Functional imaging of single cells with photothermal microscopy

Photothermal technique for light microscopy is reported as the new tool for quantitative analysis of single living cell structural and functional properties. Probe laser illumination with phase contrast method allows to visualize thermal field being induced in cell due to absorption of pump laser radiation. In our set up the cell is pumped by the pulsed laser at 532 nm, 10 ns, 0.01-0.4 ml. The source of probe beam is a pulsed dye laser ( 630 nm, 10 nJ, 10 ns) which forms cell image. Fully automated image cytometer with acquisition rate 1 cell/s is described. An acquired cell image is considered as spatially (0.7 um) and temporally (10 ns) resolved cell response to non-specific load being induced with a pump laser. Thus we introduce photothermal image cytometry which may be applied for investigation of mechanisms of photodamage, single cell dosimetry, cell functions and structure and thermal phenomena at cell level. The advantages of photothermal cytometry are: - study of a single living cell in its natural state without pre-staining; - monitoring of living cell population parameters during action of some other factors; - distribution of natural chromophores inside single cell can be measured. In experiments in vitro with living human leukocytes we have found that the changes of cell functional features membrane stabilization or damage, lipids peroxidation, ion balance alternation, reaction to the drugs - cause the changes of cell PT-images. For the different biological factors we have found specific PT-image parameters.