Microscopy
- Early compound microscope credited to Z. jansen around 1590.
- Later improved by Anton Van Leeuwenhoek in mid 17th century.
- Electron microscopy developed in 1930s
Resolution
The ability to discern two distinct points in space.
Factor influencing resolution
Particle wavelength (gamma)
Refractive index of the medium (n)
Angular aperture.
Characteristics of Light Microscopy
- Can magnify images up to about 1400x
- Uses visible light for illumination
- Resolution down to ~200nm (w/ oil)
- Objects ~500 nm can be studied (bacteria, mitochondria)
Advantages of light microscopy
- Inexpensive
- easy preparation
- Can use living cells
Disadvantages of light microscopy
- Lower resolution
- difficult to see translucent cells
Microscopy methodology (light microscopy)
- Sometimes, tissues are fixed to prevent cell degradation.
- Tissues are embedded to allow slicing
- Sections are cut using a microtone
Types of Light Microscopy
- Bright-field microscopy - normal white light is passed through a specimen, light is absorbed (or transmitted) and the image is based on light.
- Phase-contrast microscopy - refracts light differently as it travels at different rates through materials of different composition
- Differential Interference contrast (DIC) - polarized light passes through material differently due to different index of refraction.
Fluorescence
fluorochromes are molecules that are excited by light at one wavelength and then emit light at a difference wavelength.
(special dyes attached to abs can identify individual molecules within living cells).
Fluorescence microscopy
excites a chemical at one wavelength that fluoresces back at a separate, longer wavelength.
Green fluorescent protein (GFP) labeling
- A common fluorochrome
- Gene for fluorescent protein can be inserted into a cell’s genome.
Indirect immunocytochemistry
use of secondary abs to generate immunofluorescence.
Confocal Microscopy
- Light from a laser is passed through a confocal pinhole onto the specimen
- Fluorescence from the specimen is passed to the detector through a second pinhole.
- The pinhole blocks out the blurred light emitted from areas that are different plane focus.
(Blurring from light out of the plane of focus reduces resolution.
If the light from the layers are not in the plane of focus is removed)
Electron Microscopy
- Uses electron beams rather than light beams to inimunate the specimen.
- electron wavelength is very small leading to a higher resolving power (0.1 -2.0 nm)
Two types of Electron Microscopy commonly used:
- Transmission electron microscopy (TEM): resolution 0.1 - 2.0 nm
- Scanning electron microscopy (SEM): resolution 3 - 10nm.
TEM (traditional)
- Sliced specimen is stained with electron-dense material (heavy metals).
- Electrons projected through a vacuum to strike the specimen.
- Electrons striking the stained material are scattered and not collected.
- Electrons that are transmitted through
SEM
- Electrons strike the surface and are scattered based on surface angles.
- or absorbed energy causes the release or a secondary electron.
- electrons scatter is measure by detector to create surface image.
- SEM has less resolution than TEM
- TEM has to be sliced very thin
- Negative of electron microscopy
