Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells.
Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.
Koopman
M
M
Applied Optics group, Faculty of Science and Technology and MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands. m.koopman@utwente.nl
Cambi
A
A
de Bakker
B I
BI
Joosten
B
B
Figdor
C G
CG
van Hulst
N F
NF
Garcia-Parajo
M F
MF
D015815:Cell Adhesion Molecules / Q000032:analysis*; D002454:Cell Differentiation; D003713:Dendritic Cells / Q000166:cytology; D037181:Lectins, C-Type / Q000032:analysis*; D021962:Membrane Microdomains / Q000737:chemistry*; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D018613:Microscopy, Confocal / Q000295:instrumentation; D011956:Receptors, Cell Surface / Q000032:analysis*; D012680:Sensitivity and Specificity; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015815:Cell Adhesion Molecules; D037181:Lectins, C-Type; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin