Monitoring of apoptosis in intact single cells with photothermal microscope

BACKGROUND: The methods for detection of apoptosis, cannot as a rule be applied to intact single cells and do not allow monitoring of the cell population. The photothermal (PT) method was evaluated for detection and monitoring of apoptosis in single intact cells--lymphocytes and blasts. METHODS: PT microscopy is based on optical registration of cell response to the thermal impact that is induced in a cell due to absorption of a short laser pulse (532 nm, 10 ns) by cellular hem-proteins. With no pretreatment of cells, optical detection of cellular response with this method may allow monitoring of apoptosis. The PT method was applied for in vitro studies of lymphocytes and K562 blast cells during drug-induced apoptosis. Dexamethasone in 10 microM concentration was used and cell properties were monitored for 6 h. RESULTS: PT parameters of cells deviated from control after incubation with Dexamethasone. Control measurements with fluorescent microscopy (using the Annexin-V-FLUOS kit) verified the development of apoptosis in drug-treated cells, while there was no development of necrosis. Also necrotic cells had PT properties similar to those of control cells. The PT method allowed the detection of the influence of Dexamethasone at earlier stages (2-hr incubation) than the fluorescent technique (4 h). CONCLUSIONS: Results obtained confirmed the applicability of PT microscopy for detection and monitoring of apoptosis in single intact cells. The sensitivity of the suggested method may exceed the sensitivity of fluorescent methods.