Single molecule detection

 

In contrast to ensemble measurements that yield information about the averaged properties of the sample, single molecule studies yield information about individual molecular entities, bypassing the ensemble averaging effect. These molecular properties vary from molecule to molecule and with time for individual molecules. As a result, single molecule studies on a statistically relevant number of molecules yield distributions of parameters that in the limit of very large numbers of molecules approach ensemble data, but which contain a great deal more detailed information about subensembles and individual properties than ensemble measurements can hope to provide.

Schematic of our setup and typical single molecule emission image. The image is recorded by rasterscanning the sample. Periods during which the fluorophore does not emit (dark or off states) are the reason for the noncontinuous appearance of most of the emitters.

We have realized a custom-built setup for optical single molecule detection comprising fluorescence excitation wavelengths over the whole range of visible light plus different detection modalities. Two spectrally integrating detectors (APDs) allow for the recording of single photon arrival times for photon antibunching experiments and to construct fluorescence lifetime decay histograms. Further, the emission spectra of single emitters can be recorded by dispersing the light in a prism-based spectrometer and recording the spectrum by using a sensitive camera. Fluorescence emission spectra and lifetimes are characteristic for certain fluorophores and fluorophore states and are hence especially valuable parameters in the research of the photophysics of single fluorescent probes.

Intensity timetrace and decay characteristic from a single quantum dot.

We use single molecule spectroscopy to research the photophysical properties of molecules used for external labeling and of visible fluorescent proteins used for genetic labeling of cells.

Selected publications

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Schleifenbaum, F., C. Blum, V. Subramaniam, and A.J. Meixner, Single Molecule Spectral Dynamics at Room Temperature. Molecular Physics, 2009. 107(18): p. 1923-1942, doi:10.1080/00268970802635004.

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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.

PhD student: Niels Zijlstra, Martijn Stopel
Project leader: Christian Blum, Ine Segers-Nolten