Image analysis and measurement of biological samples

1. A method of identifying a cell in a sample containing a plurality of cells, said cell in said sample comprising a cell-surface antigen, the sample being contained within a single sample holder, the method comprising:

(a) Illuminating a sample chamber of said sample holder with light from a single illumination source, said sample chamber containing the sample comprising the plurality of cells,

Wherein the sample holder comprises an optically transmissive material, said optically transmissive material comprising an optically transmissive surface and a reflective surface;

wherein said single illumination source is configured to provide light that illuminates and passes through said optically transmissive surface of the sample holder effective to illuminate the sample by a direct light path to said sample chamber and by an indirect light path to the sample chamber;

wherein said optically transmissive surface and said reflective surface are shaped and positioned to simultaneously provide:

i) said direct light path from the single illumination source into the sample chamber without reflection at a surface of the sample chamber, effective that light travelling along the direct light path encounters the sample chamber and sample before encountering the reflective surface, and

ii) said indirect light path from the single illumination source to the sample chamber comprising a point of internal reflection at the reflective surface of the optically transmissive material of the sample holder, wherein said indirect light path passes through the optically transmissive surface and to the reflective surface and then to the sample chamber, wherein light traveling along the indirect light path is reflected at the reflective surface before encountering the sample chamber, and then, following said reflection, light traveling along the indirect light path encounters the sample chamber effective to illuminate the sample therein;

effective that light from the single illumination source simultaneously provides both epi-illumination and trans-illumination to the sample in the sample holder, where epi-illumination comprises light traveling along the direct light path from the single illumination source to the sample without reflection at the reflective surface of the sample holder, and where trans-illumination comprises light from the single illumination source traveling along the indirect light path within the optically transmissive material and to the sample comprising at least one reflection within the sample holder at the reflective surface of the optically transmissive material; and assaying for a plurality of attributes, said assaying comprising:

(b) assaying said cell of the plurality of cells within the sample holder for said cell surface antigen, wherein said assaying comprises assaying for the presence of the cell surface antigen or assaying for the amount of the cell surface antigen, wherein said assaying for the cell surface antigen comprises contacting the cell with an antibody directed against the cell surface antigen or an antigen binding fragment thereof;

(c) assaying the cell of (b) within the sample holder for a morphological attribute; and

(d) assaying the cell of (b) within the sample holder for scatter, wherein said assaying comprises quantitative cell light scatter,

wherein the combination of information from steps (b), (c), and (d) is used to identify the cell in the sample containing the plurality of cells.

2. The method of claim 1 , comprising assaying for two or more antigens on the cell, said antigens comprising a first antigen and a second antigen, wherein said assaying for two or more antigens comprises the use of a first type of antibody or antibody fragment that specifically binds to said first antigen, and a second type of antibody or antibody fragment that specifically binds to a said second antigen, wherein said first type of antibody or antibody fragment is different than the second type of antibody or antibody fragment.

3. The method of claim 1 , wherein assaying the cell of (a) for a morphological attribute comprises assaying the cell for one or more of nuclear size; nuclear shape; nuclear area; nuclear lobularity; cell size; cell shape; cell area; cell lobularity; mitochondrial size; mitochondrial shape; mitochondrial area; mitochondrial lobularity; ratio of nuclear area to total cell area; and ratio of nuclear volume to cell volume.

4. The method of claim 3 , wherein said assaying the cell of (c) for a morphological attribute comprises i) assaying the cell for cell size and ii) comprises computing a ratio of nuclear area to total cell area for said cell.

5. The method of claim 1 , wherein said sample holder is configured to receive light from the single illumination source to provide both trans-illumination and epi-illumination to the sample, wherein the single illumination source is positioned to illuminate substantially only one side of the sample holder.

6. The method of claim 5 , wherein total internal reflection is used to provide said trans-illumination to the sample.

7. The method of claim 6 , wherein said total internal reflection used to provide trans-illumination to the sample comprises total internal reflection within the sample holder.

8. The method of claim 1 , wherein said antibody or antigen binding fragment thereof comprises a fluorescently labeled antibody or fluorescently labeled antibody fragment.

9. The method of claim 1 , wherein assaying the cell of a plurality of cells comprises use of fluorescence microscopy, and assaying said cell for light scatter comprises dark field microscopy.

10. The method of claim 1 , wherein the sample remains separate from a detector in a fluid circuit fully confined in the sample holder, where the sample holder is movable relative to the detector.

11. The method of claim 1 , wherein a portion of the sample is held in a static, non-flowing manner during imaging.

12. The method of claim 1 , wherein a portion of the sample is held in a flowing manner during imaging.

13. The method of claim 1 , wherein a portion of the sample is held in a flowing manner during imaging, and a portion of the sample is held in a static, non-flowing manner during imaging.

14. The method of claim 1 , wherein said assaying the cell of (a) for a morphological attribute comprises determination of the ratio of nuclear area to total cell area of the cell.

15. The method of claim 1 , wherein said identifying the cell in the sample comprises use of fluorescence microscopy and of dark field microscopy.

16. The method of claim 1 , wherein said identifying the cell in the sample comprises use of a spectrophotometer.