Ultrasensitive microscopy and spectroscopy of fluorescent markers


Fluorescence microscopy and spectroscopy is a primary research tool in many areas of research, especially in the life sciences. This, together with the trend to ever more complex and more quantitative analysis, makes the detailed understanding of the photophysics of the fluorescent probes used mandatory to discriminate between the properties of the probe and the processes illuminated by the probe.

We are especially interested in Visible Fluorescent Proteins (VFPs), which are fluorescent markers that allow for genetic labeling and have revolutionized cellular biology (Nobel Prize in Chemistry 2008).

General structure of VFPs. The light emitting chromophore is encapsulated within the protein barrel.

The photophysics of VFPs are exceptionally complex and cannot be understood from ensemble spectroscopic methods alone. We use ultrasensitive time and spectrally resolved single molecule spectroscopy to gain a deeper understanding of the emission properties of these unique fluorescent labels. We identify and characterize coexisting subensembles, research the influence of the chromophore nanoenvironment formed by the protein backbone on the exact emission properties, and analyze the energy transfer coupling of different chromophores within protein oligomers.

Monitoring Förster resonance energy transfer (FRET) efficiency of single FRET pairs

Monitoring the energy transfer between two fluorophores forming a FRET pair is an excellent tool to monitor distances between fluorophores on the nanometer scale, since the FRET efficiency is exquisitely sensitive to the distance between the fluorophores. By measuring the FRET efficiencies for labeled, freely diffusing proteins distance information can be gained that give insights into preferred conformations of e.g. intrinsically disordered proteins.

Subensemble identification in EGFP using single molecule emission spectra. Different forms can be identified and transitions between these forms can be followed.

Selected publications


Blum, C., A.J. Meixner, and V. Subramaniam, Dark proteins disturb multichromophore coupling in tetrameric fluorescent proteins. Journal of Biophotonics, 2010, doi:10.1002/jbio.201000075.


Blum, C. and V. Subramaniam, Single-molecule spectroscopy of fluorescent proteins. Analytical and Bioanalytical Chemistry, 2009. 393(2): p. 527-541, doi:10.1007/s00216-008-2425-x.


Blum, C., A.J. Meixner, and V. Subramaniam, Single oligomer spectra probe chromophore nanoenvironments of tetrameric fluorescent proteins. Journal Of The American Chemical Society, 2006. 128(26): p. 8664-8670, doi:10.1021/ja060726g

PhD student: Niels Zijlstra, Martijn Stopel
Project leader: Christian Blum


Student projects:

Master / Bachelor assignments:

Characterization of the switching of photochromic fluorescent proteins

By illuminating certain visible fluorescent proteins with light of defined wavelength, the optical properties of these proteins can be switched, which is the basis of some of the newly developed superresolution microscopy techniques. The exact dependence between the used switching wavelength and the switching efficiency will be determined, also the possible influence of e.g. the pH will be researched.

Monomeric alpha-synuclein structure assessed by FRET mapping

Alpha-synuclein is an intrinsically disordered protein that is supposed to contain some structure in the monomeric form dependent on sample conditions. To access this structure, the protein will be labeled with fluorophores that form a Förster resonance energy transfer (FRET) pair. By measuring the FRET efficiencies of single proteins, distance information between the labels will be obtained which will give indications for a preferred structure of the monomer under certain conditions.

Structural difference between alpha-synuclein in monomeric and oligomeric form

Alpha-synuclein labeled with a FRET fluorophore pair (see project above) will be used to prepare oligomers which are believed to be a key species in the development of Parkinsons disease. The FRET efficiency distributions in oligomers will be measured and will provide information on conformational differences between monomeric and oligomeric alpha-synuclein.

Contact: Christian Blum, ZH 157, c.blum@tnw.utwente.nl, +31 53 489 4622

Other team members involved in this research: Niels Zijlstra, Martijn Stopel, Ine Segers-Nolten, Vinod Subramaniam