Method for counting cells in section

Abstract

The invention discloses a method for counting cells in a slice, which is characterized by comprising the following steps of: preparing an additional slice in a direction vertical to a slice to be counted, measuring the cell length on the vertical slice by using a microscale of a microscope to obtain an average length L, and carrying out conventional cell counting on the slice to be counted to obtain a cell count K, and measuring the slice thickness T, and calculating the actual cell number S according to a formula. According to the invention, the accuracy of cell counting is obviously improved, the method is suitable for various tissue types, is particularly suitable for the condition that the cell size is similar to the slice thickness, and is convenient for cell density comparison of different tissues or under different conditions.

Description

Technical Field

[0001] The invention belongs to the field of histology and cell biology, and in particular relates to a method for accurately estimating the number of cells in a tissue section. Background Art

[0002] In histological research, it is of great significance to accurately estimate the number of cells in tissues. However, the traditional method of counting cells in slices has a significant flaw: because some cells will be cut into fragments and distributed in adjacent slices, when counting cells in adjacent slices, the same cell will be counted repeatedly, resulting in an overestimation of the actual number of cells. This error is particularly significant when the cell size is similar to the slice thickness. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for counting cells in a slice, which solves the problem that the same cell is easily counted repeatedly in the existing cell counting method.

[0004] In order to solve the above technical problems, an embodiment of the present invention provides a method for counting cells in a slice, comprising the following steps:

[0005] Prepare additional slices in a direction perpendicular to the slice to be counted, use a microscope micrometer to measure the cell length on the vertical slice to obtain the average length L, perform a conventional cell count on the slice to be counted to obtain the cell count K, measure the slice thickness T, and calculate the actual cell number S according to the formula.

[0006] Among them, it is achieved through the following steps:

[0007] a) Take the same tissues from two animals with the same characteristics and fix them with formaldehyde; cut the slice plane for counting cells on a plane corrector from one tissue and cut the slice plane for measuring cell length from the other tissue in a vertical direction, dehydrate, slice and stain them;

[0008] b) Using a microscope micrometer ruler, measure the cell length on vertical slices to obtain the average length L;

[0009] c) observing the slices under a microscope, determining the area where the cells to be counted are located, determining the optimal magnification, ensuring that the area where the cells to be counted are located is complete in each image, and using a camera to acquire images at the optimal magnification;

[0010] d) Manually mark each stained cell in the area in an image software (such as Photoshop) and manually count to obtain K;

[0011] e) obtaining a slice thickness T according to the slice;

[0012] f) Calculate the actual cell number S according to the formula to obtain the cell number of the tissue section.

[0013] Wherein, the formula is:

[0014]

[0015] Where S is the actual number of cells in the slice, K is the number of cells observed in the slice, T is the slice thickness (μm), and L is the average cell length (μm).

[0016] The beneficial effects of the above technical solution of the present invention are as follows:

[0017] The present invention significantly improves the accuracy of cell counting through the formula, is applicable to various tissue types, and is particularly suitable for situations where the cell size is similar to the slice thickness, and is convenient for comparing cell densities in different tissues or under different conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of tissue sections in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0020] An embodiment of the present invention provides a method for counting cells in a slice, comprising the following steps:

[0021] a) Take the same tissues from two animals with the same characteristics and fix them with formaldehyde; cut the slice plane for counting cells on a plane corrector from one tissue and cut the slice plane for measuring cell length from the other tissue in a vertical direction, dehydrate, slice and stain them;

[0022] b) Using a microscope micrometer ruler, measure the cell length on vertical slices to obtain the average length L;

[0023] c) observing the slices under a microscope, determining the area where the cells to be counted are located, determining the optimal magnification, ensuring that the area where the cells to be counted are located is complete in each image, and using a camera to acquire images at the optimal magnification;

[0024] d) Manually mark each stained cell in the area in an image software (such as Photoshop) and manually count to obtain K;

[0025] e) obtaining a slice thickness T according to the slice;

[0026] f) Calculate the actual number of cells S according to the formula to obtain the number of cells in the tissue section. The formula is as follows:

[0027]

[0028] Where S is the actual number of cells in the slice, K is the number of cells observed in the slice, T is the slice thickness (μm), and L is the average cell length (μm).

[0029] like Figure 1 As shown in the figure, the slice is between the two lines. First, two spherical cells with the same radius are placed on the slice surface, touching at points A and B respectively, that is, they are cut on the slice surface. Any cell with the same radius will appear in the slice if its center is between points C and D. Since the cells are randomly distributed between points C and D, the proportion of cells whose centers are located inside the slice among all the cells presented in the slice is equal to AB / CD, that is, slice thickness / (slice thickness + average cell length).

[0030] Therefore, prepare additional sections in a direction perpendicular to the section to be counted, use a microscope micrometer to measure the cell length on the perpendicular section to obtain the average length L, perform a conventional cell count on the section to be counted to obtain the cell count K, measure the section thickness T, and calculate the actual cell number P according to the following formula:

[0031]

[0032] Where S is the actual number of cells in the slice, K is the number of cells observed in the slice, T is the slice thickness (μm), and L is the average cell length (μm).

[0033] The present invention is applicable to any homogeneous particles in the slices, including cells. If the particles cannot be seen clearly under a microscope, the particles need to be stained. The key to using this method is to chop the particles into pieces during the slicing process so that the same particle is distributed in two or even more slices.

[0034] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for counting cells in a slice, characterized in that: The following steps are involved: Prepare additional slices in a direction perpendicular to the slice to be counted, use a microscope micrometer to measure the cell length on the vertical slice to obtain the average length L, perform a conventional cell count on the slice to be counted to obtain the cell count K, measure the slice thickness T, and calculate the actual cell number S according to the formula.

2. The method for counting cells in a slice according to claim 1, characterized in that: This is accomplished by following these steps: a) Take the same tissues from two animals with the same characteristics and fix them with formaldehyde; cut the slice plane for counting cells on a plane corrector from one tissue and cut the slice plane for measuring cell length from the other tissue in a vertical direction, dehydrate, slice and stain them; b) Using a microscope micrometer ruler, measure the cell length on vertical slices to obtain the average length L; c) observing the slices under a microscope, determining the area where the cells to be counted are located, determining the magnification to ensure that the area where the cells to be counted are located is complete in each image, and acquiring the image by photography; d) Manually mark each stained cell in the area and manually count to obtain K; e) obtaining a slice thickness T according to the slice; f) Calculate the actual cell number S according to the formula to obtain the cell number of the tissue section.

3. The method for counting cells in a slice according to claim 2, characterized in that: The formula is: Where S is the actual number of cells in the slice, K is the number of cells observed in the slice, T is the slice thickness, and L is the average cell length.

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