Microscopy

• How many nanometres in a micrometer


  1000
• How many micrometers in a millimeter


  1000
• What power should you begin to view a sample with


  Low power- so that the whole specimen is visible, and the depth of focus is greater. You are then able to focus in on the specimen with a higher power lens
• How to create a temporary slide

  First collect a specimen that is one cell thick. Place the specimen on a slide, using tweezers. Add water or a stain to the specimen, you may wish to use a fluorescent stain or a normal stain, like iodine. Lower a cover slip ontop of the specimen, slowly and carefully to avoid air bubbles. If there is any excellent water, you may place the entire slide in a paper towel
• Calibrating an eyepiece graticule using a stage micrometer

  An eyepiece graticule is a measurement on the eyepiece of the microscope. To calibrate, first you must place the stage micrometre on the stage of the microscope. Then line up the eyepiece graticule with the scale on the micrometre. Once they are lined up you can identify scale, (For every x stage micrometre divisions there are y eyepiece graticule divisions). Repeat for different magnification lenses.
• Magnification


  Remember IAM to calculate magnification, image size, and actual size. Ensure all of the numbers are in the same units, either millimeters, micrometers or nanometers. Each is 1000x the next.
• Finding the diameter of a field of veiw

  The field of view is everything that is visible under the microscope. Multiply the image size of the FOV in low power by the magnification of the low power, then divide that by either high or medium power, to obtain the desired high or medium power FOV diameter
• Electron Vs light microscopes


  Light microscopes use a beam of light to view specimens

  Light microscopes have a resolution of 200 nm

  Light microscopes can view live specimens

  Light microscopes can produce coloured images

  Electron microscopes use an electron wavelength (shorter than light)

  Electron microscopes have a resolution of 1 nm

  Electron microscopes kill the specimens

  Electron microscopes can only produce black and white images

  Electron microscopes have higher magnifications than light microscopes
• Fluorescent stains


  Are used with fluorescent microscopes that have light sources such as lasers or LEDS that emit a single wavelength. The fluorescent stains absorb the light from the microscope and them re-emit it at a longer wavelength, which produces a very bright image. The stain bonds to some chemicals, and not other, therefore producing a bright image in which specific chemicals are clearly visible
• Immunofluorescence


  Fluorescent markers are linked to specific antibodies that bind to specific antigens (chemicals). These antibodies are part of a fluorescent stain which is placed on the specimen. A multicoloured, bright image is then produced, in which specific antigens may be located. This method is how proteins in the cell membrane were discovered
• Freeze fracture electron microscopy


  A sample is plunged into liquid propane and frozen. A steel blade fractures the sample at its weakest point, and the surface is vaporised to enhance the surface texture, known as etching. A carbon or platinum vapour is fired onto the surface at around 35°, to create a coating. This coating is removed from the sample and observed using an electron microscope. It looks as if it is 3D due to shadowing. This process gives a unique image of the middle of the two layers of phospholipid in the cell membrane. This method led to the development of the fluid mosaic model of the cell membrane.
• Cryo-EM


  A thin layer of pure protein solution is applied to a grid and then flash frozen using liquid ethane, to prevent the formation of water crystals. The frozen grid is observed under an electron microscope with detectors that record the patterns of electrons emitted by individual protein molecules. Computerised algorithms use these patterns to create virtual 3D images of the protein molecules. Cryo em allows the observation of proteins at the specific point that they were flash frozen, which is especially useful for proteins that change their form. The resolution of cryo em is extremely high, each atom is visible. Cryo em allowed scientists to develop a COVID- 19 vaccine