WO2009133980A1

WO2009133980A1 - Raman microscope

Info

Publication number: WO2009133980A1
Application number: PCT/KR2008/002592
Authority: WO
Inventors: Yong Bum Kim
Original assignee: Yong Bum Kim
Priority date: 2008-05-01
Filing date: 2008-05-08
Publication date: 2009-11-05
Also published as: KR100936645B1; KR20090115276A

Abstract

The present invention relates to a Raman microscope. The Raman microscope comprises: (i) an image selection device AP in a variable iris form selectively passing only desired portions among the inverted real images of the sample obtained by the objective lens Ll; (ii) a beam expander lens L2 expanding the beam diameter of the Raman scattering light and the interference visible light passing the image selection device AP; (iii) a long pass filter FL removing a short wavelength interference visible light among the Raman scattering light and the interference visible light passing the beam expander lens L2; and (iv) a condensing lens L3 removing the interference visible light due to a focal length difference between the Raman scattering light and the interference visible light. Also, the present invention further includes a visible light blocking plate BL positioned on a focus of visible light passing through the condensing lens L3. The present invention can obtain a spectrum with excellent ratio of signal to noise S/ N using a very small sample without being affected from a used illumination light environment while lowering a measuring limit size of a sample to 1 to 10 microns.

Description

RAMAN MICROSCOPE

TECHNICAL FIELD

The present invention relates to a Raman microscope, and more specifically to a Raman microscope capable of obtaining a spectrum with excellent ratio of signal to noise (S /N) upon measuring an extremely small amount of a sample without using a separate dark room or dark box by minimizing visible light interference from an external illumination environment, that is, under illumination of a general laboratory.

BACKGROUND ART

A Raman spectroscopy is a device for qualitatively and quantitatively analyzing substances using a Raman scattering. Reviewing a measuring principle of the Raman spectroscopy, when monochromatic light, such as laser light with a frequency VO, is irradiated to a molecular binding site vibrating at a frequency Vl, most light is scattered as it is without changing its frequency, but some light gives (VO-Vl) the frequency Vl corresponding to a binding energy of a molecule bound to the molecular binding site or receives (V0+V1) energy from the molecular binding so that it is scattered, thereby lengthening or shortening its wavelength. The change in the wavelength is referred to as a Raman scattering. A degree of the change in the wavelength corresponds to an infrared region and all substances have their inherent variations. Therefore, the qualitative and quantitative analysis can be performed on substances using the change in the wavelength in a similar way to a person's fingerprint. Raman spectroscopy qualitatively and quantitatively measures variations.

Reviewing quantum mechanics, since the intensity of stock-line is much larger than the intensity of antistock-line according to Boltzmann distribution, the Raman spectroscope analysis uses the analysis of the stock- line excepting a special case.

At this time, variation Vl of a frequency shows different values according to substances to measure the changed degree and intensity, making it possible to qualitatively and quantitatively analyze an unknown sample. Therefore, the analysis method using the Raman scattering has lower sensitivity than other spectroscope analyzing methods, but has many advantages of convenience of a sample preparation, rapid analysis, and selection of various media, etc. if there are a sufficient amount of samples. In this case, the measurement can be performed at a state where external light is blocked by being trapped in a small glass bottle, a sample partition or a probe sampler box so that the analysis spectrum can be obtained without largely paying attention to an indoor illumination. However, if the sample size is small, completely other situations occur. In particular, if the sample size is confirmed only through a microscope, a general Raman spectroscope method is difficult to measure the sample. Therefore, the Raman microscope should be used to measure the sample so that the sample is necessarily exposed to the external environment. As the Raman measuring method of the most general extremely small sample, an optical microscope is used as the Raman microscope by connecting the optical microscope to a Raman adapter as shown in FIG. 2. A size relation between an image and an objective lens of a microscope being a convex lens is as follows.

When an image with a size Sl is formed at a position spaced by a apart from the front of the convex lens with a focal length f, an image with a size S2 formed at the rear of the convex lens is formed at a place spaced apart by b obtained from the following equation (I) and the size

S2 of the image depends on the following equation (II). l/a+l/b=l/f (I)

Sl/S2=b/a (II)

However, the above case is applied to a case of monochromatic light. In a case of multi-color light such as white light, refractive index of light is varied according to a wavelength of light so that a distance at which an image is generated is varied. When light passes through substances with large density like a medium, such as a glass lens in air, the shorter the wavelength of light, the larger the refractive index becomes so that the position forming the image is shorter. This is referred to as chromatic aberration and becomes larger upon using a single lens. Therefore, if the image of light with a short wavelength is S2¹, a distance is b¹ the image of light with a long wavelength is S2", and a distance is b", S2'<S2" and b'<b".

A conventional Raman microscope is used by attaching the Raman adapter to the general optical microscope as shown in FIG. 2. The operating principle thereof is as follows.

FIG. 2 is a schematic view showing a basic structure of the conventional Raman microscope.

A sample S is positioned at a focal length of an objective lens L4 and light from the sample becomes parallel light. This parallel light is formed on a focus F by a condensing lens L5 as an inverted real image and is positioned just in the focal length of an ocular lens L6, making it possible to see an expanded inverted virtual image.

Therefore, the Raman adapter R is installed between the condensing lens L5 and the objective lens L4 so that although the distance between two lenses is changed, the operation of the optical microscope has not been affected.

After installing the Raman adapter R, the monochromatic laser beam is incident and is condensed and irradiated onto the sample at the focal position through the objective lens L4. Thereafter, the Raman scattering light generated from the sample is transferred to a spectroscopy via the same path and is then measured. At this time, both the Raman scattering light L by irradiated laser light as well as illumination light v of a device peripheral environment reflected from the sample S is generated in the sample S. They are also transferred to the Raman spectroscopy so that they serve as the hindering light for obtaining the Raman spectrum. At this time, if the sample size is smaller than the beam diameter of the irradiated laser beam, the S/ N of the sample is degraded and the influence of external light is larger. In particular, when the sample size is extremely small to about 1 to 10 microns, it is impossible to obtain the spectrum of the sample.

Therefore, in order to exclude the influence of the peripheral illumination light, the sample size should be measured under a dark room environment without peripheral illumination light or should be measured by putting a device in a dark box so as not to irradiate the sample with peripheral light.

DISCLOSURE OF THE INVENTION

(TECHNICAL PROBLEM)

In order to solve the interference problem of external illumination light that is the largest problem of an existing Raman microscope, the present invention provides a user-friendly Raman microscope with excellent ratio of signal to noise (S/ N) while lowering a measuring limit size of a sample to 1 to 10 microns under a general laboratory environment where normal illumination is performed, without using a dark room or a dark box.

(TECHNICAL MEANS TO SOLVE THE PROBLEM)

In accordance with the present invention, there is provided a Raman microscope obtaining inverted real images RI of a sample by an objective lens Ll using a laser beam and then transferring them to a selected one of a Raman spectroscopy and a device for visibly confirming a sample state, the Raman microscope comprising: (i) an image selection device AP in a variable iris form selectively passing only desired portions among the inverted real images of the sample obtained by the objective lens Ll; (ii) a beam expander lens L2 expanding the beam diameter of the Raman scattering light and the interference visible light passing the image selection device AP; (iii) a long pass filter FL removing a short wavelength interference visible light among the Raman scattering light and the interference visible light passing the beam expander lens L2; and (iv) a condensing lens L3 removing the interference visible light due to a focal length difference between the Raman scattering light and the interference visible light.

(ADVANTAGEOUS EFFECTS) The present invention uses a very small sample to obtain a spectrum with excellent ratio of signal to noise S/ N and lower a measurable limit size of a sample to 1 to lOμm.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features, aspects, and advantages of preferred embodiments of the present invention will be more fully described in the following detailed description, taken in conjunction with the accompanying drawings. In the drawings: FIG. 1 is a schematic view of a basic structure of a Raman microscope according to the present invention.

FIG. 2 is a schematic view of a basic structure of a conventional Raman microscope.

FIG. 3 is a diagram schematically showing a process wherein external illumination light enters from an objective lens.

FIG. 4 is a diagram of a very small sample on a slide glass covered by a cover glass that is a first embodiment of the present invention.

FIG. 5 is a Raman spectrum of polyphenylene sulfide measured in the first embodiment of the present invention.

FIG. 6 is a Raman spectrum of acrylic resin measured in a second embodiment of the present invention.

FIG. 7 is the Raman spectrum of an ammonium sulfate sample measured in a third embodiment of the present invention.

[Description of symbols for major parts in drawings]

S: Sample fθ: Focus of objective lens Ll, IA: Objective lens fl: Focus of laser light passing through objective lens

L: Laser light v: Visible light (external illumination light)

BS: Beam splitter FiI: Visible light primary focus

FrI : Raman scattering light primary focus

Si: Visible light inverted real image

Sr: Raman scattering light inverted real image

AP: Image selection device (Variable iris) L2: Beam expander lens

FL: Long pass filter

L3, L5: Condensing lens

Fi2: Visible light secondary focus

Fr2: Raman scattering light secondary focus BL: Visible light interrupting plate

L6: Ocular lens

N: Raman spectroscopy detector

N 1 : Optical fiber sensor of Raman spectroscopy detector F: Focus of ocular lens R: Raman adapter θ: Incidence angle and reflection angle of visible light SG: Slide glass CG: Cover glass.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the embodiments of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic view showing a basic structure of the

Raman microscope according to the present invention.

Generally, the Raman microscope uses the monochromatic light as a beam as described above to obtain the inverted real images of a sample and transfer them to the Raman spectroscopy for measuring the S/ N spectrum or transfer them to the device for visibly confirming the sample state.

As shown in FIG. 1, the present invention also uses the laser light L as a beam to obtain the inverted real images of the sample by the objective lens Ll and transfer them to a selected one of a Raman spectroscopy and a device for visibly confirming the sample state.

As shown in FIG. 1, the present invention comprises (i) an image selection device AP in a variable iris form selectively passing only desired portions among the inverted real images of the sample obtained by the objective lens Ll, (ii) a beam expander lens L2 expanding the beam diameter of the Raman scattering light and the interference visible light passing the image selection device AP, (iii) a long pass filter FL removing a short wavelength interference visible light among the Raman scattering light and the interference visible light passing the beam expander lens L2, and (iv) a condensing lens L3 removing the interference visible light due to a focal length difference between the Raman scattering light and the interference visible light.

The image selection device AP is an iris capable of changing the size continuously or stepwise, wherein the shape of the iris is a circle or a polygon, etc.

In addition, the Raman microscope of the present invention further comprises a visible light blocking plate BL positioned on a focus of visible light passing through the condensing lens L3. Preferably, the visible light blocking plate BL has any one shape selected from a circle and a polygon.

Preferably, the size of the visible light blocking plate BL is a focus size of visible light passing through the condensing lens L3.

Hereinafter, the Raman microscope according to the present invention will be described with reference to FIG.1.

A solid line L shown in FIG. 1 shows laser irradiating light and Raman scattering signal light and a broken line V shows interference light of a visible light band from the illumination of a device peripheral environment.

First, a half of monochromatic laser light L from the laser beam is transmitted and a half thereof is reflected by a beam splitter BS to be turned at an angle of 90?so that it is vertically directed downward to the objective lens Ll. The light is condensed by the objective lens Ll to be irradiated to the sample S. At this time, when the sample size is sufficiently larger than the size of laser light, there are few problems. However, when the sample size is less than 10 microns, the external illumination visible light may be reflected through the same path, as shown in FIG. 3.

More specifically reviewing, the external illumination visible light enters only from the side of the objective lens Ll, wherein the maximum incidence angle of light and the reflection angle θ are a reflection angle or an incidence angle not covered by the objective lens Ll. Therefore, the illumination visible light has difficulty to be reflected toward the objective lens, but the illumination visible light is randomly reflected from the sample surface to have the same path as the Raman scattering light, so that it has been affected. Of course, the intensity of this light is very weak, but the intensity of the Raman signal light is also very weak, thereby providing a bad influence. Therefore, in order to obtain a good Raman spectrum, the visible light reflected together with the Raman scattering light should be removed.

A process of removing the external illumination visible light is as follows.

The visible light and the Raman scattering signal light from the sample S forms the visible light inverted real image Si and the Raman scattering light inverted real image Sr at the positions of the visible light primary focus FiI and the Raman scattering light primary focus FrI, respectively, by the objective lens Ll. At this time, the stock-line with a long wavelength among the Raman scattering signal light should be used. In this case, the wavelength of the Raman scattering light is longer than that of the peripheral visible light, so that the image is formed on a higher position by the chromatic aberration.

Next, only the desired portions to be measured among the inverted real images is cut by the image selection device AP in a variable iris form to remove the visible light or unnecessary portions reflected from the circumference of the sample. Nevertheless, the peripheral illumination visible light scattered from the sample surface is different from the Raman scattering signal light only in the focal length and they are placed on the same axis line so that they cannot be removed by the image selection device AP in the variable iris form.

The beam diameter of the Raman scattering signal light and the interference visible light passing through the image selection device AP are widened by a beam expander lens L2. In other words, a distance from the visible light primary focus FiI and the Raman scattering light primary focus FrI to the beam expander lens L2 is shorter than the focal length of the beam expander lens L2 so as to continuously spread light. The visible light inverted real image Si is farther away from the beam expander lens L2 and has larger refractive index as compared to the Raman scattering light inverted real image Sr so that a degree of the spread of visible light is smaller than that of the Raman scattering light. Widening the beam diameter is to separate the focus as well as to improve the efficiency of the long pass filter FL. In other words, the beam diameter thereof is expanded and the incidence angle thereof is controlled by the beam expander lens L2, so that light with a wavelength shorter than that of the used laser light as a beam is cut and only light with a wavelength longer than that of the used laser light as a beam is passed, while effectively passing through the wide area of the long pass filter FL. At the same time, the visible light is removed.

An extremely small amount of visible light remaining even after passing through the long pass filter FL is removed through the re- condensing process of the condensing lens L3. In other words, the focuses of the Raman scattering signal light with a long wavelength and the peripheral visible light with a short wavelength are separated when they are re-condensed passing through the condensing lens L3, wherein the visible light with a short wavelength is first condensed and is then spread and the Raman scattering signal light is condensed at a higher position. An introducing part of an optical fiber sensor Nl connected to a detector is fitted in the focus position of the Raman scattering signal light using the difference in the focal length so the integration degree is better, making it possible to obtain a good signal. On the other hand, the visible light previously passes through the focus and is in an expanded state so that its optical density is low and its incidence angle is not proper, thereby removing a considerable amount of visible light.

Nevertheless, when the visible light having an effect on the Raman scattering signal light remains, it is preferable to install the visible light blocking plate BL on the focus Fi2 of visible light passing through the condensing lens L3 to further remove the visible light.

Preferably, the size of the visible light blocking plate BL is the focus size Fi2 of visible light passing through the condensing lens L3 and the shape thereof is a circle or a polygon, etc.

Hereinafter, the present invention will be described in detail with reference to the embodiment.

However, the present invention is not limited thereto.

First Embodiment The measuring result of the sample S, wherein polypheny lene sulfide of about 2 microns is placed on a slide glass SG and is covered with a cover glass CG as shown in FIG. 4, by the Raman microscope of the present invention as shown in FIG. 1 in a laboratory under a general illumination environment shows that the Raman spectrum as shown in FIG. 5 is obtained, this Raman spectrum completely meets the standard spectrum measuring a bulk sample of the same substance, and there is no influence of indoor illumination light. Second Embodiment

The measuring result of an acrylic piece of about 5 microns placed on a glass plate by the Raman microscope of the present invention as shown in FIG. 1 in a general laboratory under a general illumination environment shows that the Raman spectrum of acrylic not being affected by the illumination light as shown in FIG. 6 can be obtained.

Third embodiment

The measuring result of an ammonium sulfate particle of about 10 microns placed on a glass plate by the Raman microscope of the present invention as shown in FIG. 1 under a general laboratory environment shows that the Raman spectrum of ammonium sulfate not being affected by the illumination light as shown in FIG. 7 can be obtained.

INDUSTRIAL APPLICABILITY

The present invention can obtain a spectrum with excellent ratio of signal to noise (S/ N) upon measuring an extremely small amount of a sample without using a separate dark room or dark box by minimizing visible light interference from the external illumination environment, that is, under illumination of a general laboratory.

Although a few embodiments of the present invention have been shown and described, it would be appreciated by those skilled in the art that changes and modifications might be made in this embodiment without departing from the principles and spirit of the invention, the scope of which is defined in the claims and their equivalents.

Claims

WHAT IS CLAIMED IS;

1. A Raman microscope obtaining inverted real images RI of a sample by an objective lens Ll using a laser beam and then transferring them to a selected one of a Raman spectroscopy and a device for visibly confirming a sample state, the Raman microscope comprising:

(i) an image selection device AP in a variable iris form selectively passing only desired portions among the inverted real images of the sample obtained by the objective lens Ll; (ii) a beam expander lens L2 expanding the beam diameter of the

Raman scattering light and the interference visible light passing the image selection device AP;

(iii) a long pass filter FL removing a short wavelength interference visible light among the Raman scattering light and the interference visible light passing the beam expander lens L2; and

(iv) a condensing lens L3 removing the interference visible light due to a focal length difference between the Raman scattering light and the interference visible light.

2. The Raman microscope according to claim 1, further comprising a visible light blocking plate BL positioned on a focus of visible light passing through the condensing lens L3.

3. The Raman microscope according to claim 2, wherein the visible light blocking plate BL has any one shape selected from a circle and a polygon.

4. The Raman microscope according to claim 2, wherein the size of the visible light blocking plate BL is of a focus size passing through the condensing lens L3.

5. The Raman microscope according to claim 1, wherein the image selection device AP in a variable iris form has any one shape selected from a circle and a polygon.