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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

 

Two-photon excited autofluorescence of human skin

upper row: 5 ?m, stratum corneum Lower row: 50 ?m, stratum spinosum

 

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.

 

For details please see:

[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

 

[1] The bh TCSPC Handbook (click here)

[2] A. Liebert, H. Wabnitz, D. Grosenick, M. M?ler, R.Macdonald, H. Rinneberg, Evaluation of optical properties of highly scattering media by moments of distributions of times of flight of photons, Appl. Opt. 42, 5785-5792 (2003

[3] A. Liebert, H. Wabnitz, J. Steinbrink, H. Obrig, M. M?ler, R. Macdonald, A. Villringer, H. Rinneberg, Time-resolved multidistance near-infrared spectroscopy at the human head: Intra- and extracerebral absorption changes from moments of distribution of times of flight of photons, Appl. Opt. 43, 3037-3047 (2004)

[4] A. Liebert, H. Wabnitz, J. Steinbrink, M. M?ler, R. Macdonald, H. Rinneberg, A. Villringer, H. Obrig, Bed-side assessment of cerebral perfusion in stroke patients based on optical monitoring of a dye bolus by time-resolved diffuse reflectance, NeuroImage 24, 426-435 (2005)

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

 


 

 

Single Molecule Spectroscopy

 

 

  • 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

 

For details please see:

 

[1] The bh TCSPC Handbook (click here)

[2] W. Becker, A. Bergmann, E. Haustein, Z. Petrasek, P. Schwille, C. Biskup, T. Anhut, I. Riemann, K. Koenig, Fluorescence lifetime images and correlation spectra obtained by multi-dimensional TCSPC, Proc. SPIE 5700, 144-152 (2005)

[3] S. Felekyan, R. K?nemuth, V. Kudryavtsev, C. Sandhagen, W. Becker, C.A.M. Seidel, Full correlation from picoseconds to seconds by time-resolved and time-correlated single photon detection, Rev. Sci. Instrum. 76, 083104 (2005)

[4] M. Prummer, B. Sick, A. Renn, U.P. Wild, Multiparameter microscopy and spectroscopy for single-molecule analysis, Anal. Chem. 76, 1633-1640 (2004)

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

   

 

 


 

 

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