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Method of making biological microscope imaging technique reach increasing 3D field depth and resolution degree

A microscope and resolution technology, applied in the field of biological microscope imaging technology, can solve problems such as increasing hardware costs

Inactive Publication Date: 2004-10-06
HIVOX BIOTEK +1
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Problems solved by technology

[0004] At present, there are two methods used to increase the depth of field of the microscope scan. One is to use two microscopes to set up the front and back of the sample. After calculating the relative positions, take conjugate images from the front and back respectively. The resulting image thickness is about twice as thick as the image set that can be obtained by a single microscope, but this method greatly increases the required hardware cost

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  • Method of making biological microscope imaging technique reach increasing 3D field depth and resolution degree
  • Method of making biological microscope imaging technique reach increasing 3D field depth and resolution degree
  • Method of making biological microscope imaging technique reach increasing 3D field depth and resolution degree

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no. 1 example

[0050] Figure 9 It is the surface image of the front and back scans of the biological sample, take the bottom image of the front scan and each image of the back scan, and use the fast Fourier transform method to calculate the peak value obtained during the process of the rotation angle between the two images (peak) to make a judgment, find out the approximate position of the bottom image of the front scan image relative to the image group of the reverse scan image on the Z axis (such as Figure 7 shown). Then use the concept of Sobel edge detection mentioned above to find out the region with large edge change in the image. Using the method of correlation matching in this area, it is judged that the image in the reverse scan image is most similar to the image A in the front scan image. Determine the overlapping position of the front and back scan image groups on the Z axis. The entire process of determining the overlapping position on the Z axis is as follows Figure 8 sho...

no. 2 example

[0052] The thickness of the brain of Drosophila is about 160 μm. The brain neurons are marked with green fluorescent protein, and a 488nm laser is used to excite it to obtain a complete 3D brain image. But we can clearly find that the image obtained by laser scanning becomes blurred as it goes to the bottom. The main reason is that the biological sample is light-absorbing, and the energy of the excitation light or the energy of the emitted light is absorbed by the sample, so that the obtained stereoscopic image is very unclear below a certain depth. Using the method proposed in the present invention, it is only necessary to scan to a depth of slightly more than half of the brain to obtain a clear three-dimensional image group of the front scan and the back scan, and then perform image registration to obtain a complete and clear 3D brain image.

no. 3 example

[0054] Generally, confocal microscopic image scanning is to embed biological tissue slices in glycerol for microscopic image scanning and recording. With the method of the present invention, the thickness of the scannable biological tissue slice can be increased to nearly twice the thickness of the original biological tissue, that is, after the biological tissue is first fixed in a three-dimensional space with a sample embedding gel, it is scanned by a confocal microscope to A little more than half the depth of the brain to get a clear three-dimensional image group of the front scan and back scan, and then do the image collating to get a complete and clear 3D biological tissue image. The thickness of the three-dimensional image is about Twice the thickness of the scanned microscopic image.

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Abstract

A method for increasing the focus depth and resolution of the image formed by biomicroscope includes using gel to embed a specimen in it for fixing the specimen in 3D space, scanning the front surface and back surface of specimen, and combining the scanned front and back images in 3D mode. A method for combining said images are also disclosed.

Description

technical field [0001] The invention relates to a method, in particular to a method for increasing 3-dimensional depth of field and resolution by biological microscope image technology. Background technique [0002] The known confocal microscope obtains high-resolution microscopic images at different depths in the sample by removing the noise generated by the non-focal plane. Its main principle can be divided into three steps to explain: firstly, the laser can be focused into a single light spot through the objective lens to irradiate a specific depth of a single point of the sample; in addition, the light reflected or diverged by the focal point can also be focused into a single spot through the objective lens. The light beam completely passes through the pinhole aperture (pinhole aperture) in front of the image detector; finally, other noise photons generated by non-focus points above and below the focus point are blocked around this pinhole, thus ensuring that the detecto...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G02B21/00G06F17/14
Inventor 江安世
Owner HIVOX BIOTEK
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