CN107884388A - A kind of micro-Raman spectroscopy and its application method of fast automatic focusing - Google Patents

Abstract

The micro-Raman spectroscopy and its application method of a kind of fast automatic focusing disclosed by the invention, wherein the micro-Raman spectroscopy of the fast automatic focusing includes micro imaging system, Raman spectrum system, control device and computer system, control device is connected with computer system, micro imaging system is located above control device, and Raman spectrum system is located at micro imaging system side.The present invention is compact-sized, it is full-featured, can automatic focusing, obtain sample regional area spectral information and can obtain complete object image information under high-resolution microscope, and Raman spectral information is obtained, greatly improve the testing efficiency and test effect of Raman spectrometer.

Description

A rapid auto-focus micro-Raman spectrometer and its application method

technical field

The invention relates to the technical field of Raman spectrum detection, in particular to a fast auto-focusing micro-Raman spectrometer and a method for using the same.

Background technique

Raman spectroscopy is an analytical method that obtains information about the vibration and rotation of molecules and applies it to the study of molecular structures. Raman spectroscopy can achieve qualitative and quantitative analysis of substances through vibration and rotation information of substances. The Raman spectrometer has a simple structure and is easy to operate. The measurement is fast, efficient and accurate, and high-resolution spectral information can be obtained. Although the spectral information of the sample surface can be obtained, the Raman spectrometer collects the average spectrum of the sample surface, which cannot be obtained in the sample. Spectral information corresponding to local regions or local points.

At the same time, traditional microscopes can realize the observation of tiny objects, but they need to subjectively observe the target through the eyepiece and manually adjust the knob to focus. This manual focusing method is time-consuming and laborious and prone to manual errors. And under the high-resolution microscope, although the clear image information of the object at high resolution can be obtained, the internal structure information of the local area or local point of the object at high resolution cannot be obtained, and it cannot be obtained under the high-resolution microscope A complete object image information. Therefore, there is an urgent need for a Raman spectrometer that can automatically adjust the focus, obtain the spectral information of the local area of the sample, and obtain the complete object image information under a high-resolution microscope.

Contents of the invention

The present invention provides a micro-Raman spectrometer with rapid auto-focus and its use method to solve the problem that the existing Raman spectrometer needs to focus manually, cannot obtain the local image information of the measured object, and cannot obtain complete image information under a high-resolution microscope. The problem of object image information.

In order to solve the above technical problems, the present invention provides a fast auto-focusing micro-Raman spectrometer, which includes a micro-imaging system, a Raman spectroscopy system, a control device and a computer system, the control device is connected to the computer system, and the The microscopic imaging system is located above the control device, and the Raman spectroscopy system is located on one side of the microscopic imaging system.

Preferably, the control device includes a stage, a first motor, a second motor, an autofocus device, a coarse adjustment knob, a control box and a base, the control box is connected to a computer system, the first motor, the second motor The two motors and the autofocus device are respectively connected with the control box circuit, the object stage is connected with the first motor and the second motor and installed above the base, and the coarse adjustment knob is connected with the object stage.

Preferably, the control box includes a communication module, a single-chip microcomputer connected to the communication module, a driver connected to the single-chip computer, and a power supply connected to the driver.

Preferably, the microscopic imaging system includes an LED lamp, a first collimating mirror, a first beam splitter, a second beam splitter, a pipeline, a CCD camera and a microscopic objective lens, and the microscopic objective lens is located above the stage , the first beam splitter is located above the microscope objective lens and forms an angle of 45 degrees with the horizontal plane, the second beam splitter is located above the first beam splitter and forms an angle of 90 degrees with the first beam splitter, and the pipeline is located Above the second beam splitter, the CCD camera is located directly above the pipeline, the first collimator is vertically arranged on the lower side of the second beam splitter, and the LED light is arranged on the first collimator away from the second beam splitter. slice side.

Preferably, the Raman spectroscopy system includes a processing device, a reflector, a third beam splitter, a second collimating mirror, a coupling lens, a first optical fiber probe, a second optical fiber probe and an optical filter, and the processing device and the The computer system is connected, the third beam splitter is located on the side of the lower side of the first beam splitter, and the filter, coupling lens and first optical fiber probe are sequentially arranged on the side of the third beam splitter away from the first beam splitter , the reflector is located directly above the third beam splitter, the second collimator and the second fiber optic probe are sequentially arranged on the side of the lower side of the reflector, the first fiber optic probe and the second fiber optic probe are connected to the processing device connection.

Preferably, the computer system includes a display screen and a computer connected thereto.

In order to solve the above-mentioned technical problems, the present invention also provides a method for using a micro-Raman spectrometer with fast auto-focus as described above, comprising the following steps:

Step 1: Place the sample on the stage, start the computer and corresponding software, and select the image acquisition auto-focus mode;

Step 2: Start the LED light and CCD camera;

Step 3: automatically collect image information, and the computer judges whether the evaluation function value of the image is the largest; if not, enter step 4; if so, enter step 5;

Step 4: PLC drives the stage to move, and enters into Step 3;

Step 5: Store the best focus image, and obtain a complete image by splicing multiple best focus images;

Step 6: Select a region of interest or a point from the complete image as a measurement region, and drive the stage to move the measurement region under the microscope objective;

Step 7: Start the Raman spectroscopy system to collect the Raman spectroscopy information of the measurement area.

A rapid auto-focus micro-Raman spectrometer provided by the present invention mainly includes a micro-imaging system, a Raman spectroscopic system, a control device and a computer system. The medium autofocus microscope objective lens obtains high-resolution clear images as the research object, and can quickly and easily obtain the corresponding spectral information of the sample by selecting a local area or local point through the image. The spectral information obtained through the autofocus microscope can be accurately The Raman signals of different layer depths and focal planes of the sample are detected, which has good spatial resolution and can precisely locate the specific position of the sample through the control device to control the auto-focusing microscope objective, and collect the Raman signals of this area, thereby The combination of high-resolution image acquisition and spectral analysis of the sample under the microscope is realized. The present invention has compact structure, comprehensive functions, can automatically focus, obtains the spectral information of the local area of the sample and can obtain the complete object image information under a high-resolution microscope, and conducts Raman spectral analysis, which greatly improves the performance of the Raman spectrometer. Test the effect.

Description of drawings

Fig. 1 is the structure schematic diagram of a kind of fast auto-focus micro Raman spectrometer provided by the present invention;

FIG. 2 is a flowchart of a method for using a fast auto-focusing micro-Raman spectrometer provided by the present invention.

Among them, microscopic imaging system 1, Raman spectroscopy system 2, control device 3, computer system 4, LED lamp 11, first collimating mirror 12, first beam splitter 13, second beam splitter 14, pipeline 15, CCD camera 16. Microscopic objective lens 17, processing device 21, reflector 22, third beam splitter 23, second collimating mirror 24, coupling lens 25, first optical fiber probe 26, second optical fiber probe 27, optical filter 28, carrier Object stage 31, first motor 32, second motor 33, autofocus device 34, coarse adjustment knob 35, control box 36, base 37, display screen 41, computer 42, sample a.

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Please refer to shown in Fig. 1, a kind of microscopic Raman spectrometer of rapid autofocus provided by the present invention comprises microscopic imaging system 1, Raman spectroscopy system 2, control device 3 and computer system 4, control device 3 and computer system 4 connections, the microscopic imaging system 1 is a high-resolution microscopic imaging system, located above the control device 3, and the Raman spectroscopy system 2 is located on the side of the microscopic imaging system 1. Wherein, the control device 3 includes a stage 31, a first motor 32, a second motor 33, an autofocus device 34, a coarse adjustment knob 35, a control box 36 and a base 37, the control box 36 is connected with the computer system 4, the first Motor 32, second motor 33 and autofocus device 34 are respectively connected with control box 36 circuits, stage 31 is connected with first motor 32 and second motor 33 and installed on the top of base 37, coarse adjustment knob 35 is connected with load The object table 31 is matched and connected.

Further, the control box 36 includes a communication module, a single-chip microcomputer connected with the communication module, a driver connected with the single-chip microcomputer, and a power supply connected with the driver.

Microscopic imaging system 1 comprises LED lamp 11, first collimating lens 12, first spectroscopic sheet 13, second spectroscopic sheet 14, pipeline 15, CCD camera 16 and microscopic objective lens 17, and microscopic objective lens 17 is positioned at stage 31 Above, the first beam splitter 13 is located above the microscope objective lens 17 and forms an angle of 45 degrees with the horizontal plane, the second beam splitter 14 is located above the first beam splitter 13 and forms a 90 degree angle with the first beam splitter 13, and the pipeline 15 Located above the second beam splitter 14, the CCD camera 16 is positioned directly above the pipeline 15, the first collimating mirror 12 is vertically arranged on one side of the lower side of the second beam splitter 14, and the LED lamp is arranged on the first collimating mirror 12 away from the second beam splitter. One side of the beam splitter 14.

The Raman spectroscopy system 2 includes a processing device 21, a reflector 22, a third spectroscopic plate 23, a second collimating mirror 24, a coupling lens 25, a first fiber probe 26, a second fiber probe 27 and an optical filter 28, and the processing device 21 is connected to the computer system 4, the third beam splitter 23 is located at the side of the lower side of the first beam splitter 13, and the optical filter 28, the coupling lens 25 and the first optical fiber probe 26 are sequentially arranged on the third beam splitter 23 away from the first beam splitter 13 side, the reflector 22 is located directly above the third beam splitter 23, and the two are arranged in parallel, the second collimating mirror 24 and the second fiber optic probe 27 are successively arranged on the side of the lower side of the reflector 22, and the first fiber optic probe 26 and The second fiber optic probe 27 is connected to the processing device 21 .

The computer system 4 includes a display screen 41 and a computer 42 connected thereto.

In order to solve the above-mentioned technical problems, the present invention also provides a method for using a micro-Raman spectrometer with fast auto-focus as described above, comprising the following steps:

Step 1 S1: Place the sample on the stage, start the computer and corresponding software, and select the image acquisition autofocus mode;

Step 2 S2: Start the LED light and CCD camera;

Step 3 S3: Automatically collect image information, and the computer judges whether the evaluation function value of the image is the largest; if not, proceed to step 4; if so, proceed to step 5;

Step 4 S4: PLC drives the stage to move, and enters into step 3;

Step 5 S5: storing the best focus image, and obtaining a complete image by splicing multiple best focus images;

Step 6 S6: Select a region of interest or a point from the complete image as a measurement region, and drive the stage to move the measurement region under the microscope objective;

Step 7 S7: Start the Raman spectroscopy system to collect the Raman spectroscopy information of the measurement area.

The specific work process of the present invention is as follows: firstly open the autofocus mode, click the autofocus mode of the computer system of the display screen, start the LED light 11, and the incident light passes through the first collimating mirror 12, the second beam splitter 14 and the first beam splitter After 13 reflection enters the microscope objective lens 17, the light source converges to the sample surface, and the reflected light generated passes through the microscopic object 17, and after being transmitted through the first beam splitter 13 and the second beam splitter 14, it enters the CCD with a high-pass filter through the pipeline 15 The camera 16 carries out instantaneous image acquisition, and the image signal collected is processed by the computer 42 and displayed on the computer display screen 41; the computer 42 evaluates the value according to the sharpness function of the image, the larger the evaluation value, the clearer the image, and the computer 42 transmits the instruction to the control panel. box 36 and drives the autofocus device 34 to drive the stage 31 carrying the sample a to move upward or downward. At this time, the microscopic imaging system 1 collects the image of the sample and transmits it to the computer system 4 for calculation to generate an image function evaluation value. Through the function evaluation Value comparison, the computer 42 transmits instructions to the control device 3 to drive the stage 31 carrying the sample to move up and down to obtain different focus planes, and find the focus plane position with the largest function evaluation value to obtain the best focus image , and the focus mode is turned off at this time, and the auto focus ends. The acquisition of the best focal plane of multiple different images is to transmit instructions to the single-chip microcomputer in the control box 36 through the computer 42, and the single-chip microcomputer drives the first motor 32 and the second motor 33 to drive the stage 31 carrying the sample a to move left and right, At this time, the computer 42 sends an instruction to the microscopic imaging system 1 to collect the best focus image under the microscope and save it through the computer 42 .

Next, turn on the image stitching mode: click to select the image stitching mode of the computer system on the display screen 41, use the computer image stitching function to stitch multiple best-focus images saved by the computer into a complete high-resolution image information.

Finally, open the spectral acquisition mode: click to select the spectral acquisition mode of the computer system on the display screen 41, select a local area or point on the image through the image information collected in the previous two steps, and the computer 42 outputs signals and instructions to transmit to the user. Man spectrum system 2 and control device 3, control box 36 composed of communication module, single-chip microcomputer, driver and power supply in control device 3 receives computer instructions to drive motor 19 and motor 20 to drive the stage to select a local area or point on the image The position moves to under the microscope objective lens 17. At this time, the processing device 21 composed of the Raman module and the spectrometer in the Raman spectroscopy system 2 outputs continuous laser light through the second optical fiber probe 27 and is collimated by the second collimating mirror 24, and After 22 reflections, it is reflected by the third beam splitter 23 and the first beam splitter 13 and enters the microscope objective lens 17 to converge on the surface of the sample a. After passing through the third spectrometer 23, it passes through the optical filter 28 and the coupling lens 25 to reach the first optical fiber probe 26, and finally enters the processing device 21 composed of the Raman module and the spectrometer, and the acquired spectral information is transmitted to the computer 42, and the computer 42 processes the displayed on the display screen 41 to complete the collection of spectral information.

Since the traditional microscopic autofocus is that the operator manually adjusts the focus through the eyepiece coarse and fine adjustment knobs, the present invention uses the computer to process and calculate the objective definition evaluation parameters of the image, and the control system can realize complete and fast autofocus on the sample Due to the digital processing, the device has objective accuracy and speed and does not require manual participation to achieve fully automatic focusing. The image obtained by focusing is accurate and clear, and the obtained high-resolution and clear sample image can also be saved. Prepare for subsequent analysis and processing of the study. After the best focus image is collected through autofocus, a high-resolution, clear and complete image information of the sample under the microscope is obtained through image stitching at the same time. In the present invention, except that the samples need to be loaded manually, everything from focusing, image collection to spectrum collection and image splicing can be completed automatically.

Since Raman spectroscopy can perform fast, simple, repeatable and non-destructive qualitative and quantitative analysis of organic and inorganic substances and requires a small amount of sample, although the autofocus microscope can automatically obtain high-resolution image information, it cannot obtain images. The information corresponds to the internal structure information of the sample of interest. Although Raman spectroscopy can obtain the spectral information of the sample, that is, the internal structure information, it cannot observe the image information corresponding to the sample. In the present invention, an autofocus microscope device is combined with a Raman spectrometer, and Raman spectroscopy system 2, high-resolution microscopic imaging system 1, control device 3, and computer system 4 operate in coordination with each other, that is, it can be acquired through an autofocus microscope. The high-resolution clear image is used as the research object, and the region of interest or the point of interest can be selected as the sample through the Raman spectroscopy system 2, and the corresponding spectral information of the sample can be obtained quickly and easily, so that the spectral information obtained by the autofocus microscope can be Raman signals of different layer depths and focal planes of the sample are accurately detected. The invention has good spatial resolution and can accurately locate the specific position of the sample through the autofocus microscope adjustment system, and collect the Raman signal of the micro area, thereby realizing the collection and processing of high-resolution images of the sample under the microscope. Combination of Spectral Analysis.

Finally, the method of the present application is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. A microscopic Raman spectrometer with fast autofocus, characterized in that it comprises a microscopic imaging system, a Raman spectroscopy system, a control device and a computer system, the control device is connected with the computer system, and the microscopic The imaging system is located above the control device, and the Raman spectroscopy system is located at one side of the microscopic imaging system. 2. a kind of microscopic Raman spectrometer of rapid autofocus according to claim 1, is characterized in that, described control device comprises stage, first motor, second motor, autofocus device, coarse adjustment knob , a control box and a base, the control box is connected to the computer system, the first motor, the second motor and the autofocus device are respectively connected to the control box circuit, and the stage is connected to the first motor And the second motor is connected and installed above the base, and the coarse adjustment knob is matched with the stage. 3. the microscopic Raman spectrometer of a kind of fast autofocus according to claim 2, is characterized in that, described control box comprises communication module, the single-chip microcomputer that is connected with described communication module, the driver that is connected with described single-chip microcomputer, and a power supply connected to the drive. 4. a kind of microscopic Raman spectrometer of rapid auto-focus according to claim 3, is characterized in that, described microscopic imaging system comprises LED lamp, the first collimating mirror, the first beam splitter, the second beam splitter , a pipeline, a CCD camera and a microscopic objective lens, the microscopic objective lens is located above the stage, the first beam splitter is located above the microscopic objective lens and forms an angle of 45 degrees with the horizontal plane, and the second beam splitter The sheet is located above the first beam splitter and forms an angle of 90 degrees with the first beam splitter, the pipeline is located above the second beam splitter, the CCD camera is located directly above the pipeline, and the first beam splitter is located above the pipeline. A collimating mirror is vertically arranged on the side of the lower side of the second beam splitter, and the LED light is arranged on the side of the first collimator facing away from the second beam splitter. 5. the microscopic Raman spectrometer of a kind of fast autofocus according to claim 4, is characterized in that, described Raman spectroscopic system comprises processing device, reflection mirror, the 3rd beam splitter, the 2nd collimating lens, coupling lens, a first optical fiber probe, a second optical fiber probe and an optical filter, the processing device is connected to the computer system, the third optical splitter is located on the lower side of the first optical splitter, and the optical filter , the coupling lens and the first optical fiber probe are sequentially arranged on the side of the third beam splitter away from the first beam splitter, the reflector is located directly above the third beam splitter, the second collimating mirror, the second optical fiber The probes are sequentially arranged on the side facing the lower side of the reflector, and the first fiber optic probe and the second fiber optic probe are connected to the processing device. 6. The microscopic Raman spectrometer of a kind of fast automatic focus according to claim 1, is characterized in that, described computer system comprises display screen and the computer that is connected with it. 7. A method for using the micro-Raman spectrometer of fast auto-focus as claimed in claims 1 to 6, characterized in that, comprising the following steps: Step 1: Place the sample on the stage, start the computer and corresponding software, and select the image acquisition auto-focus mode; Step 2: Start the LED light and CCD camera; Step 3: automatically collect image information, and the computer judges whether the evaluation function value of the image is the largest; if not, enter step 4; if so, enter step 5; Step 4: PLC drives the stage to move, and enters into Step 2; Step 5: Store the best focus image, and obtain a complete image by splicing multiple best focus images; Step 6: Select a region of interest or a point from the complete image as a measurement region, and drive the stage to move the measurement region under the microscope objective; Step 7: Start the Raman spectroscopy system to collect the Raman spectroscopy information of the measurement area.