Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.
BACKGROUND
Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching.


METHODS
A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane.


RESULTS
In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field.


CONCLUSIONS
Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.
van Munster
E B
EB
Section Molecular Cytology, Swammerdam Institute for Life Sciences, Centre for Advanced Microscopy, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands. erikvanmunster@gmail.com
Goedhart
J
J
Kremers
G J
GJ
Manders
E M M
EM
Gadella
T W J
TW
D002462:Cell Membrane / Q000648:ultrastructure; D003600:Cytosol / Q000648:ultrastructure; D004867:Equipment Design; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D011506:Proteins / Q000032:analysis
D011506:Proteins