Parkinson's disease (PD) is currently the second most common
neurodegenerative disorder in the world. Major features of cell
pathology of the disease include the presence of cytoplasmic
inclusions called Lewy bodies, which are composed of aggregated
proteins. The presence of Lewy's body is associated with more
advanced stages of the disease when considering irreversible
changes. Precise identification of the disease stage at a cellular
level presents the critical tool in developing early diagnostics
and/or prevention of PD. The aim of our work is to introduce
sensitive microscopic analysis in living cells, focused on initial
intracellular changes and thus capable to detect earlier stages of
the disease.
We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chl a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves. and O. Holub ... [et al.].