Systems and methods for counting cells and biomolecules
The invention generally relates to analytical and monitoring systems useful for analyzing and measuring cells and biological samples. More particularly, the invention relates to systems and methods for imaging, measuring, counting, analyzing, and monitoring microscopic particles such as cells and biological molecules in solution samples.
1. A system for imaging microscopic objects, comprising:
a covered sample chamber having a fixed depth ranging from about 1 μm to about 200 μm, the covered sample chamber being configured to hold about 1 μL to about 1,000 μL of a liquid suspension of microscopic objects to be imaged in a liquid sample, wherein the covered sample chamber comprising an optically clear window allowing exposure of the liquid sample;
at least one fluorescent light source capable of providing a fluorescent excitation light beam to the liquid sample through the window;
a bright-field light source capable of providing a bright-field light beam to the liquid sample; and
at least one light detection device for detecting a light signal from the liquid sample thereby forming at least one image of the microscopic objects,
wherein the fluorescent excitation light beam is at an oblique angle to the bright-field light beam and is oblique to the window's plane of surface, and
the fluorescent excitation light beam comprises a wavelength ranging from about 300 nm to about 10,000 nm.
2. The system of claim 1 , wherein the system comprises at least two fluorescent light sources capable of simultaneously providing two excitation light beams having the same or different wavelengths to the sample.
3. The system of claim 2 , wherein the system comprises four fluorescent light sources capable of simultaneously providing four excitation light beams having the same or different wavelengths to the sample.
4. The system of claim 2 , wherein the at least two fluorescent excitation light beams have different incident angles in relation to the window's plane of surface.
5. The system of claim 2 , having multiple fluorescent excitation light beams having the same incident angles in relation to the window's plane of surface.
6. The system of claim 2 , having multiple fluorescent excitation light beams having different incident angles in relation to the window's plane of surface.
7. The system of claim 1 , wherein the fluorescent excitation light beam is at an incident angle of about 10° to about 80° in relation to the window's plane of surface.
8. The system of claim 1 , wherein the fluorescent excitation light beam is at an incident angle of about 45° in relation to the window's plane of surface.
9. The system of claim 1 , wherein the microscopic objects are selected from microbeads, bacteria, algae, fungi, mammalian cells, insect cells, plant cells, proteins, DNA molecules, and surface markers.
10. The system of claim 9 , wherein the microscopic objects are selected from the group consisting of protein surface markers, weak fluorescence labels, and DNA molecules.
11. The system of claim 1 , wherein the microscopic objects comprise biomolecules.
12. The system of claim 1 , wherein the fixed depth ranges from about 10 μm to about 100 μm.
13. A system for counting cells or biomolecules, comprising:
a covered sample chamber configured to hold a liquid suspension of cells or biomolecules in a liquid sample, wherein the sample chamber comprising an optically clear window allowing exposure of the liquid sample;
two or more fluorescent light sources, each being capable of independently providing a fluorescent excitation light beam to the liquid sample through the window, wherein each fluorescent excitation light beam is at an incident angle other than normal to the window's plane of surface;
a bright-field light source capable of providing a bright-field light beam to the liquid sample;
at least one light detection device for detecting a light signal from the liquid sample; and
a shutter for controlling passage of the bright-field light beam to the liquid sample.
14. A method for detecting a biomolecule in a liquid biological sample comprising:
acquiring at least one static bright-field image of the biological sample by directing a bright-field light beam to the sample;
acquiring at least one static fluorescent image of the biological sample by directing at least two excitation light beams to the sample; and
comparing the at least one bright-field image to the at least one fluorescent image to detect the biomolecule in the biological sample,
wherein each of at least two the excitation light beams is at an oblique angle to the bright-field light beam.
15. A method for determining a concentration or number count of a type of cells in a population of cells in a liquid sample, comprising:
contacting a liquid sample comprising a suspension of cells with a fluorescently labeled agent that specifically binds the certain type of cells in the liquid sample;
loading the sample into a covered chamber having a known height, wherein the population of cells is suspended within the chamber;
acquiring at least one static bright-field image of the population of cells in the liquid sample;
acquiring at least one static fluorescent image of the population of cells in the liquid sample; and
comparing cell count from the bright-field image to cell count from the fluorescent image to determine the concentration or number count of the certain type of cells in the population of cells,
wherein the excitation light beam is at an oblique angle to the bright-field light beam.