Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe.

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.