A combined confocal/NSOM microscope for molecular biology
Bärbel de Bakker - Former member|
"The key to every biological problem must finally be sought in the cell." This sentence was written in 1925 by cell biologist E.B. Wilson to stress the importance of cellular biology. From the 17th century the structure of cells has being revealed bit by bit thanks to the improvement of the resolving power of optical instruments. Nowadays it is possible to detect individual fluorescent molecules at room temperature. This has opened new research areas in fundamental physics, chemistry and biology.
Especially in molecular biology the localization of individual proteins and observation of molecular interactions at the cell membrane are becoming new challenging fields of study. In order to study individual biomolecules in the living cell, we are developing a combined confocal/near-field scanning optical (NSOM) microscope that is capable to image under physiological conditions with single molecule sensitivity.
With this non-invasive setup it will be possible to inspect a biological sample, first with a large field of view using bright field illumination, next to zoom in on an interesting part of the sample by scanning confocal imaging and finally perform a high resolution study by NSOM. The fluorescence arising from the sample is collected with a high NA objective. The light is discriminated with respect to the wavelength and/or polarization and detected with ultrasensitive photodiodes to obtain single molecule sensitivity.
Since the excitation volume for NSOM is typically three orders of magnitude smaller than for confocal microscopy, the background signal from the cell's autofluorescence is significantly reduced. This reduction allows the detection of individual molecules at the cell membrane.
This project is carried out in collaboration with the Tumor Immunology Laboratory at the University of Nijmegen and financed by The Technology Foundation STW.
The following articles have been published regarding this project:
Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells(abstract) (full pdf)
Bärbel I. de Bakker, Andrea Bodnár, Erik M. H. P. van Dijk, György Vámosi, Sándor Damjanovich, Thomas A. Waldmann, Niek F. van Hulst, Attila Jenei, and María F. Garcia-Parajo
Journal of Cell Science
vol 121 issue 5 p627-633 feb 20 2008
Nanoscale Organization of the Pathogen Receptor DC-SIGN Mapped by Single-Molecule High-Resolution Fluorescence Microscopy(abstract) (full pdf)
Bärbel I. de Bakker, Frank de Lange, Alessandra Cambi, Jeroen P. Korterik, Erik M. H. P. van Dijk, Niek F. van Hulst, Carl G. Figdor, and Maria F. Garcia-Parajo
vol 8 issue 10 p1473-1480 July 16, 2007
Near-Field Fluorescence Microscopy An Optical Nanotool to Study Protein Organization at the Cell Membrane(abstract) (full pdf)
María F. García-Parajó, Bärbel I. de Bakker, Marjolein Koopman, Alessandra Cambi, Frank de Lange, Carl G. Figdor, and Niek F. van Hulst
Volume 1 Number 1 ISSN: 1551-1286 (Print) ISSN: 1551-1294 (Online) july 2005
Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells(abstract) (full pdf)
Alessandra Cambi, Frank de Lange, Noortje M. van Maarseveen, Monique Nijhuis, Ben Joosten, Erik M.H.P. van Dijk, Bärbel I. de Bakker, Jack A.M. Fransen, Petra H.M. Bovee-Geurts, Frank N. van Leeuwen, Niek F. Van Hulst and Carl G. Figdor
The Journal of Cell Biology
Volume 164, Number 1, 145-155, 5 January 2004
Cell biology beyond the diffraction limit: near-field scanning optical microscopy(abstract) (full pdf)
de Lange F, Cambi A, Huijbens R, de Bakker B, Rensen W, Garcia-Parajo M, van Hulst N, Figdor CG
JOURNAL OF CELL SCIENCE
vol 114 issue 23: p4153-p4160 DEC 2001
Single molecule studies of living colors and transmembrane proteins on cells.
Garcia-Parajo MF, de Bakker BI, Koopman M, Figdor C, van Hulst NF
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
vol 221: p379-PHYS part 2 APR 1 2001
Study of individual transmembrane proteins using a combined confocal/near field optical microscope.(full pdf)
de Bakker BI, Garcia-Parajo MF, Rensen WHJ, van Hulst NF, de Lange F, Cambi A, Figdor CG
vol 80 issue 1: p655.13 part 2 JAN 2001