Photon budget analysis for fluorescence lifetime imaging microscopy

Photon budget analysis for fluorescence lifetime imaging microscopy

Zhao, Q.
Young, I.T.
De Jong, J.G.S.

Applied Sciences

IST/Imaging Science and Technology

2011-08-17

We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon “budget.” These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

fluorescence microscopy
fluorescence lifetime imaging microscopy
photon efficiency
signal-to-noise ratio
light power

http://resolver.tudelft.nl/uuid:b06d651b-71f3-4a46-9ed7-a853389150af

https://doi.org/10.1117/1.3608997

Society of Photo-optical Instrumentation Engineers (SPIE)

1083-3668

https://doi.org/10.1117/1.3608997

Journal of Biomedical Optics, 16 (8), 2011

Institutional Repository

journal article

(c) 2011 The Author(s)
SPIE