BIO 3153: Lecture 2 - Microscopy Microscopy There are several types of microscopy that divide into two main divisions: ●Light microscope ●Electron microscope Focusing on light microscopy, there are four sub-types of microscopes: ●Conventional (ex: bright field and phase contrast) ●Fluorescence ●Confocal ●Two-photon The typical vision field of a light microscope ranges from a little bigger than 1mm, which is the equivalent to a frog egg, to a little smaller than 100 nm which is the size of a virus ribosome. Furthermore, the conventional resolution limits to above 100 nm, while the limit of super resolution is 30 nm. The scale with all the limits is in the diagram below:
Light Microscopy The light microscope utilises a basic light path. It can be used for live or fixed cells and tissue. Tissues must use an upright microscope. Isolated cells must use an inverted microscope There are four types of light microscope: ●Bright field (A) ●Phase contrast (B) ●Differential interference (C) ●Dark field (D) Bright field uses transmitted light which gives low contrast imaging of cells. Phase contrast converts phase differences into changes of brightness using wave properties, giving an extremely contrasted imaging of cells. In an unstained cell, a phase shift will occur as light travels through the cells. Phase alignment will increase brightness, while an increase in misalignment will increase darkness.
Differential interference contrast uses similar principles of phase contrast, however its polarised light is separated and recombined to give definition. Dark field uses a lateral light source to show scattered light only. Fluorescence Microscopy In fluorescence microscopy, there are a variety of fluorescent molecules used: ●DAPI ●GFP ●FITC ●Cy3 ●Cy5 The opticals used for this microscopy are similar to light microscopy. High energy lamps of mercury are used to provide a bright light source. Unwanted wavelengths are filtered using a first and second barrier filter. Chromophores are excited at specific wavelengths, which are atoms or groups whose presence is responsible for the colour of a compound.