Calibrating differential interference contrast microscopy with dual-focus fluorescence correlation spectroscopy.
We present a novel calibration technique for determining the shear distance of a Nomarski Differential Interference Contrast prism, which is used in Differential Interference Contrast microscopy as well as for the recently developed dual-focus fluorescence correlation spectroscopy. In both applications, an exact knowledge of the shear distance induced by the Nomarski prism is important for a quantitative data evaluation. In Differential Interference Contrast microscopy, the shear distance determines the spatial resolution of imaging, in dual-focus fluorescence correlation spectroscopy, it represents the extrinsic length scale for determining diffusion coefficients. The presented calibration technique is itself based on a combination of fluorescence correlation spectroscopy and dynamic light scattering. The method is easy to implement and allows for determining the shear distance with nanometer accuracy.
Institute of Physical Chemistry II, RWTH-Aachen University, Landoltweg 2, 52056 Aachen, Germany. mueller@pc.rwth-aachen.de
Claus B
CB
Müller
Kerstin
K
Weiss
Walter
W
Richtering
Anastasia
A
Loman
Jöerg
J
Enderlein
D000465:Algorithms; D002138:Calibration; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation* / Q000592:standards*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000592:standards*