Multi-Wavelength FLIM


Multi-Wavelength FLIM

The principle of TCSPC FLIM can be extended to simultaneously detect in 16 wavelength channels. The optical spectrum of the fluorescence light is spread over an array of 16 detector channels. The TCSPC system determines the detection times, the channel numbers in the detector array, and the position, x, and y, of the laser spot for the individual photons. These pieces of information are used to build up a photon distribution over the time of the photons in the fluorescence decay, the wavelength, and the coordinates of the image. The principle of multi-wavelength FLIM is shown in the figure below.

Principle of multi-wavelength FLIM

As for single-wavelength FLIM, the result of the recording process is an array of pixels. However, the pixels now contain several decay curves for different wavelength. Each decay curve contains a large number of time channels; the time channels contain photon numbers for consecutive times after the excitation pulse. The result can also be interpreted as a number of FLIM images for different wavelength, as shown in the image below.

Multi-wavelength FLIM of a Convallaria sample. Images for 16 consecutive wavelength intervals, each 512 x 512 pixels, 256 time channels. Detected with MW-FLIM GaAsP detector, Analysis by SPCImage, Intensity-weighted lifetime of double-exponential decay model.


References for Multi-Wavelength FLIM

  1. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Klöcker, K. Benndorf, Multi-wavelength TCSPC lifetime imaging, Proc. SPIE 4620 79-84 (2002)
  2. Becker, A. Bergmann, C. Biskup, D. Schweitzer, M. Hammer, Time- and Wavelength-Resolved Autofluorescence Detection by Multi-Dimensional TCSPC, Proc. SPIE 5862, 58620S (2005)
  3. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005
  4. Becker, A. Bergmann, C. Biskup, Multi-Spectral Fluorescence Lifetime Imaging by TCSPC. Micr. Res. Tech. 70, 403-409 (2007)
  5. Becker, Fluorescence Lifetime Imaging – Techniques and Applications. J. Microsc. 247 (2) (2012)
  6. Becker, Fluorescence Lifetime Imaging Techniques: Time-correlated single-photon counting. In: L. Marcu. P.M.W. French, D.S. Elson, (eds.), Fluorecence lifetime spectroscopy and imaging. Principles and applications in biomedical diagnostics. CRC Press, Taylor & Francis Group, Boca Raton, London, New York (2015)
  7. Becker, Fluorescence lifetime imaging by multi-dimensional time correlated single photon counting. Medical Photonics 27, 41-61 (2015)
  8. Becker, Introduction to Multi-Dimensional TCSPC. In W. Becker (ed.) Advanced time-correlated single photon counting applications. Springer, Berlin, Heidelberg, New York (2015)
  9. Cheng, D. Chorvat, N. Poirier, J. Miro, N. Dahdah, A. Chorvatova, Spectrally and time-resolved study of NAD(P)H autofluorescence in cardiac myocytes from human biopsies. Proc. SPIE 6771, 677104-1 to -13 (2007)
  10. Chorvat, A. Chorvatova, Multi-wavelength fluorescence lifetime spectroscopy: a new approach to the study of endogenous fluorescence in living cells and tissues. Laser Phys. Lett. 6 175-193 (2009)
  11. Chorvatova, A. Mateasik, D. Chorvat Jr, Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells. Laser Phys. Lett. 10 125703-1 to -10 (2013)
  12. Dimitrow, I. Riemann, A. Ehlers, M. J. Koehler, J. Norgauer, P. Elsner, K. König, M. Kaatz, Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Experimental Dermatology 18, 509-515 (2009)
  13. Gerega, N. Zolek, T. Soltysinski, D. Milej, P. Sawosz, B. Toczylowska, A. Liebert, Wavelength-resolved measurements of fluorescence lifetime of indocyanine green. J. Biomed. Opt. 16(6) 067010-1 to -9 (2011)
  14. Gerega, D. Milej, W. Weigl, M. Botwicz, N. Zolek, M. Kacprzak, W. Wierzejski, B. Toczylowska, E. Mayzner-Zawadzka, R. Maniewski, A. Liebert, Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult’s brain. J. Biomed. Opt. 17(8), 087001-1 to -9 (2012)
  15. Rück, F. Dolp, C. Hülshoff, C. Hauser, C. Scalfi-Happ, FLIM and SLIM for molecular imaging in PDT, Proc. SPIE 5700 (2005)
  16. Rück, F. Dolp, C. Hülshoff, C. Hauser, C. Scalfi-Happ, Fluorescence lifetime imaging in PDT. An overview. Medical Laser Application 20, 125-129 (2005)
  17. Rück, Ch. Hülshoff, I. Kinzler, W. Becker, R. Steiner, SLIM: A New Method for Molecular Imaging. Micr. Res. Tech. 70, 403-409 (2007)
  18. Rück, C. Hauser, S. Mosch, S. Kalinina, Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells. J. Biomed. Opt. 19(9), 096005-1 to -9 (2014)


Multispectral TCSPC Detectors

FLIM System for Nikon A1 / C1 / C2

FLIM Systems for Zeiss LSM 710 / 780 / 880 / 980

FLIM System for Olympus FV1000

FLIM System for Leica SP2 / SP5 / SP8

DCS-120 Confocal FLIM System

DCS-120 MP Multiphoton FLIM System


BDS-SM Series ps Diode Lasers

SPCM Data Acquisition Software

TCSPC Package

SPCImage NG Data Analysis Software

SPC-160 TCSPC Series

FLIM System for Nikon A1+

SPC-150N TCSPC Series

SPC-180N Series

DCS-120 MP Multiphoton FLIM System

FLIM Systems for Zeiss LSM 710 / 780 / 880 / 980

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