式差动共焦cars显微光谱测试方法及装置
US20170307440A1 (en) * 2014-09-25 2017-10-26 Northwestern University Devices, methods, and systems relating to super resolution imaging

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010016267A1 (ja) * 2008-08-08 2010-02-11 独立行政法人科学技術振興機構 粒子プローブ近傍に存在する物質の分布を検出する方法、粒子プローブを用いた画像化方法およびその利用
WO2012000102A1 (en) * 2010-06-30 2012-01-05 The Governors Of The University Of Alberta Apparatus and method for microscope-based label-free microflutdic cytometry
US20130100525A1 (en) * 2011-10-19 2013-04-25 Su Yu CHIANG Optical imaging system using structured illumination
US20160231225A1 (en) * 2013-10-09 2016-08-11 Siemens Aktiengesellschaft In vitro method for the label-free determination of a cell type of a cell
US20170307440A1 (en) * 2014-09-25 2017-10-26 Northwestern University Devices, methods, and systems relating to super resolution imaging
CN106990095A (zh) * 2017-05-23 2017-07-28 北京理工大学 反射式共焦cars显微光谱测试方法及装置
CN107167455A (zh) * 2017-05-23 2017-09-15 北京理工大学 分光瞳激光差动共焦cars显微光谱测试方法及装置
CN107167457A (zh) * 2017-05-23 2017-09-15 北京理工大学 透射式共焦cars显微光谱测试方法及装置
CN107167456A (zh) * 2017-05-23 2017-09-15 北京理工大学 透射式差动共焦cars显微光谱测试方法及装置

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