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Fluorescence Lifetime Imaging


FLIM is based on the measurement of the average time fluorescent molecules spent in the excited states. Observations of the changes in the fluorescence lifetime parameters give an insight into a range of dynamic processes, such as conformation,interaction with solvent and other molecules in the system, concentration of ions in the surrounding media, and changes in refractive index, viscosity, and pH of the environment, etc
Some time different fluorophore has spectrally overlapping emission. In that case detection of fluorophore become very difficult.But every fluorophore has different life time in their exited state. FLIM technique uses the difference in life time to detect the fluorophores. Also FLIM method is the basis of FRET ( Fröster resonance energy transfer) technique. 

Based on the techniques used to acquire FLIM data, there are three major methods:
  1. Time domain: In this method, a short pulse of light excites the sample, and the subsequent fluorescence emission is recorded as a function of time by using time correlated single photon counting devices or gated cameras. This usually occurs on the nanosecond timescale. TCSPC based on the repetitive precisely timed registration of single photons of e.g. a fluorescence signal . The reference for the timing is the corresponding excitation pulse. As a single photon sensitive detector a photomultiplier tube(PMT), micro channel plate (MCP) or a single photon avalanche diode (SPAD) can be used. Provided that the probability of registering more than one photon per cycle is low, the histogram of photon arrivals per time bin represents the time decay one would have obtained from a “single shot” time-resolved analog recording.

  2. Frequency domain: In this method, the sample is excited by a modulated source of light. The fluorescence emitted by the sample has a similar waveform, but is modulated and phase-shifted from the excitation curve. Both modulation (M) and phase-shift (φ) are determined by the lifetime of the sample emission; that lifetime can be calculated from the observed modulation and phase-shift.

  3. Pump probe: Alternatively, the pump-probe method extracts lifetime from the stimulated emission and is not limited by the NA of optics, and thus the working distance.
In our laboratory we are interested in the time domain and pump probe-method.

Time Correlated Single Photon Counting (TCSPC): A Time-domain Technique for Fluorescence Lifetime Measurement 

TCSPC mechanism: the concept of a very fast stop watch

TCSPC systems are naturally compatible with confocal microscope using pulsed lasers

Typical experimental setup for TCSPC based FLIM
NADH fluorescence lifetime dynamics at the impact of apoptosis inducer staurosporine (STS) (1mM) on HeLa cells.


Figure illustrates the excitation-detection technique: pulse delay (left) and reconstruction of fluorescence lifetime (right)
Experimental setup for long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay

Fluorescent lifetime image of 
ATTO 647 N mixed in gel solution injected into Y-shape microchannel (left), 
Observed by TCSPC system with accuracy ~1 ns (right) [color scale Red: Strong Blue: Weak]


FLIM FRET can measure the distance between molecular. It use donor and acceptor. When one of donor excitation spectra of acceptor emission spectra overlap, the two molecules very close to the situation, donor excited state by the energy released from a fall will turn excited acceptor, because of this energy conversion is actually a "dipole-dipole interaction " We use the characteristic and we can use optical methods, the amount photometry following scale resolution, but also very accurate, this is really a very wonderful thing. FRET has also been effectively applied to the phenomenon of single biological molecules.

As the detection, control, and understand the operating mechanism of a single biological molecules can protein engineering, organic and inorganic production of new nanomaterials and applications such as early disease detection has great value.

related publications (since 2010, for full list of publications click here )

  1. Gitanjal Deka, Kazunori Okano, Hiroshi Masuhara, Yaw-Kuen Li and Fu-Jen KaoMetabolic variation of HeLa cells migrating on microfabricated cytophilic channels studied by the fluorescence lifetime of NADH, RSC Adv., 2014,4, 44100-44104 (
  2. Jianhong Ge, Cuifang Kuang, Shin-Shian Lee, and Fu-Jen Kao, Fluorescence lifetime imaging with pulsed diode laser enabled stimulated emission,Optics Express, Vol. 20, Iss. 27, pp. 28216–28221 (2012). Also selected for the Virtual Journal for Biomedical Optics (VJBO), Editor Andrew Dunn and Anthony Durkin, Vol. 8, Iss. 1, February 4, 2013.
  3. Gitanjal Deka, Wei-Wen Wu, Fu-Jen Kao, In vivo Wound Healing Diagnosis with Second Harmonic and Fluorescence Lifetime Imaging, J. Biomed. -Opt. 18(6), 061222, 2013.
  4. Nirmal Mazumder, Rodney K. Lyn, Ragunath Singaravelu, Andrew Ridsdale, Douglas J. Moffatt, Chih-Wei Hu, Han-Ruei Tsai, John McLauchlan, Albert Stolow, Fu-Jen Kao, John Paul Pezacki, Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein, PLoS One 8(6), e66738, 2013.
  5. Po-Yen Lin, Yi-Cheng Lin, Chia-Seng Chang, Fu-Jen KaoFluorescence Lifetime Imaging Microscopy with Subdiffraction-Limited Resolution, Japanese Journal of Applied Physics 52(2), 028004-3, 2013.
  6. Thilo Dellwig, Po-Yen Lin and Fu-Jen Kao, Long-distance Fluorescence Lifetime Imaging Using Stimulated Emission, J. Biomed. Opt. 17, 011009 (2012).
  7. Po-Yen Lin, Shin-Shian Lee, Chia-Seng Chang, and Fu-Jen Kao, Long working distance fluorescence lifetime imaging with stimulated emission and electronic time delay, Optics Express, Vol. 20, Issue 10, pp. 11445-11450 (2012) Also selected for the Virtual Journal for Biomedical Optics (VJBO), Editor Andrew Dunn and Anthony Durkin, Vol. 7, Iss. 7, June 25, 2012.
  8. Tatyana Yu. Buryakina, Pin-Tzu Su, Wan-Jr Syu, C. Allen Chang, Hsiu-Fang Fan, Fu-Jen Kao*, Metabolism of HeLa Cells Revealed through Autofluorescence Lifetime upon Infection with enterohemorrhagic Escherichia coli, J. Biomed. Opt., in press (2012).
  9. Aaron D. Slepkov, Andrew Ridsdale, a Huei-Ning Wan, b Ming-Hao Wang, et al. "Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy" Journal of Biomedical Optics 16(2), 021103 (February 2011)
  10. Po-Yen Lin, Hong-Chou Lyu, Chin-Ying Stephen Hsu, Chia-Seng Chang, and Fu-Jen KaoImaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy, Biomedical Optics Express 2, pp. 149-158 (2011).
  11. Ghukasyan Vladimir, Hsu Chin-Chun, Liu Chia-Rung, F. J. Kao et al. “Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine,” Journal of Biomedical Optics, Vol- 15, 016008, 2010.