| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 4335002 | Journal of Neuroscience Methods | 2013 | 6 Pages |
Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH2) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism.Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs).Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode).LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence.
► HBO lamp of a fluorescence microscope was replaced by a combination of two LEDs. ► NAD(P)H fluorescence bleaching in cell cultures was less with LED excitation. ► LED allowed for fast-bleaching and recovery of NAD(P)H fluorescence. ► Signal to noise ratio of a photodiode, PMT and MPPC were compared. ► Combination of LED and PMT detection was suitable NAD(P)H and FAD monitoring.
