M.Sc. Biochemie - Modul Biophysical Chemistry - Spectroscopic/Biophysical Methods

The course gives a short introduction into techniques for measuring fluorescence lifetimes by using techniques established in the biomolecular photonics group.

For the course we will have the following schedule:

Schedule

Day	Topic
Feb 2017	Lecture about techniques for fluorescence lifetime measurements
March2018	Practical course - Determination of fluorescence lifetimes by time correlated single photon counting (TCSPC)
March2018	Discussion of the results
30.03.2018	Deadline for the submission of the protocols

Copies of the slides used in the lectures and manuals for the devices used in the practical course can be found here. Students interested in this practical course will receive login and password upon request.

Recommended literature

Below you will find some review articles, which will give you an introduction into fluorescence microscopy and techniques for measuring fluorescence lifetimes.

For legal reasons we are not allowed to provide you with the pdfs of the articles but only with the links to the respective publications. However, inside the compueter network of the Friedrich-Schiller-University Jena all publications are accessible.

Introduction into fluorescence microscopy

J. Lichtman, J.A. Conchello:
Fluorescence microscopy.
Nat. Methods 2, 910-919 (2005)

Review articles about techniques for labelling proteins

Introduction into labelling techniques:

B.N.G. Giepmans, S.R. Adams, M.H. Ellisman, R.Y.Tsien:
The fluorescent toolbox for accessing protein location and function.
Science 312, 217-224 (2006).

Overview over GFP variants:

N.C. Shaner, P.A. Steinbach, R.Y. Tsien:
A guide to choosing fluorescent proteins.
Nat. Meth. 2, 905-909 (2005).

On the discovery of GFP:

O. Shimomura:
A short story of aequorin
Biol. Bull. 189, 1-5 (1995).

O. Shimomura:Discovery of green fluorescent protein (GFP) (Nobel Lecture).
Angew. Chem. Int. Ed. Engl. 48, 5590-5602 (2009).

R.Y. Tsien:
Nobel lecture: constructing and exploiting the fluorescent protein paintbox.
Integr. Biol. 2, 77-93 (2010).

Please, have also a look on the website of the Nobel Prize committee

Review articles on fluorescence lifetime measurements

K. Suhling, P.W.M. French, D. Phillips:
Time resolved fluorescence microscopy.
Photochem. Photobiol. Sci. 4, 13-22 (2005).

K. Suhling, L.M. Hirvonen, J.A. Levitt, P.H. Chung, C. Tregidgo, A. Le Marois, D.A. Rusakov, K. Zheng, S. Ameer-Beg, S. Poland, S. Coelho, R. Henderson, N. Krstajic:
Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments.
Medical Photonics 27, 3-40 (2015).

C. Biskup et. al.:
Multi-dimensional fluorescence lifetime and FRET measurements.
Microsc Res Tech. 70, 442-451 (2007).

Techniques for measuring fluorescence lifetimes

C. Biskup, T. Zimmer, K. Benndorf:
FRET between cardiac Na channel subunits measured with a confocal microscope and a streak camera.
Nature Biotechnology 22, 220 - 224 (2004).

C. Biskup et. al.:
Multi-dimensional fluorescence lifetime and FRET measurements.
Microsc Res Tech. 70, 442-451 (2007).

W. Becker
The bh TCSPC Handbook. 8th ed. , Berlin 2019.

Validation of fluorescence lifetime measurements

Standards for fluorescence lifetime measurements:

N. Boens, W. Qin, N. Basarić, J. Hofkens, M. Ameloot, J. Pouget, J.P. Lefèvre, B. Valeur, E. Gratton, M. vandeVen, N.D. Silva Jr, Y. Engelborghs, K. Willaert, A. Sillen, G. Rumbles, D. Phillips, A.J. Visser, A. van Hoek, J.R. Lakowicz, H. Malak, I. Gryczynski, A.G. Szabo, D.T. Krajcarski, N. Tamai N, A. Miura:
Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.
Anal. Chem. 79, 2137-2149 (2007).

Fluorescence lifetime measurements of Coumarin 6:

U.S. Raikar, C.G. Renuka, Y.F Nadaf, B.G. Mulimani,
A.M. Karguppikar, M.K. Soudagar:
Solvent effects on the absorption and fluorescence spectra of coumarins 6 and 7 molecules: Determination of ground and excited state dipole moment.
Spectrochim Acta A 65, 673-677 (2006).

Further recources

Microscopy websites

Microscope manufacturers (in alphabetical order)

Tutorials

Dye / Indicator websites

Dye databases / fluorescence spectra viewer