Abstract
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.
Highlights
Cell auto-fluorescence due to emission from intrinsic proteins, collagen, porphyrins, lipofuscin or adenine dinucleotides has been intensively investigated in the last decades and opened up possibilities for numerous applications like tissue, organelle or cell viability imaging [1,2,3,4,5,6,7,8]
When imaging the anti-Stokes emission, where we excite the cell sample at 560 nm or 633 nm and monitor emission at shorter wavelengths, images with similar spatial distributions are obtained (Fig 1E and 1F). To confirm that both auto-fluorescence and anti-Stokes emission are originating from the mitochondria regions, HeLa cells were stained with MitoTracker-Green, a well-established commercial fluorophore used for mitochondria identification in cells
Changing the excitation wavelength for the MitoTrackerGreen stained HeLa cells to 560 nm (MitoTracker-Green does not absorb at this wavelength) yields again a good correlation between the auto-fluorescence and the anti-Stokes emission, proving that both signals originate from the same organelles
Summary
Cell auto-fluorescence due to emission from intrinsic proteins, collagen, porphyrins, lipofuscin or adenine dinucleotides has been intensively investigated in the last decades and opened up possibilities for numerous applications like tissue, organelle or cell viability imaging [1,2,3,4,5,6,7,8]. In comparison to extrinsic fluorophores that are often used to label various components of cells, the autofluorophores provide a direct relation between the emitter and its localization, while ensure that neither the morphology nor the biological function are altered in the cells due to the introduction of external molecules. This advantage is utilized for cell viability studies, where changes in the mitochondria auto-fluorescence morphology are used to monitor cell stress and viability [15,16,17].
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