Chapter 2, Problem 13RQ

Summary Introduction

To review:

The basis of dark-field, phase-contrast, and fluorescence microscopy and their applications.

Introduction:

The evolution and development of different types of microscopy originate from the simple single-lens microscope. The necessity to observe key intracellular and extracellular structures in greater detail led to the development of various kinds of microscopes. While each of them differs in terms of their use and application, the basic principles of light refraction and scattering are the same in each design.

A spider light stop is used inside the condenser lens of the dark field microscope. This spider light stop is used to detect the light that is scattered by an object. This enables the observance of objects as halos of bright light against the dark background. This technique is widely used in detecting special bacterial features like flagella and thus, bacterial motility.

The phase-contrast microscope utilises the difference in the refractive index between the inner part of cytoplasm and the surrounding medium or organelles. An annular ring near the light source is used allowing the detection of light refracted from the specimen and the outer cone of transmitted light. The eukaryotic organisms that possess numerous organelles can be effectively detected by this technique of microscopy.

Fluorescence microscopy utilises coloured fluorophore dyes that bind to the targeted specimen. They absorb and emit light at different wavelengths that are detected on a computerised screen and the output is controlled by the colour of the wavelength filter used. It is used to track events like deoxyribose nucleic acid (DNA) replication in bacteria and detection of antigen with the help of labelled antibodies.