Light microscopy – transmitted light

About this technique

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Transmission optical microscopy illuminates the sample from one side and images it from the other. Optimal illumination is vital for a good quality image. Some samples are naturally dark, or coloured, but most biological specimens will require staining. There is a huge range of stains available, many of them highly specific, though certain formulations such as haematoxylin and eosin, or Masson’s trichrome, are quite routine in histology. Typically samples will be fixed, embedded in paraffin wax or a water-miscible plastic resin, and sectioned. Paraffin sections are then washed with a solvent such as xylene to remove the wax before staining. Resin sections are stained without removal of the resin. A coverslip is affixed with a suitable mounting medium, which should match the refractive index of glass.

As well as detailed examination of stained sections under traditional light microscopes, stained histological sections or cells mounted on standard microscope slides (75mm x 25mm) can be examined by using a transmitted light slide scanner. A robotic system is used for automatic slide handling and scanning. High-resolution digital images are produced that can be examined and analysed using a standard computer and monitor. Over 100 slides can be scanned in any one session, using either a 10x, 20x or 40x objective depending on the system.  Some slide scanner systems also offer Fluorescence Microscopy. Calibrated digital images can be zoomed and captured as TIFF files for presentation or analysis. Additionally, numerous image analysis algorithms are available for handling tissue micro-arrays and for batch analysis of cellular number and/or cellular staining intensity. Higher resolution oil-immersion (100x oil objective) systems and four-colour fluorescence systems are available but these are not robotically controlled.

When staining is not an option, optical contrast techniques provide a method of observing detail in samples such as living cells. Phase contrast introduces contrast based on refractive index, while differential interference contrast (DIC) gives contrast based on the local rate of change in refractive index, which gives an artificial relief appearance to the image. Typically phase contrast is better for thin samples such as cell monolayers or bacteria, while DIC is better for thicker samples such as embryos or protozoa. Living cells will need to be in an aqueous medium and for high resolution it is therefore necessary to use water immersion lenses, which are designed for the refractive index of water.

If you sample is opaque, you can visualise it using reflected light.