Fluorophores are widely used in biology as labels because they can be measured with a high signal-to-background ratio. Most commonly, these fluorophores are excited with a steady-state bandwidth-limited excitation source. The fluorophores are excited by the excitation light and emit light at a lower energy. The weaker emission light is then measured by filtering out the excitation light with spectral interference filters. If a short pulse of excitation light is used instead, fluorophores are again excited. Each excited fluorophore has a constant probability of decaying back to the base state emitting a photon, so as the population of excited fluorophores decreases the fluorescence emission decreases with a characteristic exponential decay. Different fluorophores have different decay probabilities; so different fluorophores have different rates of exponential decay.
Fluorescence lifetime measurements have been used primarily with special fluorophores that change their fluorescence lifetime in response to their local environment. These fluorophores serve as molecular sensors able to travel within cells and report back important parameters such as pH or calcium ion concentration. We have pioneered two new applications for fluorescence lifetime imaging: fluorescence lifetime multiplexing (FLuM) and fluorescence lifetime activatable probes (FLAP). In FLuM , multiple imaging channels are measured independently within a single spectral channel using fluorescence lifetime separation. Not only does FLuM increase the number of imaging channels, but spectrally identical channels may be used to remove the effects of probe delivery and tissue absorption in whole-animal imaging. We have demonstrated that FLAP can be used to engineering fluorescence lifetime probes that are sensitive to specific enzymes.
We have designed two first-generation instruments for measuring fluorescence lifetime and are working on our second generation. First, we designed a time-domain fluorimeter for measuring the time-domain fluorescence of a sample placed in a cuvette. The circuits and performance of this instrument were presented in Transactions on Biomedical Circuits and Systems. Then we built a Small Animal Lifetime Imager (SALI) for simultaneously measuring the fluorescence lifetime in an entire image. The SALI was presented in Sensors and Actuators B: Chemical .
H. Wang, Y. Qi, T. J. Mountziariz, and C. D. Salthouse, "A portable time-domain LED fluorimeter for nanosecond Fluorescence lifetime measurements", Review of Scientific Instruments, vol. 85, no. 5, p. 055003, May 2014
Li, Shuo, and Christopher Salthouse, "Digital-to-time converter for fluorescence lifetime imaging." Instrumentation and Measurement Technology Conference (I2MTC), 2013 IEEE International. IEEE, 2013
C. Salthouse, R. Weissleder, and U. Mahmood "Development of a Time Domain Fluorimeter (TDF) for Fluorescent Lifetime Multiplexing Analysis" IEEE TBCAS, vol. 2, iss. 3, pp. 204-211, Sept. 2008
C. Salthouse, F. Reynolds, J. Tam, L. Josephson, and U. Mahmood, "Quantitative Measurement of Protease-Activity with Correction of Probe Delivery and Tissue Absorption Effects" Sensors and Actuators B: Chemical, vol. 138, no. 2, pp. 591-597, May 2009.