WO2022151469A1 - Dispositif et procédé d'imagerie confocale de lumière diffusée de nanoparticules - Google Patents

Dispositif et procédé d'imagerie confocale de lumière diffusée de nanoparticules 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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PCT/CN2021/072443
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Chinese (zh)
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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

L'invention concerne un dispositif et procédé d'imagerie confocale de lumière diffusée de nanoparticules, le dispositif comprenant une unité laser (100), un premier trou d'épingle (200), une première lentille (300), un panneau giratoire diviseur de faisceau (400), une unité de balayage (500), une plateforme de chargement électrique (600), un premier détecteur (800), un second trou d'épingle (201), un prisme dispersif spectroscopique (702) et un second détecteur (801). Dans ce dispositif, le marquage préalable des nanoparticules n'est pas nécessaire, la collecte simultanée de la lumière diffusée par les nanoparticules non marquées et de la fluorescence des biomolécules marquées par fluorescence est possible, et la réalisation de l'imagerie in situ permet d'observer directement la distribution, l'emplacement et le contenu des nanoparticules dans les cellules vivantes.
PCT/CN2021/072443 2021-01-18 2021-01-18 Dispositif et procédé d'imagerie confocale de lumière diffusée de nanoparticules WO2022151469A1 (fr)

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