Lecture 1 - Techniques for studying cells Flashcards
What are the three parameters of microscopy?
Microscopy parameters
Magnification - The ratio of the objects image size to its actual size
Resolution - minimum distance of tow distinguishable points
Contrast - Differences in brightness or colour
How do light microscopes work and give the main methods of advanced light microscopy.
The light Microscope
Light passes through the specimen which can be living. The main disadvantage is its limited resolution. There are advanced versions such as fluorescent, phase/differential contrast and confocal microscopes.
* Fluorescent - Shows the locations of specific molecules using fluorescent dyes which bind directly to the structure of interest or molecules which associate with the molecule of interest. (E.g. immunolabelling)
* Confocal - Uses lasers for optical sectioning creates 3D image of living cells
* Phase contrast - Enhances contrast using light phase shifts induced by the specimen to generate phase contrast (refracted and un-refracted light) and differential interference contrast (two light beams)
Deconvolution microscopy - removes out of focus light which improves resolution
How are samples prepared for light microscopy?
Sample preparation for light microscopy
Can be done with either:
* Whole mounts - Can be mounted directly onto slides
* Tissue sections - Need to be sectioned before being examined
§ Fixation - Chemical fixatives used to prevent cell autolysis and to preserve the structure of the tissue E.g. Formaldehyde
§ Dehydration and clearing - removes water from the tissue. Involves being passed through a series of alcohols
§ Embedding - The specimen is infiltrated with molten wax
§ Sectioning - thin sections approximately 5 microns thick are cut on a microtome
§ Staining - The wax is removed and the tissues is stained
Stains for light microscopy include
* Haematoxylin (Acidic structures Purple) and Eosin (Basic structures Pink)
Periodic acid-Shiff reaction
Give examples of light microscopes that have broken the resolution limit.
- Structured illumination microscopy
- Simulated emission depletion microscopy
- Photo-activated localization microscopy
What are electron microscopes?
Electrons have a very short wavelength so the resolution is 1000s of times better than the light microscope and the resolution is currently around 0.08nm although the theoretical limit is much less.
Electrons are kept under a vacuum and focus by magnetic fields
Describe how TEM works?
Transmission electron microscopes - Electron beam
The TEM operates within a vacuum and has a 0.2 - 0.1nm resolution limit
1. Electron gun heated by tungsten filament which produces electrons by thermionic emission
2. Electron beam passes through the specimen
3. The image is focused and magnified by magnetic objective and projector lenses
4. The image is converted into a visible image by a fluorescent screen
How are samples prepared for TEM
- Whole mounts are possible for bacteria and viruses
- Tissue sections
Fixation- usually in glyceraldehyde followed by a second fixation step in Osmium Tetroxide
Dehydration - In an ethanol series
Embedding - embedded in plastic resins
Sectioning - 50nm thick sections are cut using an ultramicrotome
Staining - Heavy metal stains such as lead to improve contrast
Describe how SEM works
Scanning electron microscopes - Electrons scattered from sample surface
Electron beam is scanned across the specimen. Electrons are reflected form the surface of the specimen and collected by an electron detector and converted into an electronic signal which is displayed on a screen. Images have a 3D appearance.
How are samples prepared for SEM?
There is no whole mounts possible
Fixation - usually in glyceraldehyde followed by a second fixation step in Osmium Tetroxide
Dehydration - The water is replaced with ethanol
Critical Point Drying - Ethanol is removed from the sample in a way that minimises shrinkage
Coating specimens - Specimen is coated with a thin layer of gold to protect from electron beam damage.
What is Cryo TEM
Cryo-TEM now has equal resolution to X-ray Crystallography and this will only improve.
