Applications

Fluorescence Lifetime Imaging Diffuse Optical Tomography
Fast Sequential MeasurementsSingle Molecule Spectroscopy

Fluorescence Lifetime Imaging

• Lifetime Imaging of Local Environment Parameters

 

The lifetime is an indicator of the pH
Lifetime image of skin tissue stained with BCECF

For details, please see

[1] The bh TCSPC Handbook (click here)

• FLIM-FRET: Single-exponential Model
   

FRET results are obtained from a single image taken at the donor wavelength.
HEK cell expressing two interacting proteins labelled with CFP and YFP

For details, please see

[1] The bh TCSPC Handbook (click here)

[2] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

[3] W. Becker, A. Bergmann, M.A. Hink, K. K?ig, K. Benndorf, C. Biskup, Fluorescence lifetime imaging by time-correlated single photon counting, Micr. Res. Techn. 63, 58-66 (2004)

[4] C. Biskup, L. Kelbauskas, T. Zimmer, K. Benndorf, A. Bergmann, W. Becker, J.P. Ruppersberg, C. Stockklausner, N. Kl?ker, Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging, J. Biomed. Opt. 9, 735-759 (2004)

• FLIM-FRET by Double-exponential FLIM

 

Double-exponential analysis separates the effect of the variable fraction of interacting proteins and the effect of the distance
	left image: right image:
HEK cell expressing two interacting proteins labelled with CFP and YFP Most of the variation in the single-exponential t comes from a variable fraction of interacting molecules, not from a variation in the distance FRET distance has to be calculated from t fast

For details, please see

[1] The bh TCSPC Handbook (click here)

[2] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

[3] W. Becker, A. Bergmann, M.A. Hink, K. K?ig, K. Benndorf, C. Biskup, Fluorescence lifetime imaging by time-correlated single photon counting, Micr. Res. Techn. 63, 58-66 (2004)

[4] C. Biskup, L. Kelbauskas, T. Zimmer, K. Benndorf, A. Bergmann, W. Becker, J.P. Ruppersberg, C. Stockklausner, N. Kl?ker, Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging, J. Biomed. Opt. 9, 735-759 (2004)

• Autofluorescence of tissue - the FLIM approach

For details please see

[1] K. K?ig, I. Riemann, High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution, J. Biom. Opt. 8, 432-439 (2003)

[2] The bh TCSPC Handbook (click here)

[3] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

[4] Jenlab GmbH, Dermainspect System.

Fast sequential measurements

• Chlorophyll transients

 

Recording non-photochemical fluorescence quenching in a living plant.
A sequence of fluorescence decay curve is recorded immediately after the laser is switched-on.	Sequences of fluorescence decay curves measured after start of illumination.
Measurement of photochemical quenching transients.
	Measurement of photochemical quenching transients: Triggered and accumulated sequential recording in the continuous flow or scan sync out mode of a bh SPC module.
	Photochemical quenching transient measured in a dandelion leaf:   Curve plot (left) and colour-intensity-plot (right). The sequence starts at the front. SPC-630 module, time per curve 100 us, 10,000 on/off cycles were accumulated.

[1] The bh TCSPC Handbook (click here)

[2] W. Becker, A. Bergmann, G. Biscotti, Recording the Kautsky Effect by Fluorescence Lifetime Detection, Application note

[3] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

Diffuse Optical Tomography

• DOT Setup for Static Brain Imaging

	DOT Setup for Static Brain Imaging
	Pulsed illumination and time-resolved detection helps to separate the absorption and reduced scattering coefficients.

For details please see:

[1] The bh TCSPC Handbook  (click here)

[2] W. Becker, A.Bergmann, A. Gibson, N. Everdell, D. Jennions, M. Schweiger, S. R. Arridge, J. C. Hebden, Multi-dimensional time-correlated single photon counting applied to diffuse optical tomography, Proc. SPIE 5693 (2005)

[3] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005

• DOT Setup for Dynamic Brain Imaging

   

Pulsed illumination and time-resolved detection helps separate the the absorption and reduced scattering coefficients
	Part of a TOF sequence recorded in the Continuous Flow mode of an SPC-134. Acquisition time 100 ms per curve, ADC resolution 1024 channels.
Left: Count rate 4.5.106 s-1. Middle: Count rate 1.8.105 s-1. Right: Intra- and extra-cerebral changes of oxy- and deoxyhemoglobin concentrations during visual stimulation obtained from DTOFs measured at 3 wavelengths and four source-detector separations. The horizontal bars indicate the stimulation period. From Liebert et. al., Proc. SPIE 5138

• Simultaneous Recording of Fluorescence Decay and FCS
   

Molecules in Laser Focus
		HEK Cell expressing a GFP-MK2 fusion protein Problem: High concentration, hundreds of molecules in focus. Fluctuation amplitude is low, high accuracy of FCS required.
Intensity Fluctuations	Fluorescence Decay		Fluorescence Correlation
GFP in solution, 10-9 mol/l, SPC-830 TCSPC Module, Two-photon Excitation

[2] W. Becker, Advanced time-correlated single-photon counting techniques. Springer, Berlin, Heidelberg, New York, 2005