In vivo fluorescence lifetimes of chlorophyll-a (chl-a) and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from the green microalgae Haematococcus pluvialis under normal and nutrient-stressed conditions (green stage and red stage, respectively), using two-photon excitation provided by a laser generating pulses in the femtosecond range, and a Leica microscope setup. Analysis of the fluorescence lifetime decay curve revealed two separate lifetime components in all our measurements. A short-lifetime component for chl-a of ?250 ps was completely dominant, contributing more than 90% of overall intensity in both green-stage and red-stage cells. Green-stage cells inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) displayed a significant chl-a lifetime increase for the short component. However, this was not the case for red-stage cells, in which DCMU inhibition did not significantly affect the lifetime. For green-stage cells, we found a short NADPH (free) lifetime component at ?150 ps to be completely dominating, but for red-stage cells, a longer component (protein bound) at ?3 ns contributed as much as 35% of the total intensity. We hypothesize that the long lifetime component of NADPH is connected to photoprotection in the cells and coupled to production of astaxanthin. DCMU does not seem to affect the fluorescence lifetimes of NADPH.
Arne S. Kristoffersen, Øyvind Svensen, Nicolausi Ssebiyonga, Svein R. Erga, Jakob J. Stamnes, and Øyvind Frette, "Chlorophyll a and NADPH Fluorescence Lifetimes in the Microalgae Haematococcus pluvialis (Chlorophyceae) under Normal and Astaxanthin-Accumulating Conditions," Appl. Spectrosc. 66, 1216-1225 (2012)