Cell viability and DNA denaturation are measured through two-photon fluorescence excitation using a single diode laser beam as the trapping and exciting source simultaneously. Two-photon fluorescence emission spectra are presented for CHO cells and T lymphocytes loaded with various fluorescent probes. This single beam method is demonstrated to be a safe tool to monitor the viability of optically trapped cells, even under intense 809 nm diode laser illumination (∼106 W/cm2). The dynamics of cellular necrosis is monitored by adding ethanol to the cell suspension during trapping. Thermal damage to heat-treated samples is assessed by recording shifts in the emission spectra from trapped cells loaded with the nucleic acid probe, acridine orange. © 1999 Society of Photo-Optical Instrumentation Engineers.