Re-evaluation of differential phase contrast (DPC) in a scanning laser microscope using a split detector as an alternative to differential interference contrast (DIC) optics.
In this paper, differential phase imaging (DPC) with transmitted light is implemented by adding a suitable detection system to a standard commercially available scanning confocal microscope. DPC, a long-established method in scanning optical microscopy, depends on detecting the intensity difference between opposite halves or quadrants of a split photodiode detector placed in an aperture plane. Here, DPC is compared with scanned differential interference contrast (DIC) using a variety of biological specimens and objective lenses of high numerical aperture. While DPC and DIC images are generally similar, DPC seems to have a greater depth of field. DPC has several advantages over DIC. These include low cost (no polarizing or strain-free optics are required), absence of a double scanning spot, electronically variable direction of shading and the ability to image specimens in plastic dishes where birefringence prevents the use of DIC. DPC is also here found to need 20 times less laser power at the specimen than DIC.
Amos
W B
WB
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. ba@mrc-lmb.cam.ac.uk
Reichelt
S
S
Cattermole
D M
DM
Laufer
J
J
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D017173:Caenorhabditis elegans / Q000648:ultrastructure; D016798:Ciliophora / Q000145:classification / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000648:ultrastructure; D004867:Equipment Design; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal / Q000295:instrumentation*; D008857:Microscopy, Interference / Q000295:instrumentation*; D008858:Microscopy, Phase-Contrast / Q000295:instrumentation / Q000379:methods*; D055095:Optics and Photonics