Nanoparticle scattered light confocal imaging device and method

Abstract

The invention discloses a nanoparticle scattered light confocal imaging device and method. The device comprises a laser unit, a first pinhole, a beam splitter turntable, a scanning focusing unit, an electric objective table, a first detection imaging unit, a second pinhole, a light splitting unit and a second detection imaging unit. The method does not need to mark nano particles, and can synchronously collect unmarked nano particles and fluorescence-labeled biomolecules; in-situ imaging can be realized, and the distribution position and content of nano-particles in living cells can be directly observed; the imaging resolution ratio is high, single nanoparticle imaging can be carried out, and fluorescence imaging can also be carried out at the biomolecule level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomaterial observation and biomolecule detection, and particularly relates to a device and method for nanoparticle scattering light confocal imaging. BACKGROUND

[0002] With more and more nanoparticles being applied to the field of biomedicine, dynamically tracking the distribution position and process of the nanoparticles in a living body has become a hot research content for evaluating the biological safety of the nanoparticles. Since the size of the nanomaterials is very small, an electron microscope, as a high-resolution microscopic imaging technology, has always been a main method for observing the distribution position and morphology state of the unlabelled nanoparticles in cells. However, although having a high resolution, the electron microscope cannot track the dynamic distribution process of the nanoparticles in living cells in situ, and the biological sample preparation process needs complex steps and a long time, which may cause the morphology change and transformation of the intracellular nanoparticles. These deficiencies of the electron microscopic imaging technology limit the in-depth study of the distribution process and interaction mechanism of the intracellular nanoparticles. Therefore, it is necessary to develop a simple and convenient microscopic imaging technology to dynamically track the intracellular nanoparticles. With the development of optical microscopic imaging technology, an opportunity is provided to solve the problem of directly and in situ dynamically tracking the distribution and interaction process of the nanoparticles in living cells.

[0003] Since most of the nanoparticles have good light scattering properties, many optical microscopic imaging technologies have been developed for unlabelled nanoparticles, such as dark field microscopy, confocal Raman microscopy, orthogonal polarization microscopy, etc. These optical microscopic imaging technologies can collect the scattering light signals of the unlabelled nanoparticles, and in situ observe the unlabelled nanoparticles in cells, which provides great convenience for the in-depth study of the cell biological effects and process of the nanoparticles. However, the deficiency is that these imaging modes will reduce the interference of the background of the transmission light, reflection light and fluorescence of the cells in order to improve the scattering light signals of the nanoparticles, and cannot simultaneously image the biological molecules such as the fine structure, subcellular organelles and proteins in the cells, so it is difficult to accurately locate the intracellular distribution position and interaction process of the nanoparticles.

[0004] In order to overcome the limitation of the means for detecting the unlabelled nanoparticles, many studies add an easy-to-detect label on the surface of the nanoparticles by modifying the surface of the nanoparticles, and track the distribution and content of the nanoparticles in living cells by detecting the label signals. For example, a fluorescent probe is modified on the surface of the nanoparticles, and the labelled fluorescent probe in living cells is detected by high-resolution fluorescence microscopy imaging, so as to observe the distribution position and content of the nanoparticles in the cells in real time.

[0005] As a kind of high-resolution fluorescence microscope, laser confocal scanning microscope can observe intracellular microstructure and specific biomolecules by detecting the fluorescence probe of labeled cell, and can detect physiological state such as pH, Ca ion and membrane potential at subcellular level, and can carry out quantitative analysis and real-time dynamic imaging, and is the most widely used molecular biology analysis instrument at present. Especially noteworthy is that laser confocal scanning microscope has a pinhole in front of its light source and detector, and only the fluorescence generated by laser on the sample focal plane can be detected through the reflection of dichroic mirror, so that the focal plane image formed has high spatial resolution. Therefore, in the field of nanobiology, many researchers use laser confocal scanning microscope to carry out high-resolution imaging of fluorescent probes labeled on nanoparticles, and combine with cell biomolecule specific fluorescent probes to realize the purpose of accurately positioning and real-time tracking the distribution position and morphology state of nanoparticles in living cells. However, such fluorescent labeling will change the surface properties of nanoparticles and affect the biological effects of nanoparticles, and long-time imaging will have problems such as fluorescence bleaching and phototoxicity. In addition, it is difficult for many nanomaterials to form stable fluorescent labeling modification on the surface, so the fluorescent labeling is easy to fall off from the nanoparticles in the cells, resulting in positive results. Therefore, this method is only suitable for a few nanomaterials.

[0006] According to the literature report, the scattering light of nanoparticles is generally several orders of magnitude higher than the fluorescence of labeled probes, and the scattering light of unlabeled nanoparticles can be collected by laser scanning confocal fluorescence microscope to accurately position the intracellular distribution position of nanoparticles. However, so far, there is no laser scanning confocal microscope that can simultaneously image the scattering light of non-labeled nanoparticles and the fluorescence of labeled biomolecules, and high-spectral imaging of nanoparticle scattering light. This is mainly because, unlike the fluorescence signal of labeled biomolecules, the scattering light of nanoparticles has the same detection wavelength as the incident laser, so it cannot pass through the filter of the fluorescence microscope, causing serious interference to single-particle scattering detection. SUMMARY

[0007] Therefore, one of the main purposes of the present application is to provide a device and method for confocal imaging of nanoparticle scattering light, in order to at least partially solve at least one of the above technical problems.

[0008] In order to achieve the above-mentioned purpose, as one aspect of the present application, a device for confocal imaging of nanoparticle scattering light is provided, comprising a laser unit, a first pinhole, a beam splitter turntable, a scanning focusing unit, a motorized stage, a first detection imaging unit, a second pinhole, a light splitting unit and a second detection imaging unit; wherein,

[0009] The laser unit emits laser light, which 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;

[0010] Part of the laser light transmitted from the sample enters the first detection imaging unit for imaging;

[0011] The mixed light emitted from the sample returns to the scanning focusing unit, and then reaches the beam splitter turntable, and the mixed light transmitted through the beam splitter turntable enters the light splitting unit for light splitting after passing through the second pinhole, and then enters the second detection imaging unit for detection imaging.

[0012] As another aspect of the present application, a method for synchronously imaging the scattering light of unmarked nanoparticles and the fluorescence of labeled biomolecules is also provided, which uses the device as described above, comprising:

[0013] Placing a sample containing unmarked nanoparticles and fluorescently labeled biomolecules on the motorized stage;

[0014] Selecting the excitation unit and the beam splitter turntable to make the scattering light signal emitted by the unmarked nanoparticles and the fluorescence signal emitted by the labeled biomolecules in the sample be detected and imaged by the second detection imaging unit at the same time, respectively.

[0015] As still another aspect of the present application, a method for hyperspectral imaging of nanoparticle scattering light is also provided, which uses the device as described above, comprising:

[0016] Placing a sample containing unmarked nanoparticles on the motorized stage;

[0017] Selecting the supercontinuum laser of the excitation unit and the flat beam splitter of the beam splitter turntable to make the scattering light signal emitted by the unmarked nanoparticles in the sample be detected and imaged by the second detection imaging unit.

[0018] Based on the above technical solutions, the device and method for confocal imaging of nanoparticle scattering light of the present application have at least one or part of the following advantages over the prior art:

[0019] (1) The nanoparticles do not need to be labeled, and unmarked nanoparticles and fluorescently labeled biomolecules can be synchronously collected;

[0020] (2) In-situ imaging can be performed, and the distribution position and content of nanoparticles in living cells can be directly observed;

[0021] (3) The imaging resolution is high, and both single nanoparticle imaging and fluorescence imaging at the biomolecule level can be performed;

[0022] (4) The operation steps are simple and convenient, and after the cells are exposed to nanoparticles, observation can be directly performed;

[0023] (5) Long time dynamic imaging, no light bleaching and phototoxicity of non-labeled nanoparticles, which can track the intracellular distribution and morphology for a long time;

[0024] (6) Low background, scanning confocal imaging mode of non-labeled nanoparticle scattering light signal, which can focus the signal collected by photomultiplier tube (PMT) on the nanoparticles;

[0025] (7) 3D scanning imaging of non-labeled nanoparticles and fluorescently labeled biomolecules can be performed simultaneously;

[0026] (8) Nanoparticle scattering light hyperspectral imaging can directly analyze the differences in nanoparticle type, size and surface modification in situ, and perform qualitative analysis of nanoparticles. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 1 is a schematic diagram of the device structure for nanoparticle scattering light confocal imaging in the embodiment of the present application;

[0028] Figure 2 Figure 2 is a schematic diagram of the top view of the beam splitter turntable in the embodiment of the present application;

[0029] Figure 3 Figure 3 is a scattering light and fluorescence synchronous imaging diagram of step (5) in the embodiment 1 of the present application.

[0030] In the above figure, the meanings of the reference signs are as follows:

[0031] 100 - laser unit, 101 - 405 nm monochromatic laser, 102 - 488 nm monochromatic laser, 103 - 543 nm monochromatic laser, 104 - supercontinuum laser, 200 - first pinhole, 201 - second pinhole; 300 - first lens; 301 - second lens; 400 - beam splitter turntable; 401 - first dichroic mirror; 402 - second dichroic mirror; 403 - third dichroic mirror; 404 - first flat beam splitter; 405 - second flat beam splitter; 500 - focusing scanning unit; 501 - x-y axis scanning galvanometer; 502 - objective lens; 600 - stage; 701 - optical fiber; 702 - beam splitter prism; 703 - grating; 800 - first detector; 801 - second detector. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below with reference to specific embodiments and the accompanying drawings.

[0033] At present, the imaging of nanoparticles on the laser confocal microscope is basically through the method of fluorescent probe modification, which not only changes the original surface modification of nanoparticles, produces phototoxicity and false positive results, but also is difficult to be synchronized with other wavelengths of fluorescent probes. These shortcomings limit the application of laser confocal microscope in dynamic tracing of nanoparticles in living cells, and a non-labeled imaging method is urgently needed. Most nanoparticles have good light scattering, so dark field microscopy is often used to collect scattered light to achieve in-situ imaging of non-labeled nanoparticles in the sample. However, dark field microscopy is basically a wide open microscope and is difficult to synchronize fluorescence imaging of the sample, and cannot realize the precise positioning of nanoparticles in space and the synchronous imaging of fluorescently labeled biomolecules. The present application is to use laser to confocally image the scattered light of nanoparticles, which can precisely position and high-resolution image non-labeled nanoparticles in space. The present application is a device and method for confocal imaging of nanoparticles scattered light generated by laser, which selects monochromatic laser according to the excitation wavelength of fluorescent probe and related long-wave pass dichroic mirror, and the narrow transition interval of its "cuton (between the cut-off wavelength and the starting wavelength)" can semi-reflect the wavelength laser, then the scattered light of nanoparticles and the fluorescence of labeled biomolecules reflected by the sample are synchronously collected by different PMT to realize synchronous confocal imaging of nanoparticles and biomolecules. At the same time, this technology can also realize confocal hyperspectral analysis of nanoparticle scattering light by using supercontinuum laser and flat beam splitter according to the difference of scattering efficiency of nanoparticles to different wavelength laser. The biomolecules can be DNA, proteins and other biological signals labeled by fluorescent probes. Compared with the prior art, the present application fills the gap in synchronous confocal imaging of non-labeled nanoparticles and fluorescently labeled biomolecules in the sample and confocal hyperspectral imaging analysis of nanoparticle scattering light, and can realize synchronous imaging of non-labeled nanoparticle scattering light and fluorescently labeled biomolecules and hyperspectral imaging of nanoparticle scattering light, with the characteristics of in-situ real-time imaging, non-labeled nanoparticle imaging, high-resolution 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 imaging and scattering light hyperspectral analysis.

[0034] The present application discloses a kind of nanoparticle scattering light confocal imaging device, including laser unit, first pinhole, beam splitter carousel, scanning focusing unit, motorized stage, first detection imaging unit, second pinhole, light splitting unit and second detection imaging unit;Wherein,

[0035] The laser emitted by laser unit reaches beam splitter carousel through first pinhole, and the light reflected by beam splitter carousel is irradiated on sample by focusing scanning unit;

[0036] Part of the laser light transmitted from the sample enters a first detection imaging unit for imaging;

[0037] The mixed light emitted from the sample returns to the beam splitter carousel via the scanning focusing unit, and the mixed light transmitted through the beam splitter carousel enters the light splitting unit after passing through a second pinhole, and then enters the second detection imaging unit for detection imaging.

[0038] In some embodiments of the present application, the laser unit comprises a tunable laser unit composed of a plurality of monochromatic lasers and a supercontinuum laser;

[0039] In some embodiments of the present application, the beam splitter carousel is configured according to the laser wavelengths of different lasers in the laser system, and comprises a tunable beam splitter carousel composed of a plurality of dichroic mirrors and a plurality of flat plate beam splitters, wherein the wavelength of the monochromatic laser is located in the narrow transition interval between the cut-off wavelength and the starting wavelength of the dichroic mirror.

[0040] In some embodiments of the present application, when the laser unit selects a monochromatic laser, the beam splitter carousel selects a dichroic mirror; the dichroic mirror can transmit 40-60% of the nanoparticle scattering light and ≥90% of the biomolecule fluorescence emitted from the sample;

[0041] In some embodiments of the present application, when the laser unit selects a supercontinuum laser, the beam splitter carousel selects a flat plate beam splitter; the flat plate beam splitter can transmit 50-70% of the nanoparticle scattering light emitted from the sample.

[0042] In some embodiments of the present application, the mixed light comprises nanoparticle scattering light and probe-labeled fluorescence emitted from the sample; the mixed light is split by the light splitting unit to form a monochromatic spectrum, and two detectors in the second detection imaging unit are used to simultaneously detect the light signals in the scattering light band and the light signals in the fluorescence band, respectively, to realize synchronous imaging of scattering light and fluorescence.

[0043] In some embodiments of the present application, the included angle between the laser passing through the first pinhole and the beam splitter carousel is 45 degrees.

[0044] In some embodiments of the present application, the scanning focusing unit comprises a scanning galvanometer and an objective lens;

[0045] In some embodiments of the present application, the scanning galvanometer comprises an x-y axis scanning galvanometer, and the scanning frequency range is 200-8000 Hz.

[0046] In some embodiments of the present application, the nanoparticles are not imaged and the detection is the scattering light signal generated by the nanoparticles on the laser;

[0047] In some embodiments of the present application, the light splitting unit comprises a light splitting prism and a grating.

[0048] In some embodiments of the present application, the first detection imaging unit comprises a photomultiplier tube.

[0049] In some embodiments of the present application, the second detection imaging unit comprises a photomultiplier tube and a spectral charge-coupled device.

[0050] The present application also discloses a method for synchronously imaging non-labeled nanoparticle scattering light and labeled biomolecule fluorescence, which uses the device as described above, comprising:

[0051] Placing a sample containing non-labeled nanoparticles and fluorescently labeled biomolecules on the motorized stage;

[0052] Selecting the excitation unit and the beam splitter carousel to enable the scattering light signal emitted by the non-labeled nanoparticles and the fluorescence signal emitted by the labeled biomolecules in the sample to be simultaneously imaged by the second detection imaging unit, respectively.

[0053] The present application also discloses a method for hyperspectral imaging of nanoparticle scattering light, which uses the device as described above, comprising:

[0054] Placing a sample containing non-labeled nanoparticles on the motorized stage;

[0055] Selecting the supercontinuum laser of the excitation unit and the flat beam splitter of the beam splitter carousel to enable the scattering light signal emitted by the non-labeled nanoparticles in the sample to be imaged by the second detection imaging unit.

[0056] In a preferred embodiment, as shown in Figures 1-2 A new device for confocal imaging of nanoparticle scattering light according to the present application comprises a laser unit 100, a first pinhole 200, a first lens 300, a beam splitter carousel 400, a scanning unit 500, a motorized stage 600, a second pinhole 201, a second lens 301, an optical fiber 701, a spectrometer dispersing prism 702, and a photomultiplier tube (PMT) for detecting light signals. The laser emitted by the laser unit 100 is incident on the beam splitter carousel 400 at an angle of 45° after passing through the first pinhole 200 and the first lens 300, is reflected by the dichroic mirror or the flat beam splitter on the beam splitter carousel 400 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. The nanoparticle scattering light and the probe-labeled fluorescence emitted from the sample return to the dichroic mirror or the flat beam splitter by the same path, pass through the second pinhole 201 and the second lens 301, enter the spectrometer dispersing prism 702 through the optical fiber 701, pass through the grating 703, and finally reach the second detector 801, thereby achieving confocal imaging of non-labeled nanoparticle scattering light. The scattering light and the fluorescence separated by the prism enter different PMTs, and the light transmitted from the sample enters the PMT of the first detector 800.

[0057] Wherein, the laser unit 100 has multiple monochromatic lasers, which can emit laser beams of different wavelengths, and each wavelength of monochromatic laser has a corresponding laser semi-reflective long-pass dichroic mirror. The wavelength is in the narrow "cut on" wavelength interval of the long-pass dichroic mirror. The monochromatic laser is incident on the long-pass dichroic mirror on the beam splitter carousel 400 at a 45° angle of incidence through the first pinhole 200. About 50% of the laser is reflected onto the sample, and the remaining 50% of the laser transmits through the dichroic mirror.

[0058] Wherein, the laser unit 100 has multiple monochromatic lasers, which can emit laser beams of different wavelengths, and each wavelength of monochromatic laser has a corresponding laser semi-reflective long-pass dichroic mirror. The wavelength is in the narrow "cut on" wavelength interval of the long-pass dichroic mirror. The monochromatic laser is incident on the long-pass dichroic mirror on the beam splitter carousel 400 at a 45° angle of incidence through the first pinhole 200. About 50% of the laser is reflected onto the sample, and the remaining 50% of the laser transmits through the dichroic mirror.

[0059] Wherein, when the laser uses a monochromatic laser, and the long-pass dichroic mirror on the beam splitter carousel is selected to be close to the "cut on" interval of the laser wavelength, so that 40-60%, preferably 50%, of the nanoparticle scattering light and more than 90% of the biomolecule fluorescence emitted by the sample transmits through the dichroic mirror, realizing the synchronous imaging of the non-labeled nanoparticle scattering light and the probe-labeled biomolecule fluorescence. When the laser uses an ultrashort supercontinuum laser 104, and a flat beam splitter is selected on the beam splitter carousel, so that 50-70% of the scattering light spectrum emitted by the sample transmits through the beam splitter, realizing the high-spectral imaging of the nanoparticle scattering light. The laser unit 100 in this embodiment has multiple monochromatic lasers and an ultrashort supercontinuum laser. The monochromatic lasers mainly include: a 405 nm monochromatic laser 101, a 488 nm monochromatic laser 102, and a 543 nm monochromatic laser 103, and an ultrashort supercontinuum laser 104. The first three monochromatic lasers are mainly used for synchronous imaging of non-labeled nanoparticle scattering light and probe-labeled biomolecules, and the ultrashort supercontinuum laser is used for high-spectral imaging of nanoparticle scattering light.

[0060] Wherein, the nanoparticle scattering light and the fluorescence of the probe-labeled biomolecules etc. generated by the monochromatic laser irradiating the sample return through the original light path, and then are incident on the long-pass dichroic mirror at a 45° angle of incidence. About 50% of the scattering light and more than 90% of the fluorescence transmits through the dichroic mirror, and reaches the beam splitter prism 702 through the second pinhole 201. The light signals in the scattering light and fluorescence bands are detected by different PMTs at the same time, realizing the synchronous imaging of the nanoparticle scattering light and the fluorescence of the probe-labeled biomolecules etc.

[0061] The spectrum of the supercontinuum laser 104 is continuous laser spectrum covering 320-2400 nm. The light emitted by the supercontinuum laser 104 is incident on the plate beam splitter at 45° through the first pinhole 200, and the plate beam splitter reflects the laser at 50% or 30% (reflection:transmission ratio = 50:50 or 30:70) in the 380-1100 nm spectral range to the sample. The nanoparticle scattering light generated by the continuous spectrum laser irradiating the sample returns through the original light path and reaches the plate beam splitter at an angle of 45°, wherein the scattering light in the 380-1100 nm range passes through the plate beam splitter at a transmittance of about 50% or 70%, reaches the spectrometer prism 702 through the second pinhole 201, and the light signals in the scattering light and fluorescence wavelength bands are detected by different PMTs at the same time, realizing high-spectral imaging of nanoparticle scattering light.

[0062] The beam splitter carousel 400 has three long-wave pass dichroic mirrors (i.e., the first dichroic mirror 401, the second dichroic mirror 402, and the third dichroic mirror 403) with different “cut on” wavelengths and two plate beam splitters (i.e., the first plate beam splitter 404 and the second plate beam splitter 405). The three long-wave pass dichroic mirrors mainly function to respectively semi-reflect 405, 488, and 543 nm laser light to the sample and semi-transmit nanoparticle scattering light and fully transmit fluorescence. The two plate beam splitters respectively function to 50% reflect laser light and 50% transmit nanoparticle scattering light, and 30% reflect laser light and 70% transmit nanoparticle scattering light.

[0063] The scanning focusing unit 500 includes an x-y axis scanning galvanometer 501 and a condenser objective 502. The frequency of the x-y axis scanning galvanometer 501 is adjustable, and the adjustable range is 200-8000 Hz.

[0064] The scanning focusing unit 500 can focus laser light in a scanning manner on the sample focal plane, irradiate the unmarked nanoparticles and fluorescently labeled biomolecules in the focal plane, and the generated scattering light and fluorescence can reach the detector through the second pinhole 201, which can not only exclude the sample background scattering light signal not in the longitudinal focal plane and the transverse observation area, but also not reduce the unmarked nanoparticle scattering light signal in the focal point.

[0065] The long-wave pass dichroic mirror of the beam splitter carousel 400 can fully transmit the fluorescence signal greater than the laser wavelength generated by the labeled biomolecules in the sample, and semi-transmit the scattering light signal equal to the laser wavelength generated by the unmarked nanoparticles in the sample, so that both wavelengths of signals can pass through the dichroic mirror.

[0066] The signal of the detected nanoparticle is scattering light, and there is no need to image and label the nanoparticle. The laser can make the nanoparticle in the sample generate a scattering light signal.

[0067] The laser unit 100 can produce laser according to the excitation wavelength of the fluorescent probe of the labeled biomolecule, which can make the non-labeled nanoparticles emit scattered light and excite the fluorescence of the labeled biomolecule, so that the scattered light spectrum of the nanoparticles and the fluorescence spectrum of the labeled biomolecule in the cell do not overlap.

[0068] The laser can make the nanoparticles scattered light and the fluorescence of the labeled biomolecule in the sample, and the composite light beams of the two light signals return to the second pinhole 201 through the dichroic mirror, and then are decomposed into dispersed spectra by the light splitting prism 702, and the scattered light spectrum signal and the fluorescence spectrum signal are respectively received by different PMTs. At the same time, the laser signal transmitted through the sample is also received by the PMT, so as to realize synchronous imaging of the non-labeled nanoparticles and the fluorescently labeled biomolecule.

[0069] The laser generator can produce monochromatic laser, and the scattered light of the non-labeled nanoparticles in the sample irradiated by the laser also has monochromaticity, and the wavelengths are the same. The PMT collects the non-labeled nanoparticle scattered light signal in the range of ±5 nm of the laser wavelength.

[0070] The laser semi-reflective long-wave pass dichroic mirror has a narrow "cut on" wavelength interval, which can transmit 50% of the non-labeled nanoparticle scattered light, so that the PMT detects and collects the non-labeled nanoparticle light signal which is scattered light.

[0071] The laser passes through the pinhole and is focused on the cell sample. The non-labeled nanoparticle scattered light and the fluorescence of the labeled biomolecule emitted by the sample return to the PMT through the second pinhole 201. The scattered light signal of the nanoparticle and the fluorescence signal of the labeled biomolecule have co-focusing property, and the nanoparticle can be imaged as a single particle.

[0072] The scattered light of the non-labeled nanoparticle and the fluorescence of the probe-labeled biomolecule have co-focusing property, so that the scattered light and the fluorescence can be imaged synchronously not only in the XY plane but also in the Z axis direction, and three-dimensional imaging can be realized.

[0073] The embodiment also discloses a method for synchronously imaging the non-labeled nanoparticle scattered light and the labeled biomolecule fluorescence in a sample by using the device.

[0074] (1) After the biomolecule of interest in the cell is labeled with fluorescent protein by genetic engineering technology (such as plasmid transfection), expose the unmarked nanoparticles, incubate in the incubator for a certain period of time, obtain the live cell sample of the nanoparticles and the fluorescently labeled biomolecule, and place the sample on the sample stage; or expose the unmarked nanoparticles to the cell, incubate in the incubator for a certain period of time, then label the cell biomolecule with a specific fluorescent probe, obtain the live cell sample of the nanoparticles and the fluorescently labeled biomolecule, and place the sample on the sample stage. Or fix the fluorescently modified nanoparticles in the gel to obtain a gel sample embedding the nanoparticles, and place the sample on the motorized stage.

[0075] (2) According to the excitation wavelength of the fluorescent probe or fluorescent protein of the labeled biomolecule, select the most suitable laser and the half-reflective dichroic mirror of the laser to reflect the laser to the scanning focusing unit 500.

[0076] (3) Adjust the scanning speed and focal length of the scanning focusing unit 500 to find the best scanning speed and the most suitable focal length to focus the laser on the observation area of interest in the sample.

[0077] (4) Select a microscope objective with an appropriate magnification, and adjust the energy of the laser to make the scattering light signal emitted by the unmarked nanoparticles and the fluorescent signal emitted by the labeled biomolecule in the sample strong enough.

[0078] (5) After setting the time and number of scans, if three-dimensional stereoscopic imaging is performed, the Z-axis imaging height and step distance also need to be set. Then turn on the three PMTs at the same time, set the range of the unmarked nanoparticle scattering light collected by PMT1 to be ±5 nm of the laser wavelength, the range of the light spectrum collected by PMT2 to be the fluorescent light emitted by the labeled fluorescent probe, and the laser signal transmitted through the sample to be collected by PMT3, so as to respectively and synchronously image the unmarked nanoparticles, the fluorescently labeled biomolecule, and the cell morphology. Among them, the cell morphology imaging is realized in the first detector 800.

[0079] (6) Analyze the intensity and position of the scattering light signal and the fluorescent signal in step (5) to obtain the relative content and distribution position of the unmarked nanoparticles and the labeled biomolecule in the live cell, and analyze the spatial relationship between the two.

[0080] Among them, the nanoparticles do not need to be labeled, and the distribution position and relative content of the unmarked nanoparticles and the fluorescently labeled biomolecule in the sample can be observed in situ and synchronously, the nanoparticle morphology change can be observed at the single particle level, and the motion trajectory of the unmarked nanoparticles in the live cell can be dynamically observed for a long time.

[0081] The embodiment also discloses a method for high-spectral imaging of nanoparticle scattering light in a sample by using the device, and the method comprises the following steps:

[0082] (1) Put the sample of cells with ingested nanoparticles or gel with embedded nanoparticles on the motorized stage, and find the nanoparticles in the sample.

[0083] (2) Turn on the supercontinuum laser, select the flat beam splitter, reflect the continuous spectrum laser to the sample, and set the spectral detection range in the 380-1100 nm interval.

[0084] (3) According to the intensity of the scattered light spectrum signal, adjust 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.

[0085] (4) After adjusting the imaging parameters, set the imaging resolution, magnification, scanning times, and spectral wavelength accuracy (generally 2 nm) according to the characteristics of the sample. If three-dimensional imaging is performed, the Z-axis imaging height and step distance also need to be set.

[0086] (5) After setting the collection parameters, perform the nanoparticle scattered light hyperspectral imaging.

[0087] (6) Analyze the nanoparticle scattered light hyperspectral signal collected in step (5) to obtain the characteristic scattered light spectrum of the nanoparticles.

[0088] The nanoparticle does not need to be labeled, and the scattered light of the nanoparticle in the sample can be imaged in situ. The nanoparticle morphology, surface modification, and species can be analyzed and compared at the single particle level.

[0089] The technical solutions of the present application will be further described and illustrated by specific examples in conjunction with the accompanying drawings. It should be noted that the following specific examples are only illustrative, and the scope of protection of the present application is not limited thereto.

[0090] The chemicals and raw materials used in the following examples are commercially available or self-made by known preparation methods.

[0091] Example 1

[0092] The structure of the in vivo label-free nanoparticle scattered light and labeled biomolecule fluorescence synchronous imaging device of the present embodiment is as follows: Figures 1-2As shown, including laser unit 100, laser half-reflective long-wave pass dichroic mirror, scanning focusing unit 500, sample stage 600 which can be mounted with live cell culture system, light splitting prism 702 and photomultiplier tube (i.e. second detector 801) for detecting optical signals. The laser emitted by the laser unit is half-reflected by the long-wave pass dichroic mirror to the scanning focusing unit 500, focused on the cell sample, and the scattered light and fluorescence of the labeled biomolecules reflected from the sample focal plane return to the dichroic mirror and then pass through the dichroic mirror. The scattered light and fluorescence separated by the light splitting prism 702 enter different avalanche diodes.

[0093] The device focuses laser on the cell sample focal plane and scans the cell in x-y direction by galvanometer, detects the local nanoparticle scattering light of the cell on the focal plane, does not reduce the scattering light of the nanoparticle, reduces the interference of the scattering light from the cell sample in the longitudinal and transverse directions, and combines PMT and computer to reduce noise of the signal, thereby improving sensitivity and signal-to-noise ratio.

[0094] The above device is used for synchronous imaging of the label-free nanoparticle scattering light and the labeled nuclear fluorescence in live cells, including the following steps:

[0095] (1) Select a glass-bottom confocal dish, transfer the macrophages grown in the logarithmic phase to the confocal dish, and place it in the incubator overnight until the cells adhere. Replace the old culture medium in the confocal dish with fresh culture medium containing 2 μg / mL of 100 nm AgNPs (silver nanoparticles), and place it back in the incubator. Add a confocal dish containing fresh culture medium without AgNPs as a blank control.

[0096] (2) After 2 hours of culture, remove the confocal dish, remove the culture medium, and wash the cells with PBS (phosphate buffer). Remove the AgNPs that have not entered the cells, then add fresh culture medium containing 10 μM SYTO Green, a nucleic acid-specific fluorescent probe, to the confocal dish, and place it back in the incubator for 10 minutes to allow SYTO Green to enter the cells and label the nuclei.

[0097] (3) After the nuclei are labeled, remove the confocal dish, remove the culture medium containing the SYTO Green fluorescent probe, wash the cells with PBS to remove excess probe, add fresh culture medium to the confocal dish, and place the sample on the device sample stage.

[0098] (4) Adjust the laser generator according to the optimal excitation light of the SYTO Green probe, select a 488 nm wavelength laser, and select a long-pass dichroic mirror that partially reflects the 488 nm laser. At this time, the 488 nm laser is in the narrow "cut on" transition region of the dichroic mirror. Then, turn on PMT1, PMT2 and PMT3 at the same time, set the receiving spectral range of PMT1 to 483-493 nm, the receiving spectral range of PMT2 to 510-560 nm, and PMT3 to receive the transmitted light of the sample. This allows for the simultaneous detection of the scattered light of AgNPs, the fluorescence of the labeled cell nuclear probe SYTO Green and the cell morphology, and the synchronous imaging of unlabeled AgNPs, fluorescently labeled cell nuclei and cell morphology.

[0099] (5) Adjust the focusing plane to focus on the unlabeled AgNPs and labeled cell nuclei in the cell, set the scanning speed and number of scans, and start using PMT1 and PMT2 to simultaneously collect the scattered light of the unlabeled AgNPs and the fluorescence signal of the labeled cell nuclei, and use PMT3 to capture the cell morphology.

[0100] (6) Analyze the intensity and location of the unlabeled AgNPs scattered light signal and the labeled cell nuclear fluorescence signal in step (5), and perform co-localization analysis on the spatial relationship between the two to accurately locate the intracellular distribution of AgNPs at the single particle level.

[0101] in, Figure 3 The image obtained in step 5, from Figure 3 As can be seen from the images, unlabeled AgNPs and fluorescently labeled cell nuclei, as well as the cell morphology of live cells, can be observed simultaneously inside exposed cells with AgNPs. In contrast, only fluorescently labeled cell nuclei can be observed in the blank control group, with no AgNPs present. This proves that the detected scattered light signal does indeed originate from unlabeled AgNPs. From the superimposed images, colocalization analysis of the light signal shows that AgNPs are located outside the cell, indicating that the device and method of this invention can accurately locate the distribution of AgNPs within cells at the single-particle level.

[0102] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A device for nanoparticle scattering light confocal imaging, comprising a laser unit, a first pinhole, a beam splitter carousel, a scanning focusing unit, a motorized stage, a first detection imaging unit, a second pinhole, a light splitting unit and a second detection imaging unit; wherein, the laser unit emits laser light, which passes through the first pinhole to the beam splitter carousel, and the light reflected by the beam splitter carousel passes through the scanning focusing unit to irradiate on the sample; part of the transmitted light from the sample enters the first detection imaging unit for imaging to image the cell morphology; the mixed light emitted from the sample returns to the beam splitter carousel through the scanning focusing unit, and the mixed light passing through the beam splitter carousel enters the light splitting unit after passing through the second pinhole, and then enters the second detection imaging unit for detection imaging; wherein the laser unit comprises an adjustable laser unit composed of a plurality of monochromatic lasers and a supercontinuum laser; the beam splitter carousel is configured according to the laser wavelengths of different lasers in the laser system, and comprises an adjustable beam splitter carousel composed of a plurality of dichroic mirrors and a plurality of flat plate beam splitters, wherein the wavelength of the monochromatic laser is located in the narrow transition interval between the cut-off wavelength and the starting wavelength of the dichroic mirror; wherein when the laser unit selects a monochromatic laser, the beam splitter carousel selects a dichroic mirror; the dichroic mirror can transmit 40 to 60% of the nanoparticle scattering light and ≥90% of the biomolecule fluorescence emitted by the sample; wherein when the laser unit selects a supercontinuum laser, the beam splitter carousel selects a flat plate beam splitter; the flat plate beam splitter can transmit 50 to 70% of the nanoparticle scattering light emitted by the sample.

2. The device according to claim 1, wherein, the mixed light comprises nanoparticle scattering light and probe-labeled fluorescence emitted from the sample; the mixed light is split by the light splitting unit to form a monochromatic spectrum, and two detectors in the second detection imaging unit are used to detect the light signals in the scattering light band and the light signals in the fluorescence band respectively and simultaneously, so as to realize synchronous imaging of scattering light and fluorescence.

3. The device according to claim 1, wherein, the included angle between the laser passing through the first pinhole and the beam splitter carousel is 45 degrees.

4. The device according to claim 1, wherein, the scanning focusing unit comprises a scanning galvanometer and an objective lens; wherein the scanning galvanometer comprises an x-y axis scanning galvanometer, and the scanning frequency range is 200 to 8000 Hz.

5. The device according to claim 1, wherein, the nanoparticles are not imaged with a marker, and the detected is the scattering light signal generated by the nanoparticles on the laser; the light splitting unit comprises a light splitting prism and a grating.

6. The device according to claim 1, wherein, the first detection imaging unit comprises a photomultiplier tube; the second detection imaging unit comprises a photomultiplier tube and a spectral charge-coupled device.

7. A marker-free nanoparticle scattering light and labeled biomolecule fluorescence synchronous imaging method, using the device according to any one of claims 1 to 6, comprising: placing a sample containing marker-free nanoparticles and fluorescently labeled biomolecules on the motorized stage; The excitation unit and the beam splitter wheel are selected to make the scattering light signal emitted by the unlabelled nanoparticles and the fluorescence signal emitted by the labelled biomolecules in the sample be imaged by the second detection imaging unit at the same time, respectively.

8. A method of nanoparticle scattering light hyperspectral imaging, using the device of any one of claims 1 to 6, comprising: placing a sample containing unlabelled nanoparticles on the motorized stage; selecting the supercontinuum laser of the excitation unit and the flat beam splitter of the beam splitter wheel to make the scattering light signal emitted by the unlabelled nanoparticles in the sample be imaged by the second detection imaging unit.

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