eTraining Introduction

Specimen Preparation

Leica Ultracut UCT

Histology

JEOL JSM-6400 SEM

Hitachi S-4700 FE-SEM

Hitachi FB-2000A FIB

Microanalysis

Veeco Dim 3000 AFM

Fluorescence Microscopes

Support

Detection and Visualization

Contrast

As light waves pass through a transparent object, the light speed changes. The light wave suffers a phase change upon entering a material having a different index of refraction to that of its medium (surrounding material). The detection and visualization of these phase changes are what compose the phenomenon of “contrast” in microscopy. The phase shifts contain many details about the specimen, but the human eye does not benefit from this information. A transparent phase-plate serves to increase the phase shift by half a wavelength. The amplified destructive interference pattern produces a specimen-to-background relative brightness which may be visible to the human eye.

Staining with colored dyes is also used to help make cells visible under a microscope.

Contrast can be enhanced by increasing the difference between the refractive index of the specimen and the refractive index of the surrounding substrate material. A compound’s refractive index is determined by its physical and chemical properties, such as the shape, the internal arrangement of light-scattering elements, and the thickness. Based on this definition of contrast, it is often very difficult to obtain satisfactory contrast with unstained or living specimens.

Differential Interference Contrast (DIC)

In DIC, the beam of transmitted light is split into two separate beams by a Wollaston prism on the DIC objective lens. Upon interaction with the sample, the two beams interfere with each other based on the differences between phases in the sample. The resultant image appears to give a three dimensional restructuring of the sample. Keep in mind, the image is formed by changes in phase, not necessarily topography. Sometimes, however, differing phases correspond to changes in topography. DIC is very useful for imaging living specimens.

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