Image analysis and measurement of biological samples
Methods, devices, systems, and apparatuses are provided for the image analysis of measurement of biological samples. Specifically, methods are provided for detecting and measuring, in a sample, cell morphology; measurement of cell numbers; detection of particles; measurement of particle numbers; and other properties and quantities of or in a sample. Some embodiments may use a sample holder comprising a sample chamber configured to hold said sample, at least a portion of said sample holder comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface.
1. A method of identifying a cell in a sample containing a plurality of cells, comprising:
preparing said cells for analysis by using a first stain which binds to nucleic acids and from this approximate amount of nucleic acid in the sample, determining an approximate number of cells in the sample;
preparing said cells by using a second stain, wherein an amount of said second stain added to the sample is calculated using the approximate number of cells determined by use of the first stain, in order that a desired ratio of second stain per cell be obtained;
(a) assaying the plurality of cells for at least one of: (i) the presence of a cell surface antigen; (ii) the amount of a cell surface antigen; or (iii) cell size;
(b) assaying the plurality of cells of (a) for at least one of: (i) nuclear size; or (ii) nuclear shape; and
(c) assaying the plurality of cells of (a) and (b) for quantitative cell light scatter, wherein the combination of optical information from steps (a), (b), and (c) is used to identify the cell in the sample containing a plurality of cells, wherein said plurality of cells is held within a sample holder, wherein said sample is held in a non-flowing manner in said sample holder during said assaying of steps (a) and (b), wherein
said sample holder comprises a cuvette comprising a sample chamber configured to hold a sample, at least a portion of said cuvette comprising an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface, wherein said optically transmissive surface and said reflective surface are configured effective that light passing through the optically transmissive surface simultaneously provides both epi-illumination and trans-illumination to said sample in the sample chamber, where epi-illumination comprises light traveling from said illumination source to the sample without reflection at a surface of the optically transmissive material, and where trans-illumination comprises light traveling within the optically transmissive material and to the sample following at least one reflection from at least one surface of said optically transmissive material
wherein at least one side of the cuvette is coated with an optically absorbent material and wherein light enters the cuvette from a side opposite the side that is coated.
2. The method of claim 1 , wherein said first sample holder comprises an elongated channel.
3. The method of claim 1 , wherein said sample holder further comprises one or more optically non-transmissive surfaces.
4. The method of claim 1 , wherein said trans-illumination in said sample holder is provided at least in part by partial internal reflection of light at a surface.
5. The method of claim 1 , wherein said trans-illumination in said sample holder is provided at least in part by total internal reflection of light at a surface.
6. The method of claim 1 , wherein the sample holder comprises two or more sample chambers for holding a sample.
7. The method of claim 1 , wherein said cuvette of said sample holder comprises a cross-sectional shape selected from a rectangular horizontal cross-sectional shape and a circular horizontal cross-sectional shape.
8. The method of claim 1 , wherein said cuvette of said sample holder comprises a cross-sectional shape selected from a saw tooth vertical cross-sectional shape and a step-shaped vertical cross-sectional shape.
9. The method of claim 1 , wherein said at least one concave or convex structure of second sample holder has a cross-sectional shape selected from rectangular, triangular, circular, and semi-circular.
10. The method of claim 1 , wherein said sample holder comprises at least one concave or convex structure to provide a pathway for internally reflected light within the sample holder.
11. The method of claim 1 , wherein said sample holder comprises at least one concave or convex structure that comprises a surface, and wherein said surface is configured to reflect light within the cuvette.
12. The method of claim 1 , wherein said cuvette of said sample holder comprises a cross-sectional shape selected from a rectangular horizontal cross-sectional shape, a circular horizontal cross-sectional shape, a saw tooth vertical cross-sectional shape and a step-shaped vertical cross-sectional shape.
13. The method of claim 1 , wherein said sample holder comprises at least one concave or convex structure that comprises a cross-sectional shape selected from rectangular, triangular, circular, and semi-circular.