CN101241069A - Dispersive multi-functional Hadamard transform microscopic imaging spectrometer - Google Patents

Abstract

Dispersion-type multifunctional Hadamard transform microscopic imaging spectrometer, including excitation light source, optical microscope, lens group, Hadamard template, monochromator and photodetector; it is equipped with a driving mechanism for driving Hadamard template; the photodetector is a linear array CCD; the lens group is a lens group that collimates, focuses and compresses the optical signal from the microscope into a light spot that matches the size of the linear array CCD. The invention integrates the functions of the fluorescence microscope and the micro Raman spectrometer, introduces Hadamard transformation technology, and successfully realizes the integration of imaging and spectrum analysis capabilities on the same instrument. Obtain sample state observation, spectral analysis, multi-spectral imaging and image analysis capabilities. The invention has good system expansibility and compatibility.

Description

Dispersive multi-functional Hadamard transform microscopic imaging spectrometer

technical field

The invention relates to the fields of spectral measurement technology and optical imaging technology, in particular to a dispersion-type multifunctional Hadamard transform microscopic imaging spectrometer.

Background technique

Fluorescence and Raman light both belong to photoluminescence. Both fluorescence spectrum and Raman spectrum can reflect the composition of substances, and can also detect the content of substances according to the luminous intensity. Therefore, in the research of fluorescence and Raman light, many kinds of special instruments have been born, and these instruments have played a huge role in promoting biomedical research. Fluorescence microscopes and micro-Raman spectrometers, which were born for the research needs of cells and tissues, have played a huge role in scientific research and medical fields.

The structure of a common upright fluorescence microscope is shown in Figure 1. Its structure is composed of halogen lamp 1, condenser lens 2, diaphragm 3, stage 4, objective lens 5, beam splitter 6, eyepiece 7, condenser lens 8, mercury lamp 9 and supporting structure. The sample is placed on the stage 4, and the illumination light of the halogen lamp 1 passes through the condenser 2 and the aperture 3 to irradiate the sample on the stage 4 from the lower side to generate a transmission image. The excitation of fluorescence generally uses a mercury lamp 9 as an excitation light source, and the excitation light is reflected from the objective lens 5 through the dichroic filter 6 to irradiate the surface of the sample on the stage 4 to generate fluorescence. After the light signal is collected by the objective lens 5 and enters the microscope, it passes through the dichroic filter 6 and enters the eyepiece 7 . The structure of the fluorescence microscope determines that the fluorescence microscope can only be used to obtain the fluorescence image of the sample, it does not have the ability of spectral analysis, and the image does not have the ability to describe the fluorescence intensity.

The structure of the micro-Raman spectrometer is shown in Figure 2. Its structure consists of halogen lamp 1, condenser lens 2, diaphragm 3, stage 4, objective lens 5, laser light source 6, beam splitter 7, reflector 8, eyepiece 9, notch filter 10, Raman spectrometer 11 and Supporting structure composition. The sample is placed on the stage 4, and the illumination light of the halogen lamp 1 passes through the condenser lens 2 and the aperture 3 to irradiate the sample on the stage 4 from the lower side, producing a transmission image, which can be observed through the eyepiece 9. The excitation light source of the micro-Raman spectrometer generally uses a laser light source 6, through the beam splitter 7, a very thin beam of laser light is irradiated through the objective lens to the sample surface on the stage 4, and the generated Raman signal is collected by the objective lens 5 , the optical signal is extracted through the beam splitter 7 and the mirror 8, and then the Rayleigh scattered light is filtered out by the notch filter 10, and finally enters the Raman spectrometer 11. The micro-Raman system has the capability of Raman spectrum analysis and the magnified image of the sample, but it cannot obtain the spectral image of Raman light.

The functions of the above two types of systems are independent of each other, but they are both based on microscope design in structure, and some structures are similar. There is a possibility of sharing this part of the instrument.

Hadamard transform technology is a modulation technology similar to Fourier transform, which has the ability of multi-channel detection and imaging. Using this technique can significantly improve the signal-to-noise ratio. Although this technology has begun to be used in the fields of spectral analysis and microscopic imaging, the functions of the instruments using this technology are relatively single, and the integration of spectral analysis and imaging capabilities is poor. For example, patent ZL94107751.9 (international patent main classification number: G01N21/31, authorized in 1998.7) discloses a Hadamard transform low-resolution imaging system, which uses a 15×17 two-dimensional Hadamard template to obtain a 15×17 pixel microscopic image. Patent 01106536.2 discloses a high-resolution Hadamard transform microscopic image analyzer, which uses a 511-dimensional template and a 512-pixel linear array CCD to obtain a high-resolution image of 511×512 pixels, but does not have the ability to obtain high-resolution spectra. Reports on spectrometers using Hadamard transform mainly use Hadamard templates to replace the slits of monochromators to achieve imaging, but multifunctional instruments with high spectral resolution and better image resolution have not yet appeared.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art, and to provide a dispersion-type multifunctional Hadamard transform microscopic imaging spectrometer, which can combine Raman spectrum with fluorescence spectrum, spectral analysis and image analysis and take into account the microscope bright light. Field of view spectroscopy and imaging, enabling multifunctional integration.

The technical scheme provided by the invention is: a dispersion type multifunctional Hadamard transformation microscopic imaging spectrometer, including an excitation light source, an optical microscope, a lens group, a Hadamard template, a monochromator and a photoelectric detector; The mechanism; the photodetector is a linear CCD; the lens group is a lens group that collimates and focuses the optical signal from the microscope into a light spot that matches the size of the linear CCD.

The above-mentioned Hadamard template is a transmissive one-dimensional circular coding template with a set of light and dark stripes, which is driven by a motor-driven screw transmission mechanism for linear motion.

The above-mentioned monochromator is a reflection grating type sinusoidal mechanism standard monochromator. The motor drives the gear and the screw rotates to drive the grating to rotate to adjust the detection wavelength.

The above-mentioned excitation light source is a high-pressure mercury lamp or a laser.

The above-mentioned optical microscope is composed of a halogen lamp lighting source, an XYZ three-dimensional stage, an objective lens group, an eyepiece lens group and a filter.

The above-mentioned objective lens group is composed of an objective lens holder, an objective lens and a color filter.

The above-mentioned eyepiece group includes an eyepiece holder and an eyepiece arranged on the eyepiece holder.

The above-mentioned eyepiece bracket is provided with a threaded interface for connecting the camera and/or a C-interface for installing the CCD.

The invention integrates the functions of the fluorescence microscope and the micro Raman spectrometer, introduces Hadamard transformation technology, and successfully realizes the integration of imaging and spectrum analysis capabilities on the same instrument. Obtain sample state observation, spectral analysis, multi-spectral imaging and image analysis capabilities.

The invention has good system expansibility and compatibility. A more comprehensive application of this system can develop a multi-functional spectral microscopy imaging system that takes into account both fluorescence and Raman, spectroscopy and imaging. And if the system structure is properly simplified, an optimized system focusing on a certain function can also be obtained, or it can be simplified as an accessory expansion part of the microscope.

Description of drawings

The optical structure diagram of Fig. 1 fluorescence microscope;

The optical structure diagram of the microscopic part of Fig. 2 micro-Raman instrument;

Fig. 3 Structural schematic diagram of Hadamard transform fluorescence and Raman spectroscopy microscopic imaging system;

Fig. 4 Schematic diagram of the principle of multi-spectral microscopic imaging;

Figure 5 Multispectral microscopic imaging of cancerous tissue labeled with fluorescent probes;

The spectral intensity-illumination time relation curve of Fig. 6 fluorescent probe;

Figure 7 Microscopic Raman spectrum and Raman imaging of β-carotene powder;

Fig. 8 is a structural schematic diagram of a miniaturized Hadamard transform fluorescence microscope imaging analyzer;

Figure 9 Hadamard fluorescence imaging of pollen cells and measurement of photobleaching.

Detailed ways

The Hadamard transformation spectrum microscopic imaging system involved in the present invention is composed of a microscope, an excitation light source, a Hadamard template, a light splitting path (monochromator), a linear array CCD detector and related control software and hardware. Figure 3 is a schematic diagram of the instrument structure.

As shown in FIG. 3 , the objective lens 8 collects various optical signals from the sample placed on the stage 6 , including transmitted light, fluorescence, and Raman light. After the light signal enters the objective lens, it can be selected by the mirror 12 to enter the eyepiece 13 for observation; or it can be selected to enter the spectral imaging system, and the light is collimated by the lenses 14 and 15 and focused on the Hadamard template 17 . The light modulated by the Hadamard template 17 is collimated by the lens group 18 and focused by the cylindrical lens groups 19 and 20 at the incident slit of the monochromator 21 into a rectangular spot suitable for the size of the incident slit. After entering the standard monochromator structure 21, the grating splits the light, and the outgoing beam is focused to the image sensitive unit of the linear array CCD 22. The computer workstation 24 regulates the stepper motors 23, 25, and receives the signal of the CCD 22, and finally obtains relevant data through program conversion. The optical signal collected by the objective lens is modulated by the Hadamard template, and the beam is focused by a cylindrical lens group, and the focused light is split by a monochromator, and the CCD is used for multi-channel data acquisition, and by decoding the data, Output data such as spectrograms, imaging pictures, and signal strengths that can be used.

According to the structure, various excitation light sources can be selected, such as mercury lamp or laser. According to the application requirements, choose different excitation wavelengths and lens combinations to become the required light source. And through the filter to eliminate the interference of excitation light.

According to this structure, various excitation illumination methods, such as epi-illumination, transmission, side-emission, etc., can be used. According to the characteristics of the sample, different excitation methods are used. For opaque samples, use epi- or side-shot excitation; for samples in tubes, side-shot lasers; for transparent samples, use whichever excitation method is more convenient.

According to this structure, the light extracted from the microscope is modulated one by one by the Hadamard template, all of which are recorded by the CCD to generate an image.

According to this structure, the light passing through the template is focused by the cylindrical lens into a linear light spot (with a size not greater than 500 microns×6000 microns).

According to this structure, the size of the linear array CCD should match the focused spot of the cylindrical mirror, and the length and width of the linear array CCD should be greater than or equal to the size of the focused spot to ensure that the light emitted from the exit slit of the monochromator can be completely captured by the linear array CCD. acquired.

According to this structure, the Hadamard template used in the Hadamard transformation is a one-dimensional cyclic template. According to the coding rules, use transparent and opaque corresponding 1 and 0 to generate a set of barcodes. The metal-plated glass sheet can be processed by etching process to make the template. When Hadamard transformation, the template should be one-dimensionally stepped along the coding direction to generate the required code elements. The step length of the step should be the same as the coding unit length, and the number of steps should be equal to the number of code elements. The liquid crystal panel can also be used as a template, and the template does not need to be moved during conversion, only the required coding barcodes are generated sequentially according to the code element structure.

According to this structure, the number of pixels in the long direction of the line array CCD is the number of pixels in the Y direction of the generated image, and the number of Hadamard coded symbols is the number of pixels in the X direction of the generated image. According to the specific requirements of the sample, the present invention can select different image resolutions, namely 128×128 pixels, 256×256 pixels and 512×512 pixels.

According to this structure, the generated Hadamard image uses 256 gray levels to measure the spectral intensity of each pixel.

According to this configuration, the Hadamard transformed image is an optical signal distribution image at a certain wavelength originating from the sample. The image obtained by the area array CCD or camera installed in the eyepiece is full-spectrum signal imaging.

According to this structure, when the CCD acquisition is associated with the grating rotation of the monochromator, the spectral curve is obtained.

According to this structure, when the signal collected by the CCD is correlated with time, the time variation curve of light intensity can be obtained.

Example 1: Hadamard Transform Fluorescence, Raman Spectroscopy Microscopic Imaging System

The structure diagram of the instrument is shown in Figure 3. The halogen lamp 1, the condenser lens 2, the aperture 3, the stage 6, the objective lens 8, the beam splitter 11, the reflector 12, and the eyepiece 13 are the structural parts of the microscope. The laser 4, the reflector 5, Optical fiber 7, mercury lamp 9, condenser 10 are peripheral excitation light sources, lenses 14, 15, diaphragm 16, Hadamard template 17, lens 18, cylindrical mirrors 19, 20, monochromator 21 etc. form the Hadamard modulation and spectroscopic Optical path, linear array CCD 22 is a photoelectric detector, grating control mechanism 23, computer workstation 24, template control mechanism 25 etc. are control mechanisms.

For this system, the optical signal collected by the objective lens 8 can come from the transmitted light of the halogen lamp 1, or the luminescence signal obtained by the sample excited by the laser 4 or the mercury lamp 9, or the non-excited signal from the sample itself. Light. If the sample is excited to emit fluorescence, the fluorescence spectrum and fluorescence image will be obtained; if the sample is excited to emit Raman light, the Raman spectrum and Raman signal will be obtained. Similarly, transmitted light and luminescent light can also be spectrum and imaged.

Microscope objective lens 8 collects the required optical signal, uses reflector 12 to guide the light out, and accurately focuses it on the one-dimensional cycle Hadamard template 17 through lenses 14 and 15, and uses stepping motor 25 to control the rotation of the screw to drive the step translation of the template to achieve For the encoding of optical signals, the encoded light is focused into a slender rectangular spot after passing through the cylindrical mirrors 19 and 20, which is the clear encoded image of the sample, and is positioned at the center of the incident slit edge of the monochromator 21 , the dispersive light split by the monochromator 21 is focused on the surface of the linear array CCD 22 again, and the Hadamard image of the field of view of the objective lens is obtained through the decoding processing of the signal output by the CCD 22 by the computer workstation 24.

The light derived from reflector 12 is not coded, and then passes through lens 18. After cylindrical mirrors 19 and 20, it also enters the monochromator with a rectangular light spot, and focuses on the surface of linear array CCD (512 pixels) 22. Workstation 24 control steps The motor 23 drives the grating in the monochromator 21 to rotate, and records the signal from the CCD 22 at the same time, and the workstation 24 converts the recorded corresponding stepping motor pulse and CCD signal data to obtain the spectrum of the optical signal collected by the objective lens 8 .

Example 2: Fluorescence Spectrum Microscopic Imaging and Spectral Analysis

Combining the imaging function and spectral scanning function in Embodiment 1, a multi-spectral microscopic imaging operation can be realized, and the implementation process is as follows:

Tissue samples labeled with two different fluorescent probes: a 550nm fluorescent probe for cell membranes and a 610nm fluorescent probe for cell nuclei. The tissue samples were imaged at different wavelengths, and corresponding pseudo-color processing was performed according to the wavelength in consideration of the visual needs, that is, a series of images from 530nm to 630nm in Fig. 4(1). Refer to the fluorescence spectrum of Fig. 4(2). For the maximum value of the spectrum, two images of 550 and 610 were selected, and the data were superimposed by software, and the final result was shown in Figure 5.

Figure 4 (2) is the fluorescence spectrum of the sample, the image of the cell nucleus (imaging at 610nm) is obtained in Figure 5 (1), the image of the cell membrane (imaging at 550nm) is obtained in Figure 5 (2), and Figure 5 (3) It is the overlay image of Figure 5(1) and Figure 5(2). Compared with the tissue fluorescence observed by the eyepiece, the interference from the autofluorescence of cells in Figure 5(3) is well eliminated.

The fluorescence intensity at the peak was continuously recorded. Figure 6 is the fluorescence intensity value-time relationship curve of the tissue, which confirms the phenomenon that the fluorescent probe gradually decays with time due to photobleaching under light irradiation.

Example 3: Raman Spectroscopy and Microscopic Imaging of β-Carotene

A 514 nm laser was used to excite the Raman signal of the β-carotene powder. After scanning the spectrum, the Raman spectrum in Figure 7(1) is obtained. At 1000 wavenumbers, the imaging picture obtained is Figure 7(3). At the same position and wavelength, the image of transmitted light imaging using halogen lamp illumination is shown in Figure 7 (2).

Example 4: Miniaturized Hadamard Transform Fluorescence Microscopic Imaging Analyzer

This embodiment is mainly aimed at fluorescence imaging with relatively single spectral components, and its optical structure schematic diagram is shown in FIG. 8 .

Halogen lamp 1, condenser lens 2, diaphragm 3, stage 4, objective lens 5, beam splitter 6, condenser lens 7, mercury lamp 8, reflector 9, eyepiece 10 constitute the structural part of the fluorescence microscope, lens 11, reflector 12, 20 , lens 13,16,17,19, diaphragm 14, one-dimensional 511 code element circular Hadamard template 15, grating 18 constitute the optical structure of Hadamard conversion imaging, and linear array CCD (512 pixels) 21 is the detection of photoelectric conversion device.

The miniaturization of the instrument is achieved by reducing the requirement for spectral resolution. Reflectors 12, 20 are used in the optical path to fold the optical path while shortening the optical path, and smaller lenses 11, 13, 16, 17, 19 and small grating 18 instruments are used to form a relatively simplified light splitting optical path to Compress the instrument volume. Hadamard template 15 also uses a one-dimensional 511-symbol cycle template, and CCD 21 also uses a 512-pixel linear array CCD. The control mechanism has been simplified accordingly, but the structure still conforms to Figure 3.

The excitation light emitted by the mercury lamp 8 is reflected by the dichroic filter 6 and passes through the objective lens 5 to irradiate the sample surface on the stage 4. The emitted fluorescence is collected by the objective lens 5 and reflected by the dichroic filter 6 The mirror 9 is turned and focused by the lenses 11, 13 onto the Hadamard template 15 surface. The light encoded by the one-dimensional Hadamard template 15 is collimated by the lens group 16-17 and split by the grating 18, and the dispersed light after the split is focused by the lens 19 on the surface of the linear array CCD 21. The workstation controls the stepping movement of the Hadamard template 15 and the data acquisition of the CCD, and the fluorescent image can be obtained through decoding.

The resolution of the grating in this embodiment is relatively poor, and it mainly serves as a simple color filter, so that certain spectral parts are strengthened in imaging. According to needs, filters can also be used to replace the split light path.

Embodiment 5: Hadamard transform fluorescence image analysis

The collected Yulian pollen was stained with acridine orange (AO) and excited by the blue light of the mercury lamp. The obtained fluorescent image of the Yulian pollen cells is shown in Figure 9(1), in the figure a, b, c three The fluorescence intensity and cell size of the cells can be accurately measured by the analysis software. The fluorescence intensity values are 559.5k, 633.9k, and 432.4k, the maximum diameters are 49.8, 48.0, and 50.4 μm, and the minimum diameters are 33.0, 35.4, and 32.4 μm. Three pollen cells were imaged every minute for 20 minutes, and the quantitative data in each image were analyzed to obtain Figure 9(2). Fig. 9(2) shows the fluorescence intensity-time relationship curve of the pollen cell b, which accurately reflects the phenomenon that the pollen fluorescence gradually decays with time due to photobleaching under light irradiation.

Claims (9)

1. the color dispersion-type multifunctional Hadamard transform microscopical imaging spectrometer comprises excitation source, optical microscope, lens combination, Adama template, monochromator and photoelectric detector, it is characterized in that: be provided with the driving mechanism that drives the Adama template; Photoelectric detector is a line array CCD; Lens combination will be for focusing on and be compressed into the lens combination of the hot spot that is complementary with the line array CCD size from microscopical light signal collimation.

2. spectrometer according to claim 1 is characterized in that: the Adama template is to be subjected to the transmission-type one dimension loop coding template drive mechanism moving linearly, that have one group of light and dark striped.

3. spectrometer according to claim 1 and 2 is characterized in that: monochromator is a reflection grating formula sine mechanism standard monochromator.

4. spectrometer according to claim 3 is characterized in that: monochromator is provided with and drives the driving mechanism that grating rotates.

5. spectrometer according to claim 1 and 2 is characterized in that: excitation source is high-pressure sodium lamp or laser instrument.

6. spectrometer according to claim 1 and 2 is characterized in that: optical microscope is made of Halogen lamp LED lighting source, the three-dimensional objective table of XYZ, objective lens, eyepiece group and filter filter disc.

7. spectrometer according to claim 6 is characterized in that: objective lens is made of objective lens support, objective lens and color filter.

8. spectrometer according to claim 6 is characterized in that: the eyepiece group comprises eyepiece support frame and is located at eyepiece on the eyepiece support frame.

9. spectrometer according to claim 8 is characterized in that: eyepiece support frame is provided with the C interface that is used to connect the hickey of camera and/or CCD is installed and constitutes.

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