Investigations on light-induced stress in fluorescence microscopy using nuclear localization of the transcription factor Msn2p as a reporter.
We utilized the nuclear localization of a stress-sensitive transcription factor, Msn2p, to study light-induced stress caused by time-lapse fluorescence imaging of green fluorescent protein (GFP) in budding yeast Saccharomyces cerevisiae. A range of exposure times, light intensities and intervals between exposures were tested in order to provide guidelines for noninvasive imaging. We found that the cellular response, revealed as an enhanced nuclear shuttling of Msn2p-GFP, is induced at significantly lower light exposures than those causing observable changes in cell morphology or cell growth. However, no stress induction was observed if the accumulated photon energy per area unit used to obtain an image was maintained at 0.16 J cm(-2) or below. Above this 'safe' level, the stress response is determined by both the intensity and the exposure time. In particular, for a given accumulated photon energy per area unit, a high intensity applied during a short exposure causes more stress than vice versa. Interestingly, no correlation was found between the degree of stress and the absolute fluorescence signal, indicating that light-induced cellular stress in the studied system is not specifically related to GFP excitation.
Logg
Katarina
K
Department of Applied Physics, Chalmers University of Technology, Göteborg, Sweden. katarina.logg@chalmers.se
Bodvard
Kristofer
K
Blomberg
Anders
A
Käll
Mikael
M
D019677:Artificial Gene Fusion; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D021381:Protein Transport / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000528:radiation effects*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013312:Stress, Physiological; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C081935:MSN2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins