KR20230099463A - Broadband label-free molecular imaging system - Google Patents

Abstract

The present invention relates to a broadband label-free molecular imaging system. The broadband label-free molecular imaging system according to an embodiment of the present invention includes a light source 10 for irradiating a laser light, and a pump light (PB) using a laser light irradiated from the light source 10. ) and the Stokes light (SB), a photonic crystal fiber (PCF, 30) extending the wavelength range of the Stokes light (SB) split in the light splitter 20, a predetermined wavelength A Stokes light splitting dichroic mirror 40 that splits the Stokes light SB passing through the photonic crystal fiber 30 into a first Stokes light SB1 and a second Stokes light SB2 according to the range, a pump light ( PB), the first Stokes light SB1, and the second Stokes light SB2 are temporally and spatially matched to generate the combined light IB, the optical coupling unit 50, CARS (Coherent anti-Stokes Raman Scattering) ), a scanning unit 60 that scans the sample with the combined light IB generated by the light coupling unit 50, and a detection unit 70 that detects a CARS signal so that a signal is generated.

Description

Broadband label-free molecular imaging system {BROADBAND LABEL-FREE MOLECULAR IMAGING SYSTEM}

The present invention relates to a broadband label-free molecular imaging system, and more particularly, to an imaging system technology capable of simultaneously acquiring two or more images in real time using two or more Raman modes based on molecular vibrational energy.

Microplastic (MP) particles are very small plastic fragments found almost everywhere on Earth. Microplastic particles ingested by microbes accumulate in the body of macroorganisms or humans through the food chain and seriously affect metabolism and reproduction, so they are considered an important ecological threat. Although various optical microscopes have been developed to identify the size and chemical composition of microplastics in environmental samples, it is difficult to directly visualize and distinguish intracellular microplastic particles because organelles of similar size and shape exist. In addition, a method of labeling and observing fluorescence on microplastic particles has been developed, but in the case of fluorescence imaging method, the fluorescence material is made of a polymer and when exposed to light for a long time, the fluorescence gradually weakens and cannot be imaged. It has unstable photo-physical properties, It is difficult to apply to biological samples because the toxicity of the fluorescence itself can cause biological damage.

Accordingly, label-free vibrational imaging technology using vibrational energy of molecules without the need for labeling is receiving great attention in the field of studying the toxicity and environment of microplastics. This label-free vibrational imaging technology enables non-destructive, non-invasive and direct chemical imaging of contaminants in biological samples. Other optical microscopes, such as the phase contrast microscope or differential interference contrast microscope, are widely used to observe biological samples that do not contain microplastic particles, but their microscopic images do not provide chemical specific information. can not do it. For example, these light microscopes cannot distinguish intracellular microplastics and lipid or protein droplets of the same size. In contrast, vibration imaging technology can identify microparticles and microplastics having different chemical compositions and structures within cells without prior labeling of microplastics.

Micro-Fourier-transform IR (FT-IR) imaging, focal array FT-IR hyperspectral imaging (HSI), near IR (NIR) HSI An imaging method using an infrared (IR) microscope such as a lamp is one of the widely used vibration imaging methods. However, such an infrared absorption or reflection-based vibration imaging method is not suitable for quantitative analysis of microplastic particles smaller than 10 μm due to limitations in basic resolution due to mid-IR wave diffraction. In addition, since water, a physiological solvent of cells and organisms, absorbs light in a wide range of infrared frequencies, its application to biological samples is further limited. Unlike infrared microscopy, Raman microscopy uses a visible laser beam that is not absorbed by water molecules, so it has improved spatial resolution and can be used to obtain images of microplastic particles. Nevertheless, spontaneous Raman scattering microscopy has limited use in biological applications due to light damage and low detection sensitivity due to a small scattering cross section.

Therefore, as disclosed in the non-patent literature of the prior art document below, a coherent anti-Stokes Raman scattering (CARS) microscope using the interaction between an ultrashort laser pulse and a multiphoton light material is used to analyze the biological properties of microplastics. It can be selected as a method for imaging. The CARS microscope has advantages such as excellent spatial resolution, high sensitivity, and fluorescence-free labeling, so it is widely used in various research fields ranging from biomedicine to environmental science. Recently, microplastic particles ingested by marine organisms such as crabs and zooplankton have been detected using a CARS microscope to reveal the distribution and accumulation of microplastic particles in living organisms. However, because monochromatic CARS microscopy provides one automatic imaging map for selected particles, it cannot simultaneously identify microplastic particles that are chemically different from each other or distinguish specific microplastic particles from other organelles. To overcome these limitations, a broadband multi-CARS microscope based on a hyperspectral imaging (HSI) approach has been developed to help characterize different chemical species simultaneously. However, although the broadband multi-CARS microscope can obtain hyperspectral images of chemically different microplastic particles from the entire vibration spectrum recorded at each image pixel, the imaging speed is slow. Ultimately, the dynamic behavior of microplastics and their attached organelles in vivo, such as cellular internalization, intracellular transport, and penetration of microplastic particles into tissues, is very important for understanding the biological responses and toxic effects of microplastic particles. Hyperspectral image-based methods for research have limitations.

On the other hand, in order to observe microplastic particles in a living body in real time, at least two Raman modes capable of distinguishing two types (cell, living body, and microplastic) are required, so a system must be built to measure two images simultaneously. However, since the current technology requires three light sources having different wavelengths, there is a problem in that the system is very complicated and a considerable cost is required to build the system.

Choi, D S; Kim, C H; Lee, T; Nah, S; Rhee, H; Cho, M Vibrational spectroscopy and imaging with non-resonant coherent anti-Stokes Raman scattering: Double stimulated Raman scattering scheme Opt Express 2019, 27, 23558-23575

The present invention is to solve the problems of the prior art described above, and one aspect of the present invention is an ultra-wideband multiplex (ultra-wideband multiplex that can simultaneously measure fingerprint and CH stretch areas by laser scanning using one femtosecond laser and a photonic crystal optical fiber) multiplex) CARS microscope and its microscope system, which provide a broadband label-free molecular imaging system.

In addition, another aspect of the present invention is a broadband label-free molecular imaging system capable of acquiring three images simultaneously using three Raman modes and real-time image acquisition by using two detectors in the front and one detector in the rear. want to provide

In addition, another aspect of the present invention is a broadband unlabeled molecule that can simultaneously acquire fluorescence images by using two detectors using Raman mode in the front and one detector using two-photon fluorescence technology in the rear. To provide an imaging system.

A broadband label-free molecular imaging system according to an embodiment of the present invention includes a light source for irradiating laser light; a beam splitter splitting the laser light irradiated from the light source into a pump light and a Stokes light; a photonic crystal fiber (PCF) extending a wavelength range of the Stokes light split in the optical splitter; a Stokes light splitting dichroic mirror for splitting the Stokes light passing through the photonic crystal fiber into first Stokes light and second Stokes light according to a predetermined wavelength range; an optical coupling unit configured to temporally and spatially match the pump light, the first Stokes light, and the second Stokes light to generate combined light; a scanning unit scanning a sample with the combined light generated by the optical coupling unit to generate a Coherent anti-Stokes Raman Scattering (CARS) signal; and a detector for detecting the CARS signal.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the light coupling unit adjusts the time of the second Stokes light so that the first Stokes light and the second Stokes light are temporally matched. light regulator; a Stokes light coupling dichroic mirror for spatially matching the second Stokes light passing through the Stokes light controller with the first Stokes light; and generating the combined light by spatially matching the pump light with the first and second Stokes light spatially matched through the Stokes light-coupling dichroic mirror, and sending the generated combined light to the scanning unit. It may include; a coupled light generating dichroic mirror that irradiates.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, in the light coupling unit, the first Stokes light and the second Stokes light, which are temporally matched in the Stokes light controller, and the pump light are temporally synchronized. The pump light controller may further include a pump light controller configured to adjust the time of the pump light to match.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the pump light band pass filter for adjusting the wavelength range of the pump light irradiated to the optical coupler; a first Stokes light band pass filter for adjusting a wavelength range of the first Stokes light irradiated to the optical coupler; and a second Stokes light band-pass filter for adjusting a wavelength range of the second Stokes light irradiated to the light coupling unit.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the Stokes light passing through the photonic crystal fiber is incident and a long pass filter for transmitting the Stokes light in a long wavelength region; can include more.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the scanning unit may include: a galvano scanner for adjusting a scan area of the coupled light with respect to the sample; and a rear objective lens for concentrating the combined light passing through the galvano scanner toward the sample from a rear side.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the detection unit may include: a front objective lens for collecting a front CARS signal generated in front by focusing the coupled light on the sample; a signal separation dichroic mirror separating the front CARS signal, a first CARS signal generated by a combination of the pump light and the first Stokes light, and a second CARS signal generated by a combination of the pump light and the second Stokes light; a first photomultiplier tube (PMT) for detecting the first CARS signal separated by the signal separating dichroic mirror; and a second photomultiplier tube for detecting the second CARS signal separated by the signal separating dichroic mirror.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the detection unit further includes a third photomultiplier tube for detecting a backward CARS signal generated from the rear when the coupled light is focused on the sample. can do.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the detection unit may further include a spectrometer for detecting a spectrum of a backward CARS signal generated from the rear when the coupled light is focused on the sample. there is.

In addition, in the broadband label-free molecular imaging system according to an embodiment of the present invention, the sample may include cells or living organisms in which microplastic particles are internalized.

Features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Prior to this, the terms or words used in this specification and claims should not be interpreted in a conventional and dictionary sense, and the inventor may appropriately define the concept of the term in order to explain his or her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

According to the present invention, ultra-wideband CARS signals capable of measuring fingerprint and CH stretch regions can be simultaneously obtained by laser scanning using one femtosecond laser and a photonic crystal fiber.

In addition, two or more images can be simultaneously acquired in real time using two Raman modes at a maximum speed of 1 frame/s through laser scanning.

Furthermore, the present invention is a non-labeling technology, and it is possible to image microplastic particles and organelles in living organisms or cells, respectively, without pretreatment of living organisms or cells.

In addition, the present invention builds a multi-modal microscope system combining two-photon fluorescence technology by arranging a rear detector using two-photon fluorescence technology in addition to a front detector using Raman mode, thereby simultaneously acquiring fluorescence images of biological samples. .

1 is a schematic configuration diagram of a broadband label-free molecular imaging system according to an embodiment of the present invention.
2 to 3 are configuration diagrams of Multi-color CARS microscopes according to experimental examples.
4 (a) to (d) are multi-color CARS-TPE images of lily pollen mixed with 8.7 μm PS beads according to the experimental example, and FIG. 4 (e) to (l) are 2 according to the experimental example. This is a multi-color CARS-TPE image of C. elegans ingesting ㎛ PS beads.
Figure 5 (a) is a wide-illuminated PC image of U2OS cells exposed to a 2 μm PS bead solution according to the experimental example, and Figure 5 (b) is a multi-color CARS of U2OS cells exposed to the PS bead solution. 5 (c) to (f) are particle tracking and migration analysis results of PS beads and organelles (LDs) in U2OS cells.

Objects, specific advantages and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In adding reference numerals to components of each drawing in this specification, it should be noted that the same components have the same numbers as much as possible, even if they are displayed on different drawings. In addition, terms such as “first” and “second” are used to distinguish one component from another component, and the components are not limited by the terms. Hereinafter, in describing the present invention, detailed descriptions of related known technologies that may unnecessarily obscure the subject matter of the present invention will be omitted.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic configuration diagram of a broadband label-free molecular imaging system according to an embodiment of the present invention.

As shown in FIG. 1, the broadband label-free molecular imaging system according to an embodiment of the present invention includes a light source 10 for irradiating laser light, a pump light PB and a Stokes light ( SB), a photonic crystal fiber (PCF, 30) extending the wavelength range of the Stokes light (SB) split in the optical splitter 20, and a photonic crystal according to a predetermined wavelength range. A Stokes light splitting dichroic mirror 40 splitting the Stokes light SB passing through the optical fiber 30 into a first Stokes light SB1 and a second Stokes light SB2, a pump light PB, a first The optical coupling unit 50 that generates the combined light IB by temporally and spatially matching the Stokes light SB1 and the second Stokes light SB2 so that a Coherent anti-Stokes Raman Scattering (CARS) signal is generated. , a scanning unit 60 that scans a sample with the combined light IB generated by the optical coupling unit 50, and a detection unit 70 that detects a CARS signal.

The present invention relates to a high-resolution, label-free imaging system using a laser, which can simultaneously acquire two or more two-dimensional images using two or more Raman modes based on molecular vibrational energy. A broadband multiplex CARS microscope system is provided. In order to obtain an image at the level of a living body or a cell, high-magnification optical imaging is generally used, or a method of labeling and observing fluorescence to observe real-time changes in a specific part or a specific organelle has been widely used until recently. In order to secure a high-resolution fluorescence image, confocal laser scanning fluorescence imaging technology can be used, which is widely used so far because it enables imaging at about 1.4 times higher resolution than general fluorescence imaging. Recently, through fluorescence imaging or laser scanning fluorescence imaging, it has been found that microplastic particles are absorbed or ingested by various types of organisms (plankton, C. elegans, zebrafish, etc.) and accumulated in the organism. However, fluorescence is composed of polymers, and when exposed to light for a long time, the fluorescence gradually weakens, making it impossible to perform imaging, and the toxicity of fluorescence itself can cause damage in vivo. Recently, microscopes that do not use fluorescence technology is being developed. In particular, in the past 20 years, various CARS (Coherent anti-Stokes Raman Scattering) spectroscopy and microscopy techniques using vibrational energy of molecules that do not require labeling have been developed for the study of various polymer materials, new materials, and living bio samples. Since CARS spectroscopy is four-wave mixing spectroscopy, a conventional CARS microscope uses at least two sub-10 ps pulse lasers of different wavelengths. For example, CARS imaging using two picosecond lasers or one picosecond laser and one femtosecond laser enables high-speed imaging, so live cells and tissues (protein, lipid droplets, nucleic acid, endoplasmic reticulum) can be monitored in real time Or you can observe the dynamics. However, since three laser lights are required to obtain two images simultaneously by inducing two Raman modes using a conventional picosecond laser, the system configuration is complicated, and the picosecond laser and the femtosecond laser are very expensive. There is a problem in terms of space, and the present invention has been devised as a solution to this problem.

Specifically, the broadband label-free molecular imaging system according to the present invention includes a light source 10, a beam splitter 20, a photonic crystal fiber (PCF, 30), a Stokes light splitting dichroic mirror, 40, a light It includes a coupling unit 50, a scanning unit 60, and a detection unit 70.

Here, the light source 10 is a laser that irradiates laser light. For example, the light source 10 may be one femtosecond laser.

The beam splitter 20 splits the laser light emitted from the light source 10 into two beams. In order to obtain the CARS signal, the pump light PB and the Stokes light SB are required, and the beam splitter 20 splits the laser light into the pump light PB and the Stokes light SB. On the other hand, a half wave plate (HWP) is disposed on the optical path of the laser light to the optical splitter 20 to adjust the polarization state of the laser light, where the polarized laser light is the polarized light splitter 20. It is incident on a polarizing beam splitter (PBS) and can be split into a pump light (PB) and a Stokes light (SB). In addition, another half wave plate (HWP) is disposed on the optical path of the Stokes light SB, so that the polarization of the Stokes light SB can be adjusted.

The photonic crystal fiber (PCF) 30 expands the wavelength range of the Stokes light (SB) split in the optical splitter (20). The Stokes light (SB) passing through such a photonic crystal fiber 30 has an ultra-wide spectrum and is irradiated to the Stokes light splitting dichroic mirror 40 .

On the other hand, on the optical path of the Stokes light SB between the photonic crystal fiber 30 and the Stokes light splitting dichroic mirror 40 so that the Stokes light SB has a predetermined wavelength range, a long pass filter, 110) may be placed. At this time, the Stokes light SB passing through the photonic crystal fiber 30 is incident on the long wave pass filter 110, and the Stokes light SB in the long wavelength region is transmitted and irradiated to the Stokes light splitting dichroic mirror 40. .

The Stokes light splitting dichroic mirror 40 splits the Stokes light SB passing through the photonic crystal fiber 30 into a first Stokes light SB1 and a second Stokes light SB2 according to a predetermined wavelength range. .

On the other hand, the CARS signal is generated when the pump light PB and the Stokes light SB having two different frequencies are temporally and spatially overlapped at the same time. In the present invention, the pump light PB and the first Stokes light SB1 A CARS signal is generated by the combination and the combination of the pump light PB and the second Stokes light SB2.

The optical coupler 50 generates the combined light IB by temporally and spatially matching the pump light PB, the first Stokes light SB1, and the second Stokes light SB2 so that the CARS signal is generated.

Meanwhile, before the combined light IB is generated for generating the CARS signal, that is, before being irradiated to the light coupling unit 50, the pump light PB passes through a pump light band pass filter 80, The Stokes light SB1 passes through the first Stokes light band pass filter 90, and the second Stokes light SB2 passes through the second Stokes light band pass filter 100, so that the pump light PB, the first Stokes light SB2, A wavelength range of each of the light SB1 and the second Stokes light SB2 may be adjusted. The frequency difference between the pump light (PB) and the first Stokes light (SB1) and the frequency difference between the pump light (PB) and the second Stokes light (SB2) whose wavelength ranges are adjusted in this way match the vibration frequency of the target Raman active mode in the sample. CARS signal is generated when As a result, by independently adjusting the frequencies of the first Stokes light SB1 and the second Stokes light SB2 with respect to the pump light PB having a fixed frequency, two or more vibration modes are used to generate two or more vibrations. images can be obtained at the same time.

In one embodiment, the optical coupling unit 50 may include a Stokes light controller 51, a Stokes light coupling dichroic mirror 52, and a combined light generating dichroic mirror 53 to generate combined light IB. there is.

Here, the Stokes light controller 51 is disposed on the optical path of the second Stokes light SB2, and adjusts the time of the second Stokes light SB2, so that the first Stokes light SB1 and the second Stokes light ( SB2) in time. The Stokes light controller 51 adjusts the optical path of the second Stokes light SB2 by extending or shortening the optical path of the second Stokes light SB2 to shorten the time of the second Stokes light SB2. can be changed

The second Stokes light SB2 and the first Stokes light SB1 passing through the Stokes light regulator 51 are incident on the Stokes light coupling dichroic mirror 52, respectively, and are coaxially overlapped, that is, spatially matched, to generate combined light. It is irradiated with a dichroic mirror (53). Meanwhile, as the pump light PB is also irradiated with the coupled light generating dichroic mirror 53, the pump light PB, the first Stokes light SB1, and the second Stokes light SB2 are spatially matched to form the combined light IB. generate The combined light IB generated through the combined light generating dichroic mirror 53 is irradiated to the scanning unit 60 . Meanwhile, the pump light PB irradiated toward the coupled light generating dichroic mirror 53 may pass through the pump light controller 54 . The pump light controller 54 is disposed on the optical path of the pump light PB passing through the coupled light generating dichroic mirror 53 from the light splitter 20, and delays the time of the pump light PB, so that the Stokes light controller 51 The first Stokes light SB1 and the second Stokes light SB2, which are temporally matched, and the pump light PB may be temporally matched. Here, the pump light controller 54 may adjust the time of the pump light PB by adjusting the optical path of the pump light PB by extending or shortening the optical path of the pump light PB.

The scanning unit 60 scans the sample with the combined light IB. The scanning unit 60 may include a galvano scanner 61 and a rear objective lens 62 . Here, the galvano scanner 61 is a scanning device that adjusts the scan area of the coupled light IB for the sample. The combined light IB passing through the galvano scanner 61 is irradiated to the rear objective lens 62, and the rear objective lens 62 focuses the combined light IB from the rear of the sample toward the sample, thereby CARS signals are generated forward and/or backward.

Here, when a plurality of materials having different chemical compositions are included in the sample, the first CARS signal for any one material is generated by a combination of the pump light PB and the first Stokes light SB1, The second CARS signal for the other material may be generated in the front by a combination of the light and the second Stokes light SB2 . As an example, the sample may be a biological sample including cells or living organisms in which microplastic particles are internalized, wherein the first CARS signal is a Raman mode for microplastic particles and the second CARS signal is a Raman mode for organelles within cells. As , each can be detected and imaged in the front.

In this way, the Stokes light SB passing through the photonic crystal fiber 30 is converted into the first Stokes light SB1 and the second Stokes light SB2 using two dichroic mirrors 40 and 52 with almost no light energy loss. By separating and combining and matching temporally and spatially in the focused sample, ultra-wideband CARS signals covering the fingerprint area (500 ~ 1800 cm ^-1 ) and the CH stretch area (2800 ~ 3200 cm ^-1 ) can be simultaneously measured.

Meanwhile, the combined light IB passing through the galvano scanner 61 sequentially passes through a scan lens (SL) and a tube lens (TL) and is irradiated to the rear objective lens 62. there is.

The detection unit 70 detects the CARS signal, forward to the sample, a front objective lens 71, a signal separation dichroic mirror 72, a first photomultiplier tube (PMT, 73), and a second photoelectric A multiplier tube 74 may be disposed.

Here, the front objective lens 71 collects the front CARS signal generated in front of the sample by focusing the combined light IB from the rear of the sample toward the sample. At this time, the front CARS signal includes the first CARS signal and the second CARS signal. The collected front CARS signal is irradiated with the signal separation dichroic mirror 72 and separated into a first CARS signal and a second CARS signal. Here, the pump light PB, the first Stokes light SB1, and the second Stokes light SB2 as well as the front CARS signal are collected by the front objective lens 71, and the front objective lens 71 and the signal separation dichroic mirror A short pass filter (SPF) is disposed on the optical path between 72 to block the pump light PB, the first Stokes light SB1 and the second Stokes light SB2, and to block the first CARS light. Only the signal and the second CARS signal may be irradiated with the signal separation dichroic mirror 72 . The first CARS signal separated by the signal separation dichroic mirror 72 is detected by the first photomultiplier tube 73, and the second CARS signal is detected by the second photomultiplier tube 74, so that two images can be obtained. there is. Here, the first CARS signal and the second CARS signal may pass through a band pass filter (BPF) and be detected by the first photomultiplier tube 73 and the second photomultiplier tube 74, respectively.

In addition, the detector 70 may further include a third photomultiplier tube 75 for detecting a rear CARS signal generated from the rear by focusing the combined light IB on the sample. The backward CARS signal is moved inversely along the optical path where the coupled light IB is focused on the sample, and is detected and imaged by the third photomultiplier tube 75 . Here, the rear CARS signal may pass through a band pass filter (BPF) and be detected by the third photomultiplier tube 75.

In this case, according to the present invention, three images can be simultaneously acquired by the front CARS signal, that is, the first CARS signal, the second CARS signal, and the rear CARS signal.

Meanwhile, the detector 70 may further include a spectrometer 76 that detects the spectrum of the rear CARS signal. Here, a detector array such as a charge coupled device (CCD) may be mounted on the spectrometer 76 . At this time, the rear CARS signal may pass through a short pass filter (SPF) and be detected by the spectrometer 76.

Here, the rear CARS signal moving inversely along the optical path of the combined light IB can be deviated from the optical path by a dichroic mirror (DM), and can be deviated from the optical path by another dichroic mirror (DM). It can be separated and detected in the third photomultiplier tube 75 and the spectrometer 76, respectively.

Overall, according to the present invention, ultra-wideband CARS signals capable of measuring fingerprints and CH stretch regions can be simultaneously obtained by laser scanning using one femtosecond laser and a photonic crystal fiber. In addition, two or more images can be simultaneously acquired in real time using two Raman modes at a maximum speed of 1 frame/s through laser scanning. Furthermore, the present invention is a non-labeling technology, and it is possible to image microplastic particles and organelles in living organisms or cells, respectively, without pretreatment of living organisms or cells. In addition, the present invention builds a multi-modal microscope system combining two-photon fluorescence technology by arranging a rear detector using two-photon fluorescence technology in addition to a front detector using Raman mode, thereby simultaneously acquiring fluorescence images of biological samples. .

Hereinafter, the present invention will be described in more detail through specific experimental examples.

1. Multi-color CARS microscope

2 and 3 are configuration diagrams of a multi-color CARS microscope according to experimental examples. Referring to FIGS. 2 and 3, the broadband label-free molecular imaging system according to the present invention was implemented with a multi-color CARS microscope. The broadband label-free molecular imaging system according to the present invention has been described above, and will only be briefly described below. In FIG. 2, fs-OPD is a femtosecond optical parametric oscillator, PBS is a polarizing beam splitter, PCF is a photonic crystal fiber, λ/2 is a half-wave plate, and BPF is Bandpass filter, SPF is a shortpass filter, DM is a dichroic mirror, PMT is a photomultiplier tube, CCD is a charge coupled device, and SL is a charge coupled device. Scan lens (scan lens), TL represents a tube lens (tube lens), respectively.

The main goal of this experimental example is to simultaneously label-free vibrational imaging of microplastics (PS microbeads) and cytoplasmic organelles (LDs) internalized in living cells using a high-speed laser scanning multicolor CARS imaging system. Conventional monochromatic CARS microscopes use two microwave laser lights, such as a Raman pump light and a Stokes light, whereas in this experimental example, the Raman pump light (ω _p ), the first Stokes light (ω _S1 ) and the second Stokes light (ω _S2 3 different picosecond (ps) lights. The wavelengths of the two Stokes lights can be adjusted using band-pass filters (BPFs). The wavelength of the first Stokes light is adjusted in the range of 815 to 940 nm, and the wavelength of the second Stokes light is adjusted in the range of 993 to 1043 nm. , the wavelength of the pump light is fixed at 783 nm. The CARS signal is generated when a frequency difference between the pump light and the Stokes light coincides with the vibration frequency of the target Raman-active mode. In this experimental example, by properly adjusting the frequency of two independently adjustable Stokes lights for a fixed pump light frequency, two or more vibration modes are simultaneously used to image different microplastic particles and organelles within cells. can be obtained simultaneously. In this experimental example, the wavelength of the first Stokes light was adjusted to 850 nm and the wavelength of the second Stokes light to 1008 nm to obtain two-color CARS imaging of the PS beads and LDs. Here, the frequency difference between the pump light and the first Stokes light (Δω ₁ = ω _P -ω _S1 ) is the ring breathing mode frequency of the PS bead (Δω ₁ = ω _PS = 1000 cm ^-1 ), and the frequency difference between the pump light and the second Stokes light (Δω ₂ = ω _P - ω _S2 ) is the CH stretch mode frequency of LD (Δω ₂ = ω _LD = 2850 cm ^{- 1} ). Three lights (pump light, first and second Stokes light) are combined with each other and focused on the sample through an objective lens, thereby generating a corresponding CARS signal. Here, CARS 1 is λ _CARS1 = 726 nm (ω _CARS1 = _ωP + ω _PS ) is generated by the combination of the pump light and the first Stokes light, and CARS 2 is λ _CARS2 = 640 nm (ω _CARS2 = _ωP + ω _LD ), which is generated by the combination of the pump light and the second Stokes light, and corresponds to the CARS signals of PS beads and LDs, respectively. The two CARS signals are separated by a dichroic mirror (DM), spectrally filtered by a band pass filter (BPF), and then detected separately in two photomultiplier tubes (PMTs).

Multi-color CARS microscope according to this experimental example is a femtosecond laser light source and a photonic crystal fiber (PCF), using a 2000 cm ^-1 distance fingerprint and CH stretch vibration mode. Two or more CARS images can be obtained there is. In order to simultaneously acquire two or more images using a conventional picosecond laser, three or more laser lights are necessarily required, but the multi-color CARS microscope according to this experimental example does not require multiple complex and expensive laser light sources.

In addition, the CARS microscope according to the present experimental example is a high-speed laser scanning method, and can image organelles (LDs) moving in cells and microplastic particles internalized in real time. The CARS microscope according to this experimental example may be used with a spectrometer equipped with a detector array such as a charge coupled device (CCD). Similar to other hyperspectral imaging microscopes, while scanning the sample stage, all CARS spectral data is recorded at each image pixel, and two or more CARS images are reconstructed from that spectral data cube. The conventional multi-CARS method has an excellent advantage in extracting full spectrum information of chemically different substances, but has a disadvantage in that the imaging speed is slow due to sample scanning and CCD detection speed. The CARS microscope according to this experimental example adjusts incident light using a galvano-scanner for two or more CARS imaging. This significantly increases the speed of data acquisition, enabling real-time label-free bio-imaging of microplastic particles.

Furthermore, the CARS microscope according to this experimental example measures the rear CARS signal (CARS3 in FIG. 1) or two-photon fluorescence (TPF) by using another photomultiplier tube (PMT), and three CARS Alternatively, a CARS-TPF image can be obtained. In addition, the posterior CARS spectrum recorded through the spectrometer can provide information obtainable in epi-CARS microscopy. These additional imaging means can contribute to imaging studies of living cells or chemically different particles in vivo.

2. Biological sample preparation

C. elegans (a wild type N2 strain) was cultured on a nematode growth medium (NGM) plate containing a bacterial layer of E. coli strain OP50. Before the experiment, 2 μm PS beads (78452-5ML-F, Sigma-Aldrich) suspended in 1 ml water were inoculated into the NGM plate and incubated with the nematode (C. elegans) for 2 hours, so that C. elegans could be treated with PS beads. was induced to consume. Then, a number of nematodes were fixed with a 4% formaldehyde solution and transferred to a thin glass chamber. The glass chamber is formed of two overlapping cover glasses spaced apart from each other with a parafilm spacer having a thickness of 100 μm or less. For multi-color CARS live cell imaging, U2OS cells were cultured in a 35 mm confocal dish coated with 0.1 mg/ml poly-D-lysine for cell attachment on the bottom. After incubation for 1 day in RPMI medium supplemented with growth supplement (10% FBS), 2 μm PS beads (0.002 wt.%) were mixed in a sample dish to induce cell uptake. As a result, a large amount of PS beads were internalized into U2OS cells by phagocytic activity within 4 hours. Residual PS beads were removed by exchanging the medium, and a sample dish was installed in a mini incubation mounted on a motorized stage, and multi-color CARS imaging was performed. At this time, the temperature was maintained at 37°C and CO ₂ was maintained at a level of 5%.

3. Multi-color CARS Imaging of Lily Pollen and C. elegans

To demonstrate that simultaneous imaging of chemically different particles in biological samples is possible, multi-color CARS microscopy was performed on Lily Pollen and C. elegans using PS microbeads, and Results are shown in FIG. 4 . 4 (a) to (d) are multi-color CARS-TPE images of lily pollen mixed with 8.7 μm PS beads according to the experimental example, and FIG. 4 (e) to (l) are 2 according to the experimental example. This is a multi-color CARS-TPE image of C. elegans ingesting ㎛ PS beads. Referring to (a) to (d) of FIG. 4, ω _ν = 1605 cm ^-1 (green), ω _ν = 3050 cm ^-1 (red), and ω _ν = 1524 cm ^-1 (blue) obtained CARS images of three different vibration modes for lily pollen mixed with 8.7 μm PS beads. Lily pollen grains and sticky pollenkitts each have ω _ν = 1605 cm ^-1 and ω _ν = 1524 cm ^-1 , which are related to the C=CC aromatic ring stretching mode and the C=C stretching mode of lily pollen, respectively. The first Stokes light, which is combined with the pump light and has a broad spectrum, excites the Raman active mode of lily pollen at frequencies of 1605 and 1524 cm ^-1 (see (a) and (c) in FIG. 4 ).

Using spectral filters, CARS signals were separately measured through photomultiplier tubes (PMTs) placed in the front and rear. The CARS image in (b) of FIG. 4 is related to the CH stretch vibration mode (ω _ν = 3050 cm ^-1 ) of the PS bead, generated by the pump light and the second Stokes light, and another photomultiplier tube (PMT) is detected through Due to the spectral overlap between PS beads, lily pollen grains, and the Raman peaks of pollen, CARS images can appear together spatially. For example, ω _ν = 1605 cm ^-1 CARS image (Fig. 4 (a)) shows both PS beads and pollen grains. ω _ν = 3050 cm ^-1 CARS image (Fig. 4 (b)) provides spatial distribution information of PS beads and pollen kitts. On the other hand, ω _ν = 1524 cm ^-1 In the CARS image (FIG. 4(c)), pollen kitts appear predominantly. Therefore, in the merged CARS image of FIG. 4(d), clear CARS contrasts of PS beads (orange), pollen grains (green), and pollen (purple) are shown in different combinations of similar colors. In addition, the microstructure of pollen surrounded by pollen grains can be detected at sub-micrometer resolution.

On the other hand, C. elegans was observed 2 hours after ingestion of 2 μm PS beads using a multi-color CARS microscope according to this experimental example. 4(e) to (l) show a two-color CARS image and an auto-TPF image. In PS-specific CARS images (Fig. 4 (f) and (j), red) and lipid-specific CARS images (Fig. 4 (e) and (i), green), the first 850 nm wavelength A Stokes light and a second Stokes light having a wavelength of 1008 nm were used. The two CARS images are related to the ring breathing mode of PS beads (ω _ν = 1000 cm ⁻¹ ) and the CH stretch mode of lipids (ω _ν = 2850 cm ⁻¹ ), respectively. The chemically different PS beads and lipids and their spatial distribution in C. elegans are well represented in PS-specific CARS images, lipid-specific CARS images, and merged CARS images. The auto-TPF images (blue) of (g) and (k) of FIG. 4 were obtained by detecting autofluorescence in the wavelength range of 550 to 600 nm, and show the overall appearance and intestinal structure of C. elegans. Here, PS microbeads are located along the intestine of C. elegans, but no trace of penetration into adjacent tissues is observed. In contrast, it can be confirmed that many lipid storages of different sizes and shapes are widely distributed in the extra-intestinal tissues.

4. Label-free live cell imaging with embedded PS beads and organelles

It has been described above that chemically different particles can be simultaneously imaged in static biological samples (lily pollen and C. elegans) using the CARS microscope according to the present experimental example. Hereinafter, real-time CARS imaging was performed on U2OS cells to investigate the intracellular dynamics of PS beads and organelles, and the results are shown in FIG. 5 . Figure 5 (a) is a wide-illuminated PC image of U2OS cells exposed to a 2 μm PS bead solution according to the experimental example, and Figure 5 (b) is a multi-color CARS of U2OS cells exposed to the PS bead solution. 5 (c) to (f) are particle tracking and migration analysis results of PS beads and organelles (LDs) in U2OS cells.

5(a) and (b) show conventional wide-illuminated PC images and two-color CARS images of U2OS cells exposed to PS beads. In the PC image of FIG. 5 (a), many bright spherical particles were observed in U2OS cells. Particles outside the cell represent PS beads drifting in the out-of-focus plane by convection of the buffer solution. As can be seen in the enlarged PC image at the bottom of FIG. 5 (a), PS beads and organelle particles are not well distinguished. This is considered to be because the PC image contrast is very insensitive to the chemical composition of the target. Ultimately, the presence of organelles with indistinguishable sizes and shapes within cells makes it impossible to characterize particles through size, shape or image contrast. In addition, wide-illuminated PC images are also not useful for distinguishing PS beads inside cells from those outside cells due to limited axial resolution. In contrast, the multi-color CARS image (Fig. 5(b)) shows a clear chemical contrast between PS beads internalized in cells and organelles (LD), where PS-specific ring breathing vibrations (ω _ν = 1000 cm ^-1 : orange) and organelle (LD) specific CH stretching vibration (ω _ν = 2850 cm ^-1 : green) were measured simultaneously. Indeed, the relatively weak Raman activity peaks of PS associated with the CH stretch vibrational mode are at the same vibrational frequency _ων = 2850 cm ^-1 partially overlaps with the much stronger CH stretch-induced Raman peak of the organelle (LD). ω _ν = 2850 cm ^-1 to remove the small contribution of the PS-derived CARS signal, ω _ν = 2850 cm ^-1 in the appropriately factored CARS signal, ω _ν The CARS signal factored at = 1000 cm ^-1 was subtracted. like this ω _ν = 2850 cm ^-1, the CARS image with PS contribution removed is ω _ν = merged into the PS specific CARS image at 1000 cm ⁻¹ . This post-processing can improve image quality as well as chemical differentiation between PS beads (orange) and organelles (green). A large number of spherical particles appeared in the CARS image of U2OS cells, but unlike the PC image, 2 μm PS beads (orange) and organelles (green) were easily distinguished in the merged CARS image.

High-speed laser scanning two-color CARS imaging, which individually and simultaneously tracks PS beads and organelles, allows tracking the intracellular movement of PS beads and organelles in real time. 52 PS bead trajectories were obtained from time-lapse images taken every 30 seconds for 6 minutes, and it was determined that the average speed was 14.0 ± 6.4 nm/s. Among them, a representative trajectory of a predetermined PS bead moving in a specific direction is shown in FIG. 5(d). On the other hand, some of the PS beads showed a Brownian-like behavior with diffusion motion and random direction conversion. It was also observed that a plurality of PS beads adjacent to each other near the cell boundary moved together in the same direction toward the cell body. This correlative movement is thought to be the passive movement of the PS beads locally trapped on the plasma membrane surface according to the plasma membrane flow commonly observed in cell movement. In addition, as can be seen from the different trajectories of organelles with reference to FIG. 5(c), multiple organelles can be simultaneously tracked. The average velocity of the 21 organelles was 18.9 ± 11.3 nm/s, approximately 35% faster than that of the internalized PS beads.

6. Comparison of multicolor CARS imaging method with other label-free imaging methods

Phase imaging methods such as PC microscopy and DIC microscopy, which map brightness changes by interference of light generated by different refractive indices and thicknesses of samples, have been widely used to analyze transparent biological samples such as live cells in which microplastics are internalized. However, this refractive index-based imaging approach cannot provide direct information on the chemical composition of the analyte and cannot distinguish chemically different microparticles.

Referring to (a) of FIG. 5 , as can be seen from the live cell PC image, the shape of the PS beads in the cells internalized with the PS beads shows an image contrast similar to that of the spherical organelles of the cells not exposed to the PS beads. That is, microparticles that are chemically different, such as PS beads and organelles, have an indistinguishable phase difference in PC images. Therefore, it is very difficult to accurately identify particles using only PC images. On the other hand, in the case of multi-color CARS imaging, chemical bond-specific vibration signals are separately detected, and PS beads and organelles, which are chemically different but have difficult to distinguish, can be spatially and spectroscopically resolved in a two-dimensional vibration image map. can Conventional HIS-based vibration imaging techniques such as Raman microscopy and multiplex CARS microscopy can characterize multiple microparticles simultaneously and fairly stably and accurately, but have a low signal-to-noise ratio (SNR) due to their reliance on the sample scanning method. , the imaging speed is relatively slow, generally reaching several tens of minutes per frame, so that rapid bioimaging cannot be performed. In addition, since light irradiation time is long due to slow sample scanning and signal averaging, phototoxicity may be induced in biological samples.

On the other hand, the CARS microscope according to the present experimental example is a high-speed laser scanning CARS microscope, and improves the imaging speed to less than several tens of seconds per frame, enabling live cell imaging in real time.

Multi-color CARS microscopy is basically a detection method that does not require a fluorescence background, but in conventional CARS microscopy, a vibrational non-resonant (NR) background signal that creates non-chemical image contrast is a problem. In contrast, the multi-color CARS microscope according to the present experimental example can effectively remove unwanted NR contrast by taking the difference between two CARS images simultaneously obtained at resonance and non-resonance vibration frequencies.

On the other hand, stimulated Raman scattering (SRS) microscopy is known to be useful for rapidly and reliably characterizing environmental microplastics. Since the SRS signal has no NR background and is linearly proportional to the concentration, accurate spectral measurement and chemical measurement analysis of microparticles are possible. However, the SRS microscope requires additional optical devices such as a laser light modulator and a lock-in amplifier to distinguish a weak SRS gain or loss signal from very strong incident light. In addition, in order to simultaneously image fingerprint and CH stretch vibration signals, in principle, a plurality of picosecond laser systems capable of irradiating one pump light and two or more Stokes lights should be constructed. The requirements for such SRS imaging inevitably complicate the setup of optical equipment. On the other hand, the multi-color CARS microscope according to this experimental example uses a single oscillator coupled with a photonic crystal fiber (PCF) as a light source, and can configure a system relatively inexpensively and simply. Real-time imaging of microplastic particles and organelles within a variety of living cells can be achieved.

Although the present invention has been described in detail through specific examples and experimental examples, this is for explaining the present invention in detail, the present invention is not limited thereto, and within the technical spirit of the present invention, those skilled in the art It is clear that modification or improvement is possible by the person.

All simple modifications or changes of the present invention belong to the scope of the present invention, and the specific protection scope of the present invention will be clarified by the appended claims.

10: light source 20: light splitter
30: photonic crystal fiber 40: Stokes light splitting dichroic mirror
50: optical coupling unit 51: Stokes light controller
52: Stokes light coupling dichroic mirror 53: coupled light generating dichroic mirror
54: pump light regulator 60: scanning unit
61: galvano scanner 62: rear objective lens
70: detection unit 71: front objective lens
72: signal separation dichroic mirror 73: first photomultiplier tube
74: second photomultiplier tube 75: third photomultiplier tube
76: spectrometer 80: pump light bandpass filter
90: first Stokes optical band-pass filter 100: second Stokes optical band-pass filter
110: long-wave pass filter PB: pump light
SB: Stokes mine SB1: Stokes mine 1
SB2: Second Stokes light IB: Combined light
HWP, λ/2: half-wave plate DM: dichroic mirror
BPF: band pass filter SPF: short pass filter
SL: Scan Lens TL: Tube Lens
PBS: beamsplitter PCF: photonic crystal fiber
PMT: photomultiplier tube

Claims (10)

a light source for irradiating laser light;
a beam splitter splitting the laser light irradiated from the light source into a pump light and a Stokes light;
a photonic crystal fiber (PCF) extending a wavelength range of the Stokes light split in the optical splitter;
a Stokes light splitting dichroic mirror for splitting the Stokes light passing through the photonic crystal fiber into first Stokes light and second Stokes light according to a predetermined wavelength range;
an optical coupling unit configured to temporally and spatially match the pump light, the first Stokes light, and the second Stokes light to generate combined light;
a scanning unit scanning a sample with the combined light generated by the optical coupling unit to generate a Coherent anti-Stokes Raman Scattering (CARS) signal; and
A broadband label-free molecular imaging system comprising a detector for detecting the CARS signal.
The method of claim 1,
The optical coupling unit,
a Stokes light controller that adjusts a time of the second Stokes light so that the first Stokes light and the second Stokes light are temporally matched;
a Stokes light coupling dichroic mirror for spatially matching the second Stokes light passing through the Stokes light controller with the first Stokes light; and
The combined light is generated by spatially matching the first Stokes light and the second Stokes light, which are spatially matched through the Stokes light-coupling dichroic mirror, and the pump light, and the generated combined light is irradiated to the scanning unit. A broadband label-free molecular imaging system comprising a; coupled light-generating dichroic mirror.
The method of claim 2,
The optical coupling unit,
A broadband label-free molecule imaging system further comprising a pump light controller for adjusting the time of the pump light so that the pump light is temporally matched with the first Stokes light and the second Stokes light synchronized in time by the Stokes light controller. .
The method of claim 1,
a pump light band pass filter for controlling a wavelength range of the pump light irradiated to the optical coupler;
a first Stokes light band pass filter for adjusting a wavelength range of the first Stokes light irradiated to the optical coupler; and
The broadband label-free molecule imaging system further comprising a second Stokes light band pass filter for controlling a wavelength range of the second Stokes light irradiated to the optical coupler.
The method of claim 1,
The broadband label-free molecular imaging system further comprising a long pass filter into which the Stokes light passing through the photonic crystal fiber is incident and transmits the Stokes light in a long wavelength region.
The method of claim 1,
The scanning unit,
a galvano scanner for adjusting a scan area of the combined light with respect to the sample; and
A broadband label-free molecular imaging system comprising: a rear objective lens for concentrating the combined light passing through the galvano scanner toward the sample from the rear side.
The method of claim 1,
The detecting unit,
a front objective lens for collecting the front CARS signal generated from the front by focusing the coupled light on the sample;
a signal separation dichroic mirror separating the front CARS signal, a first CARS signal generated by a combination of the pump light and the first Stokes light, and a second CARS signal generated by a combination of the pump light and the second Stokes light;
a first photomultiplier tube (PMT) for detecting the first CARS signal separated by the signal separating dichroic mirror; and
A broadband label-free molecular imaging system comprising: a second photomultiplier tube for detecting the second CARS signal separated by the signal separating dichroic mirror.
The method of claim 7,
The detecting unit,
The broadband label-free molecular imaging system further comprising a third photomultiplier tube for concentrating the combined light on the sample and detecting a rear CARS signal generated from the rear.
The method of claim 7,
The detecting unit,
The broadband label-free molecular imaging system further comprising: a spectrometer configured to focus the combined light on the sample and detect a spectrum of a backward CARS signal generated backward.
The method of claim 1,
The sample is
A broadband label-free molecular imaging system that is a biological sample containing cells or living organisms in which microplastic particles are internalized.

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