Internal focus reference beads for imaging cytometry

1. A method for detecting the presence of a biological material in a sample, comprising:

staining a sample to be tested for the presence of the biological material with a dye capable of fluorescent emission at a detection wavelength selected from about 510 nm to about 600 nm upon excitation at an excitation wavelength selected from about 470 nm to about 550 nm;

mixing the sample to be tested with a pre-determined amount of micro-particles having a substantially uniform diameter selected from the range of about 3 μm to about 50 μm in diameters capable of fluorescent emission both at the detection wavelength and at a reference wavelength selected from about 350 nm to about 400 nm upon excitation at the excitation wavelength;

acquiring a fluorescent image of the sample at the reference wavelength;

acquiring a fluorescent image of the sample at the detection wavelength; and

determining the presence of the biological material in the sample, wherein the sample to be tested comprises less than about 10 8 cells/ml of the biological material.

2. The method of claim 1 , wherein the micro-particles are microspheres.

3. The method of claim 2 , wherein the micro-particles are polystyrene, or polymethylmethacrylate.

4. The method of claim 2 , wherein the microspheres have substantially uniform diameters.

5. The method of claim 1 , wherein the microspheres are about 5 μm to about 15 μm in diameters.

6. The method of claim 1 , wherein the dye is selected from the group consisting of: acridine orange, propidium iodide, ethidium bromide, and other nucleic acid stains.

7. The method of claim 6 , wherein the dye is propidium iodide.

8. The method of claim 1 , wherein the excitation wavelength is selected from about 480 to about 525 nm.