Microscopes

Microscopes

1. TYPES

   • Light
     • Max Mag= x1500
     • Max Res: 200nm
       • light has wavelength of range 400- 750nm)
       • can only distinguish between 2 points of light beam can pass thru them
         • if 2 objs closer together than 1/2 wavelength of light (250nm)- cannot be distinguished
           • will appear as 1 object
     • Lenses
       • Four objective lenses
         • x4, x10, x40, x100 (oil immersion lens)
       • 1 eyepiece lens
         • x10
       • USING LIGHT MICROSCOPE
         • Light passes thru multiple lenses to produce image at eyepieces
           • light
             • condenser lens
               • thru specimen
                 • obj lens
                   • projector lens
                     • eyepiece lens
                       • eye
   • Electron
     • TEM
       • Electron beam PASSES through sample and give contrast
         • E's pass thru denser bits less easily- contrast
           • Final image: 2D
             • Max Mag: x500 000
     • SEM
       • Electron beam directed at sample
         • Electrons bounce off sample
           • Final image: 3D view of surface
             • Max Mag: x100 000
     • Max Res: 0.1nm

2. MAGNIFICATION AND RESOLUTION

   • MAG= no. of times image is larger than object
   • RES= ability to distinguish between 2 objects v.close together
   • MAG= image size/actual size

3. Advantages of light microscope

   • No skills/training required
   • Can view many (living) specimens
   • Easy to use
     • PREPARATION
       • STAINING
         • Coloured stains- chemicals that bind to chemicals in/on specimen
           • allows specimen to be seen
           • some stains bind to specific cell structures
             • Acetic orcein- DNA dark red
             • Gentian violet- bacterial cell walls
         • because a lot of biological material is not coloured- no detail
       • SECTIONING
         • Specimens embedded in wax
           • Thin sections cut w/out distorting structure
         • Useful for making sections of soft tissue (brain)
         • because material distorts when you cut it thinly
   • Cheap

4. Disadvantages of light microscope

   • Lower res
     • Cannot view cell's ultrastructure
   • Lower mag

5. ADVANTAGES OF ELECTRON MICROSCOPE

   • Higher res
     • Can view cell's ultrastructre
     • more detailed image

6. DISADVANTAGES OF E MICROSCOPE

   • EXPENSIVE
   • HARD TO USE- SKILLS/TRAINING
     • PREPARATION
       • 1) Fix Specimen in glutaraldehyde to make tissue firm
         • 2) dehydrate it- replace water w ethanol
           • 3) fix this into solid resin
             • 4) cut v.thinly with diamond knife
               • 5) stain using lead salts- scatter e's differently= contrast
                 • 6) mount it on copper grid
                   • place specimen in vacuum
       • "False colour" electron micrograph
         • sometimes colour added because electron micrograph produced is B&W
   • REQUIRES A VACUUM
     • SPECIMEN MUST ALWAYS BE DEAD
       • NOT ACCURATE REPRESENTATION OF LIVING SPECIMEN
   • tissue observed undergoes several stages of prep
     • each stage may alter real appearence
   • PREP may introduce artefacts
     • are structures not originally present/alter specimen from original condition

7. UNITS

   • 1mm= 1000 micrometers
   • 1 micrometer= 1000 nanometres (nm)
   • Measuring cells/organelles w light microscope
     • microscope eyepiece fitted with eyepiece graticule
       • transparent w ruler etched on
       • scale is arbitrary- represents diff lengths at diff mags
         • must be CALIBRATED for each diff obj lens
           • using a STAGE MICROMETER
             • a microscopic rule on a slide placed on stage
               • ruler is 1mm long and divided in 100 division
                 • each division is 0.01nm/10 um
                 • with x4 obj lens and x10 eyepiece- mag= x40
                   • 40 epu= 1000um --> 1epu=25 um
             • shows 'true' lengths
       • graticule remains constant at any mag

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