Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics.
Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.
Esposito
Alessandro
A
European Neuroscience Institute, Göttingen, Cell Biophysics Group, Germany. aesposito@quantative-microscopy.org
Gerritsen
Hans C
HC
Oggier
Thierry
T
Lustenberger
Felix
F
Wouters
Fred S
FS
D001690:Biological Science Disciplines / Q000295:instrumentation; D003952:Diagnostic Imaging / Q000295:instrumentation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation* / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods; D008403:Mass Screening / Q000295:instrumentation; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation*; D018511:Systems Integration; D014159:Transducers