An iBiology lecture about birefringence in calcite crystals from none other than the late Dr. Shinya Inoué (RIP, Professor). Prof. Inoué was a pioneer in the field of Polarized Light Microscopy and considered by many to be the co-inventor of Video Microscopy. His book, titled Video Microscopy (1986: Plenum Press, 1997: Springer) is as relevant today as it was when first published. For a wonderful tribute to a scientist whose contributions changed the way in which we image live cells, please visit the Marine Biological Laboratory website.
This iBiology lecture by Prof. Edward Salmon, University of North Carolina at Chapel Hill covers four important transmitted light imaging techniques that are useful in imaging live cells: Dark Field, Phase Contrast, Polarization and Differential Interference Contrast (DIC).
Transmitted Light imaging modalities:
Darkfield imaging provides high sensitivity, since the black background provides high contrast to see scattered light from small (~25nm) objects and is excellent for low magnification outlines of individual cells such as sperm and flagella.
Phase Contrast Microscopy overcomes the lower resolution of Darkfield microscopy, which occurs because Darkfield microscopy requires that the NAcondenser > NAobjective.
Polarized Light Microscopy is a quantitative method for measuring birefringent retardation, which enables the quantification of the anisotropic structural detail in a sample.
Differential Interference Contrast leverages the fact that a polarization microscope converts birefringence retardation of two orthogonally vibrating waves into contrast. DIC overcomes one of the issues in Phase Contrast Microscopy which produces “haloes” at the edges of features in the sample. These haloes accumulate through the depth of the sample and are a particularly noticeable effect with thicker specimens. This limits the effectiveness of Phase Contrast Microscopy in optical sectioning. DIC highlights edges but is less sensitive to the mass of objects.
Camera selection for Darkfield, Phase Contrast, Polarization and DIC applications:
In general, these transmitted light imaging modalities are shot noise limited. It is not particularly useful to use sCMOS or other low read-noise cameras, which tend to be more expensive. Exposures are usually short, therefore there is unlikely to be any discernable benefit from using TE-cooled cameras.
A CMOS camera with good Quantum Efficiency and a reasonable dynamic range and an imager size that is well matched to the optical format of the microscope will typically produce good quality images. In live cell experiments, it is important to use a camera with a frame rate that is matched to the dynamics of the experiment. For example, if the experiment involves tracking faster movement (e.g. movement of flagella or cilia), then a camera with Global Shutter and a higher frame rate would be desirable (see table below).
CMOS cameras may be considered as good, cost-effective choices for transmitted light applications. There is a wide range of FOV and framerates from which to choose a cost-effective camera that meets the requirements of the application. The Cameras and Objectives table may be used to estimate the FOV and the limiting resolution that can be achieved with a particular camera on a microscope with a specific objective.
