KR20100002742A - Method for imaging raman spectroscopy of biologic tissues and cells and an apparatus using the same - Google Patents

Abstract

PURPOSE: A method and an apparatus for imaging raman signal of cell or biological tissue is provided to diagnose deformation and use for early diagnosis of cancer and biochemical variation. CONSTITUTION: A method for imaging raman signal of cell or biological tissue comprises: a step of emitting light of visible light wave length range; a step of make incidence of light to a point of the cell or biological tissue; a step of filtering the light of light source wave; a step of sending the light to a first charge-coupled device(CCD) and detecting raman signal; a step of converting position of point to detect raman signal; and a step of collecting raman signal obtained from each point and converting in an image of cell or biological tissue. An apparatus for imaging the raman signal of biological tissue or cell comprises: a first light source generating laser; a spatial filter enhancing laser bean diameter; a beam splitter which is optically connected to the spatial filter; a motion controller controlling position of the sample; a filter which filters light of light source wave length; and a monochromator which separats spectrum of light through the filter.

Description

Method for imaging Raman spectroscopy of biologic tissues and cells and an apparatus using the same

The present invention relates to a method and apparatus for imaging Raman signals of cells or biological tissues. The present invention provides a method for imaging and monitoring a Raman signal, thereby identifying phenomena caused by modification of biomaterials or normal, abnormal tissues, and cellular accurate biochemical information. A method and apparatus for Raman signal imaging of cells or biological tissues is provided.

The development of spectroscopy technology over the last few decades has had a significant impact on the biotechnology sector. Spectroscopic techniques, such as confocal fluorescence microscopy, FT-IR and Raman, have been used to elucidate the root and progress of phenomena resulting from the modification of biomaterials. These techniques have been used to diagnose biochemical changes in cells and tissues following biomaterial modifications. Among these spectroscopy techniques, Raman spectroscopy is a relatively simple and non-destructive preparation of samples and provides detailed information on the biochemical structure of normal and non-normal tissues. Has attracted considerable attention for As a result, research is being actively conducted in the clinical field with the development of a light source capable of obtaining high-quality Raman spectra with little tissue damage, a technique for collecting, analyzing and imaging high-quality Raman spectra, and a computer for spectrum analysis.

Raman spectroscopy provides detailed information on the biochemical structure of biomaterials. When light of monochromatic light is incident on a sample, light scattered by the light is generated. When this light is detected, the frequency variation between incident light and scattered light is known as Raman variation, which represents the rotational or vibrational energy state of a particular molecule. It is unique to the molecules that make up this substance, and it can extract biochemical information about biomaterials. In other words, external influences or internal deformations can be detected according to inherent values. This can be used to identify the cause of a substance's transformation and to diagnose diseases, particularly cancers, caused by the modification.

In addition, much research has been conducted on the diagnosis of various diseases using Raman. In particular, the diagnosis of cancer has been progressed a lot, and the possibility of the diagnosis of skin cancer using the resonant Raman has been suggested, and the autofluorescence signal reduction method has been developed and applied to the diagnosis of skin cancer using a visible light source. However, these techniques only cover the local part of the sample.

Imaging technology in the field of biotechnology has primarily been driven by confocal fluorescence microscopy. However, in the case of fluorescence microscopy, many other molecules of various molecules fluoresce in a wide frequency band, making it difficult to determine the presence of a specific substance. In the case of the imaging technique using Raman spectroscopy, near infrared rays have been developed mainly because of the difficulty of detecting the Raman signal due to the magnetic fluorescence signal even though the signal intensity decreases in inverse proportion to the square of the wavelength of the light source. Recently, the autofluorescence signal reduction method has been suggested using confocal micro Raman. However, this method also reduced the fluorescence signal to obtain a Raman spectroscopic signal and failed to image the Raman signal.

The first problem to be solved by the present invention is to provide a method for imaging a living cell or tissue using confocal micro Raman to diagnose the deformation of the biomaterial. A second object of the present invention is to provide an apparatus for Raman signal imaging of cells or biological tissues.

The present invention to solve the first object of the present invention comprises the steps of (a) generating and providing light of the visible region wavelength by the first light source; (b) injecting the light at one point of the cell or biological tissue sample; (c) filtering light at a light source wavelength from light scattered from the sample; (d) sending the filtered light to a spectrometer and sending the light passing through the spectrometer to a first charge-coupled device (CCD) to detect a Raman signal; (e) transforming the points to detect the Raman signal; And (f) collecting the Raman signal obtained at each point and converting the Raman signal into an image of the cell or living tissue.

The cell may be a cell of a single cell organism.

The visible light may be a wavelength of 400 to 700nm.

In one embodiment of the present invention, the visible light may be a wavelength of 514.5nm.

The point position may be converted by a motion controller.

In order to solve the second object of the present invention, the first light source 10 for generating a laser; A spatial filter 20 for increasing the beam diameter of the laser; A beam splitter 21 optically connected to the spatial filter; A motion controller 50 for controlling a change in position of the sample; A filter (40) for filtering light of the light source wavelength from the light scattered from the sample; A spectroscope (111) for separating the spectrum of light passing through the filter (40); A first CCD (100) receiving a Raman signal of the spectrometer; And a device 110 for collecting Raman signals obtained from a point located by the motion controller 50, converting the Raman signals into an image of the cell or biological tissue, and displaying the converted CCD image. Provides a Raman signal imaging apparatus.

The Raman signal imaging apparatus includes a second light source 70 for obtaining an optical image; A second CCD camera 80 which detects light transmitted through the sample by injecting light of a second light source into the sample of the cells and biological tissues; And the light scattered by incident light from the first light source to the sample is sent to the first CCD 100, and the light obtained by transmitting the light from the second light source 70 to the sample is transferred to the second CCD camera. It may further include a flip mirror 60 to be sent to 80, and at this time may further include an objective lens 30 between the second light source 70 and the second CCD camera (80).

The first light source 10 may be an argon (Ar) ion laser.

The filter 40 for filtering the light of the light source wavelength from the light scattered from the sample may be an edge filer.

A zoom lens 90 may be installed in front of the second CCD camera 80 to adjust the magnification.

Since the present invention can provide accurate biochemical information on normal, abnormal tissues, and cells, it is possible to identify the source of the deformation of the biomaterial or to diagnose the deformation, and to diagnose or identify the condition or cause of the disease. It is anticipated that this may be used in the early diagnosis of cancer and biochemical changes, particularly in cancer-causing cells and tissues.

In the present invention, a Raman image is measured by applying confocal micro Raman imaging spectroscopy using a visible light source to living tissue and single cell organisms. In order to increase the intensity of the signal, confocal technology is used to reduce the magnetic fluorescence signal.In particular, unlike the conventional confocal micro Raman system, a flip mirror is used to distinguish the Raman system from the optical microscope system. It can prevent the reduction. In addition, a zoom lens is installed in front of the optical microscope CCD camera to improve the magnification of the optical microscope.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. However, the following drawings and examples are only intended to illustrate the invention, the invention is not limited or limited thereto.

The present invention provides a method comprising the steps of: (a) generating and providing light having a visible wavelength range by a first light source; (b) injecting the light at one point of the cell or biological tissue sample; (c) filtering light at a light source wavelength from light scattered from the sample; (d) sending the filtered light to a spectrometer and sending the light passing through the spectrometer to a first charge-coupled device (CCD) to detect a Raman signal; (e) transforming the location of the point to detect the Raman signal; And (f) collecting the Raman signal obtained at each point and converting the Raman signal into an image of the cell or living tissue. The above method will be described in detail through the following apparatus.

In one embodiment of the cell, the cell may be a cell of a single cell organism. Raman signal imaging techniques are useful as a means of detecting variance differences between normal and abnormal tissues, and are also a good tool for observing cells in unicellular organisms.

The visible light may have a wavelength of 400 to 700 nm, in one embodiment the visible light may be a wavelength of 514.5nm. Auto-fluorescence occurs in the case of using a visible light source in the bio field, and the Raman image experiment using a near-infrared light source has been mainly performed because the magnetic fluorescence causes a decrease in the Raman signal. However, since the Raman signal is inversely proportional to the square of the wavelength of the light source, the intensity of the Raman signal may be increased when using a visible light source. Therefore, if the magnetic fluorescence can be reduced, optical devices are more developed in the case of visible light than near infrared light, and it is advantageous to optimize the optical system when using a visible light source. Therefore, the present invention uses a light source having a wavelength in the visible light region that can increase the intensity of the Raman signal by applying a method capable of reducing the magnetic fluorescence.

The point position may be converted by a motion controller. When the Raman signal is obtained at one point of the sample, the Raman signal of this point is measured by moving to another point of the sample through the control of the motion controller. The Raman signal collected in this way is used for imaging the entire sample.

The present invention, the first light source 10 for generating a laser; A spatial filter 20 for increasing the beam diameter of the laser; A beam splitter 21 optically connected to the spatial filter; A motion controller 50 for controlling a change in position of the sample; A filter (40) for filtering light of the light source wavelength from the light scattered from the sample; A spectroscope (111) for separating the spectrum of light passing through the filter (40); A first CCD (100) receiving a Raman signal of the spectrometer; And a device 110 for collecting Raman signals obtained from a point located by the motion controller 50, converting the Raman signals into an image of the cell or biological tissue, and displaying the converted CCD image. Provides a Raman signal imaging apparatus.

The Raman signal imaging apparatus includes a second light source 70 for obtaining an optical image; A second CCD camera 80 which detects light transmitted through the sample by injecting light of a second light source into the sample of the cells and biological tissues; And the light scattered by incident light from the first light source to the sample is sent to the first CCD 100, and the light obtained by transmitting the light from the second light source 70 to the sample is transmitted to the second CCD camera. It may further include a flip mirror 60 to be sent to 80, and at this time may further include an objective lens 30 between the second light source 70 and the second CCD camera (80).

1 is a schematic diagram of a confocal micro Raman scanning system using a first light source in a visible light region, a spatial filter, and a zoom lens. Referring to FIG. 1, a light source having a wavelength of 514.5 nm in which light generated from the first light source 10 passes through the spatial filter 20 increases, the beam diameter is increased, and is cleaned up. Pass 30 to focus on the sample to a size of about 1 μm. The position of the sample is changed using the XY transition stage system, and the light excited at each position is detected as the Raman signal using the edge filter 40 and the first CCD 100. A second CCD camera 80 is additionally mounted to obtain an optical image, and a zoom lens 90 is installed in front of the second CCD camera 80 to improve the magnification of the optical microscope. The Raman signal passed through the first CCD camera 100 is sent to an apparatus 110 for converting and displaying the converted CCD image. One embodiment of the conversion and display device may be a personal computer including a monitor.

A flip mirror 60 is installed to prevent the reduction of the Raman signal by distinguishing the confocal Raman system from the optical microscope. In a typical Raman system, a CCD for detecting a Raman signal and a CCD camera for measuring an optical image are divided through a beam splitter 21. In this case, since the Raman signal and the optical image signal are separated due to the characteristics of the beam splitter, the Raman and optical signals are inevitably reduced. When the flip mirror 60 is used as in the present invention, the Raman system and the optical microscope system can be selected and used. This does not reduce the Raman signal or the optical image signal.

The first light source 10 may be an argon (Ar) ion laser. This is because the argon laser generates a light source having a wavelength of 514.5 nm.

The filter 40 for filtering the light of the light source wavelength from the light scattered from the sample may be an edge filer. When measuring the Raman signal of the present invention, the light scattered from the sample is composed of excitation light (514.5 nm) and light having Raman information, and the edge filter serves to remove the excitation light. The edge filter passes frequencies smaller than the reference frequency and does not allow large frequencies to pass, ie passes wavelengths larger than the reference wavelength and does not pass small wavelengths. Therefore, if the reference wavelength is 514.5 nm, the 514.5 nm wavelength is removed and a larger wavelength, i.e., light having Raman information, can be passed to detect the Raman signal with the CCD.

Magnification may be adjusted by installing a zoom lens 90 in front of the second CCD camera 80. This enhances the optical microscope's ability to produce optical images, allowing the optical images to be viewed in greater detail.

Example  1: Sample production

Chlamydomonas reinhardtii CC124 bacteria were incubated at 30 ° C. in TAP (Tris-Acetate-Phosphate) medium in the presence of ampicillin (50 μg / ml). (Provided by Chlamy Core Collection Center, USA) was incubated to the mid-log phase, diluted with TAP medium, and then dropped in a drop glass (10 uL) on a slide glass, and dried in vacuum to be used for measurement.

Example  2: Raman signal and optical image measurement

 Confocal micro Raman spectroscopy was performed using a confocal Raman scanning system. An argon ion laser (Coherent 307C) with a wavelength of 514.5 nm was used as excitation light, and the laser light passing through the objective lens was focused to a size of about 1 μm. Raman signals were detected using an edge filter and a charge coupled device (CCD). A CCD camera was additionally mounted to obtain an optical image of the sample, and a zoom lens was used in front of the CCD camera to improve magnification of the optical microscope.

Experimental Example  1: Raman signal from unicellular organism (Clamidomonas) and Imaging

2 is the main Raman spectrum of Chlamydomonas, a single cell organism. 2, the vibration modes associated with a confocal micro-Raman spectroscopy and single-celled organisms, carotenoids of Chlamydomonas are each 1015cm ^-1, 1159cm ^-1, 1524cm ^{- 1.} 3 is an optical microscope image of Chlamydomonas, a single cell organism. 4 is a single-celled organisms of 1524cm ^- a confocal Raman image of a Chlamydomonas measured for the ^first. Comparing FIG. 3 with FIG. 4, the difference between the optical microscopic image and the Raman image can be found. In other words, it is possible to identify nuclei and chloroplasts, which are components of cells, which cannot be identified by an optical microscope from Raman images. Based on this technology, biochemical changes in cells or tissues can be 2D imaged to diagnose biomaterial modifications.

Experimental Example  2: rat lung normal tissue Raman signal and Imaging

5 is the major Raman spectrum of normal lung tissue of rats. Referring to FIG. 5, vibration modes of lipid and protein, Amide I, and CH bonding of biological tissues using confocal micro Raman spectroscopy are respectively ~ 1450 cm ^-1 , ~ 1650 cm ^-1 , 2800 ~ Observed at 3000 cm ^-1 . 6 is an optical microscopic image of rat lung normal tissue. Figure 7 is a lung normal tissue Raman image of the rat obtained on the basis of 2800 ~ 3000 cm ^-1 (CH bonding mode). Comparing FIG. 6 with FIG. 7, it can be seen that the Raman image exactly matches the optical microscope image.

1 is a schematic diagram of a confocal micro Raman scanning system using a first light source in a visible light region, a spatial filter, and a zoom lens.

2 is the main Raman spectrum of Chlamydomonas, a single cell organism. 3 is an optical microscope image of Chlamydomonas, a single cell organism.

4 is a confocal Raman image of Chlamydomonas, a single cell organism.

5 is the major Raman spectrum of normal lung tissue of rats.

6 is an optical microscopic image of rat lung normal tissue.

7 is a confocal Raman image of rat lung normal tissue.

<Explanation of symbols for the main parts of the drawings>

10: first light source 20: spatial filter

21: light splitter 30: objective lens

40: edge filter 50: motion controller

60: flip mirror 70: second light source

80: second CCD camera 90: zoom lens

100: first CCD 110: CCD image display device

111: spectrometer

Claims (11)

(a) generating and providing light having a visible wavelength range by the first light source; (b) injecting the light at one point of the cell or biological tissue sample; (c) filtering light at a light source wavelength from light scattered from the sample; (d) sending the filtered light to a spectrometer and sending the light passing through the spectrometer to a first charge-coupled device (CCD) to detect a Raman signal; (e) transforming the location of the point to detect the Raman signal; And (f) collecting Raman signals obtained at each point and converting the Raman signals into images of the cells or living tissues.  The method of claim 1, wherein the cell is a cell of a single cell organism. The method of claim 1, wherein the visible light has a wavelength of 400 to 700 nm. 4. The method of claim 3, wherein the visible light has a wavelength of 514.5 nm. The method according to claim 1, wherein the point position is converted by a motion controller. A first light source 10 for generating a laser; A spatial filter 20 for increasing the beam diameter of the laser; A beam splitter 21 optically connected to the spatial filter; A motion controller 50 for controlling a change in position of the sample; A filter (40) for filtering light of the light source wavelength from the light scattered from the sample; A spectroscope (111) for separating the spectrum of light passing through the filter (40); A first CCD (100) receiving a Raman signal of the spectrometer; And Raman signal imaging of the cell or biological tissue, characterized in that it comprises a device 110 for collecting the Raman signal obtained from the point located by the motion controller to convert the image of the cell or biological tissue and display the converted CCD image Device. The apparatus of claim 6, wherein the Raman signal imaging apparatus comprises: A second light source 70 for obtaining an optical image; A second CCD camera 80 which detects light transmitted through the sample by injecting light of a second light source into the sample of the cells and biological tissues; And The light from the first light source is incident to the sample and scattered to the first CCD 100, and the light obtained by transmitting the light from the second light source 70 to the sample is transmitted to the second CCD camera 80. Raman signal imaging device of the cell or biological tissue, characterized in that it further comprises a flip mirror (60) for sending. 8. The Raman signal imaging apparatus according to claim 7, wherein the Raman signal imaging apparatus further comprises an objective lens 30 between the second light source 70 and the second CCD camera 80. . 7. The Raman signal imaging apparatus according to claim 6, wherein the first light source (10) is an argon (Ar) ion laser. 7. The Raman signal imaging apparatus according to claim 6, wherein the filter (40) for filtering the light of the light source wavelength in the light scattered from the sample is an edge filer. 7. The Raman signal imaging apparatus according to claim 6, wherein a zoom lens (90) is provided in front of said second CCD camera (80).

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