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A simple method allowing DIC imaging in conjunction with confocal microscopy
Author(s) -
CODY S. H.,
XIANG S. D.,
LAYTON M. J.,
HANDMAN E.,
LAM M. H. C.,
LAYTON J. E.,
NICE E. C.,
HEATH J. K.
Publication year - 2005
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2005.01452.x
Subject(s) - confocal , differential interference contrast microscopy , optics , confocal microscopy , bright field microscopy , contrast (vision) , microscope , materials science , microscopy , optical sectioning , computer science , detector , phase (matter) , optical microscope , phase contrast microscopy , artificial intelligence , computer vision , physics , scanning electron microscope , quantum mechanics
Summary Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark‐field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast‐enhanced images from archival data. The technique described here allows for the creation of contrast‐enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z ‐drive and a TLD, no hardware or optical modifications are required. The contrast‐enhanced images are calculated with software using the quantitative phase‐amplitude microscopy technique (Barone‐Nugent et al. , 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain‐free objective lenses.