Multi-photon lifetime imaging (FLIM)

Numerous intracellular molecules respond to a light stimulus with the emission of light of a distinct wavelength termed autofluorescence. Prominent examples of these include the abundant metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavins in their reduced states. NADH is the key electron transporter that feeds the electron transport chain of the mitochondria for ATP production. Fluorescence is generally characterized by a wavelength as well as an intensity. It is also hallmarked by a a temporal duration termed its lifetime which is sensitively dependent on the environment of each fluorophore. Differences in pH, viscosity, temperature, protein interactions and other chemical or physical characteristics of the surrounding medium affect the decay of a fluorescence. This phenomenon explains the existence of different lifetime fractions of NAD(P)H in living cells. Short lifetimes represent free NAD(P)H, associated with glycolysis, while longer and more variable lifetimes are characteristic for protein-bound NADH in complexes of the electron transport chain and others, associated with oxidative phosphorylation. The ratios and distinct lifetimes of these two fractions are used to define the metabolic state of biological samples. Yet, the biophysical basis of these fluorescence lifetime patterns is not yet understood.


FLIM of the bedbug ectospermalege (aquisition and analysis with SPCM/SPCI software)

Other strongly autofluorescent molecules include structural components such as melanin, chitin and dityrosine. These represent key architectural components of insects such as the cuticle. Structural aspects may therefore be approached by FLIM without the need of invasive staining techniques. The lifetime information of autofluorescent molecules improves the distinction between different molecules with similar excitation and emission characteristics. The example image displays the external part of the spermalege, the copulation organ of female bedbugs. It contains a distinct external structure strongly excitable with UV light, hypothetically rich in resilin [1, 2].


1] Michels, J.; Gorb, S. N. & Reinhardt, K.: Reduction of female copulatory damage by resilin represents evidence for tolerance in sexual conflict. Journal of The Royal Society Interface 12 (2015), p. 20141107–20141107.

2] Reinhardt, K.; Breunig, H. G. & Koenig, K.: Autofluorescence lifetime variation in the cuticle of the bedbug Cimex lectularius. Arthropod Structure & Development 46 (2017), p. 56–62.