Microscopes

Microscopes

1. Compound Light microscope

   • Lower resolution - 0.2 um
   • Maximum magnification is 1500x
   • Monocular one eyepiece
   • Binocular two eyepiece lens
   • What limits the microscope is the wavelength of light
   • Image created on retina
   • Preparing a specimen
     • Always put on a glass slide
     • Light must be able to travel through the specimen so it needs to be very thin
     • Water can be used for temporary specimens - to last longer you have to use something other than water
     • A method of making something thin enough is squashing it
     • Another way is cutting it
     • Making a section slide
       • Fixation
         • Put in chemical called fixative which preserves it
       • Dehydration
         • Transfer specimen to dehydration chemical such as alcohol
       • Clearing
         • Put it in another chemical to drive out the alcohol
       • Embedding
         • Put the specimen in molten wax and let it solidify so you can cut it
       • Sectioning
         • Cut the specimen using a microtome
       • Staining
         • Coloured chemical which will bind to specific chemicals in the cell
       • Mounting
         • Put it in the slide, put clear liquid and coverslip
       • Fat Dudes Can Eat Subway So Much
     • Artifacts are parts seen down the microscope which were not there before

2. Magnification = image size / object size

3. Confocal laser scanning microscope

   • Called optical sectioning
   • Uses laser to focus at different levels at tissue
   • Uses fluorescent stains
   • Image is 3D
   • Clearer than light microscope

4. Calibrating light microscope

   • There is a different eyepiece with a scale that has 100 small measurements of epu
   • Stage micrometer - 1cm - 10mm - 10 000 um
   • To calibrate you need to find out how many small measurements of the stage micrometer are in 100 epu
   • Then work out the um it would equal
   • Then divide the um by 100 to get the size of 1 epu

5. Electron microscope

   • Transmission electron microscope (TEM)
     • Electrons pass through specimen
     • Works like X-ray
     • Electrons have bad penetration
     • Even air molecules will block electrons so a vacuum is needed
     • An electromagnetic lens is used to focus the beam of electrons
     • Specimen has to go through double door air lock to prevent a loss of vacuum
     • Image is called electron micrograph
     • Electron hits electron sensitive fluorescent sheet - under is another sheet that will create a permanent image
     • Has to be bolted to concrete base to prevent vibrations - also has to be on bottom floor of building
     • Magnification is from 500 000x to 2 000 000x
     • Resolution is 0.2-05nm
     • Specimen has to be dead
     • Preparing a section
       • First 3 steps same as light - Fixation Dehydration and Clearing - Fat Dudes Can
       • Embedding
         • Resin instead
       • Sectioning
         • Glass or diamond blade because they have to be nm thick
       • Staining
         • Heavy metal stains to block electrons - mercury lead silver
       • Mounting
         • Copper grid
     • More artifacts
   • Uses an electron beam made by electron gun instead of light
   • Scanning electron microscope (SEM)
     • Creates 3D image of surface of specimen
     • Not using sections
     • Beam of electrons scans the specimen
     • Needs to be coated in gold to make it more reflective
     • The specimen is surrounded in electron detectors which create an image on a computer
     • The specimen has to be frozen in liquid nitrogen and is carefully broken and will fracture through the weakest point in the cell - allowing you to look inside

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