Intrinsic anti-Stokes emission in living HeLa cells

Intrinsic fluorescence of biological material, also called auto-fluorescence, is a well-known phenomenon and has in recent years been used for imaging, diagnostics and cell viability studies. Here we show that in addition to commonly observed auto-fluorescence, intrinsic anti-Stokes emission can also be observed under 560 nm or 633 nm excitation. The anti-Stokes emission is shown to be spatially located on/in the mitochondria. The findings presented here show that sensitive imaging experiments e.g. single molecule experiments or two-photon excitation imaging can be compromised if intracellular anti-Stokes emission is not accounted for. On the other hand, we suggest that this anti-Stokes emission could be exploited as an additional modality for mitochondria visualization and cell viability investigation even in systems that are already labeled with commonly used fluorophores that rely on normal Stokes-based detection.