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.
Müller
Claus B
CB
Institute of Physical Chemistry II, RWTH-Aachen University, Landoltweg 2, 52056 Aachen, Germany. mueller@pc.rwth-aachen.de
Weiss
Kerstin
K
Richtering
Walter
W
Loman
Anastasia
A
Enderlein
Jöerg
J
D000465:Algorithms; D002138:Calibration; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation* / Q000592:standards*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000592:standards*