Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains.
We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 106 91, Stockholm, Sweden.
Jonas
J
Mücksch
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 106 91, Stockholm, Sweden.
Thiemo
T
Spielmann
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 106 91, Stockholm, Sweden.
Evangelos
E
Sisamakis
Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, Albanova University Center, 106 91, Stockholm, Sweden. jerker@biomolphysics.kth.se.
Jerker
J
Widengren
D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes
fluorescence; live cell imaging; redox state; single plane illumination microscopy; transient state imaging; triplet state