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Method for producing light field or wide field fluorescence light section

A fluorescent light and slicing technology, applied in the field of optical slicing, can solve the problems of toxic damage to biological samples, affecting imaging speed, sample limitation, etc., and achieve the effects of wide application prospects, increased imaging speed, and simple settings.

Inactive Publication Date: 2009-07-22
JINAN UNIVERSITY
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Since the laser confocal microscope needs to scan on the X-Y plane before imaging, it greatly affects its imaging speed, so it is generally impossible to perform dynamic tracking measurement on living cells
In addition, laser confocal microscopy can only image the part of the sample that is excited by fluorescence, and the laser will cause toxic damage to biological samples due to fluorescence bleaching when exciting fluorescence
Coupled with the use of a highly monochromatic laser to excite fluorescence, the available fluorescent probes are limited, so the detectable samples are also greatly limited
[0003] Although multiphoton absorption microscopy can reduce the toxic damage of fluorescence bleaching compared with laser confocal microscopy, it still has the above-mentioned problems of laser confocal microscopy

Method used

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  • Method for producing light field or wide field fluorescence light section
  • Method for producing light field or wide field fluorescence light section
  • Method for producing light field or wide field fluorescence light section

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Observation of corneal slices and imaging of bright-field light slices are realized on an inverted microscope with the technology that can generate wide-field light slices. The objective lens used is a 40× plan achromatic objective lens. The specific method is: a 25 / mm sinusoidal grating is installed in front of the microscope tungsten halogen lamp (wide-field white light source), and the image square Fourier spectrum plane behind the microscope objective lens is Set a pass filter on it. The tungsten-halogen lamp projected the grating on the image of the corneal slice to obtain an optical slice image with a thickness of 0.7 microns. Adjust the focal length of the microscope objective lens to obtain the optical section images of each slice. A bandpass filter removes the grating fringes into a clear image of the light slice. Specific images such as figure 1 shown by figure 1 It can be seen that the resulting corneal slice images not only have clear boundaries, but als...

Embodiment 2

[0027] The tomographic wide-field optical slice imaging of pollen spores is realized on a fluorescence microscope by using the optical spatial filtering method that can generate three-dimensional images. The objective lens used is a 60× plan achromatic objective lens. The specific method is: set a 100 / mm sinusoidal grating in front of the microscope mercury lamp (wide-field ultraviolet light source). The mercury lamp projects the grating onto the sample for imaging, and the band-pass filter filters out the grating stripes to form a clear light slice image. Adjust the focal length of the microscope objective lens to obtain optical slice images of 30 slices, each with a thickness of 0.3 μm. Specific images such as figure 2 As shown, the images of each optical slice are clear, and the images of each layer transition gradually. figure 2 At the same time, the 3D stereoscopic image obtained after 3D reconstruction of the 30-layer optical slice image is given with our 3D image r...

Embodiment 3

[0029] The tomographic wide-field optical slice imaging of fingerprints is realized on a polarizing microscope by means of an optical spatial filtering method that can generate three-dimensional images. The objective lens used is a 20× objective lens. The specific method is: set a 150 / mm sinusoidal grating in front of the microscope tungsten-halogen lamp (wide-field white light source), and the tungsten-halogen lamp projects the grating on the sample slide with fingerprints for imaging, and the resulting image is then processed by digital image to perform spatial filtering. Specific images such as image 3 as shown, image 3 a is an image superimposed with grating stripes, and the frequency spectrum of the image after Fourier transform after digital image processing is as follows: image 3 c see. image 3 d is to remove the part of the spectrum corresponding to the grating stripes by means of digital image processing, image 3 b is the tomographic optical slice image of t...

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Abstract

The invention provides a method capable of producing bright-field or wide-field fluorescent light slices, that is, a tomographic imaging technique and method. This method can use the white light or wide-field ultraviolet light of an ordinary optical microscope as a light source to obtain clear tomographic images of microscopic samples. It can be scanned at one time and can be imaged at one time, which can realize the dynamic tracking and monitoring of samples (including living cells). Based on this method, on the basis of obtaining each tomographic image, three-dimensional image reconstruction, three-dimensional display and analysis of the sample can also be realized. This method is suitable for the imaging of various tiny object samples, which can be transparent or total reflection samples, and can be applied to the observation of tissue embryos, blood, nerves, microorganisms, cell biology, three-dimensional biology, and various organic and inorganic substances measurement field.

Description

technical field [0001] The present invention relates to optical sectioning technology, in particular to a method for producing bright-field or wide-field fluorescence optical sectioning, that is, realizing fast tomographic imaging of tiny samples under a microscope under white light or wide-field ultraviolet light source illumination. Background technique [0002] In biomedical research, people need to perform tomographic detection on tiny samples such as cells. This process is often vividly called optical sectioning. Because the clear image of each layer of the sample can be obtained without actual mechanical slicing of the sample, and the three-dimensional image can also be obtained by three-dimensional reconstruction, so it will soon become a powerful method for the study of various samples, especially biological samples. Laser confocal microscopy is currently the main means of microscopic light sectioning. Laser confocal microscopy and similar technologies use laser lig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 黄耀雄李金
Owner JINAN UNIVERSITY
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