Microscopy

• Electron Microscope


  • Organelles (e.g. mitochondria, ER, and membranes) seen in detail
  • Transmission:
  • Transmits electron beam through thin specimen, on screen/film
  • Magnification = x1,000,000
  • Resolution limit = 0.5nm
  • Scanning:
  • Scans electron beam + collects scattered by surface; 3D images
  • Magnification = 10x-500,000x
  • Resolution limit = 3-10nm
  • Benefits: Large field depth, high resolution + magnification
  • Flaws: Vacuum (damage specimen) + stain is electron-dense chemical (heavy metals; dead)
• Light Microscope

  • Most common
  • Use several lenses for high mag
  • Illuminate specimen below
  • Differential staining; show specific cell parts, e.g. DNA
  • No mag limit, but higher blur due to resolution - 200nm limit
  • Sample Prep
  • Squash Slide: Gently press down coverslip or a 2nd slide
  • Smear Slide: Create thin, even coat using dif slide's edge
  • Uses
  • Inexpensive/small
  • Specimen can be original colour + alive or dead
  • Flaws
  • Resolution limit - can't see organelles
  • Specimens must be stained
• Magnification + Resolution

  Magnification

  • How many times larger an image is than real life
  • Magnification = Image size/Actual size

  Resolution

  • Detail degree seen - min distance between 2 separate points seen clearly
  • Limited by light diffraction, light waves' tendency to spread as they get close to physical structures

  Units

  • Most cells too small to be measured by international SI unit (metres)
  • So micrometres (μm) used; 1mm = 1000μm, 1μm = 1/1000mm
  • Some biological structures still small so nanometres (nm) used; 1μm = 1000nm, 1nm = 1/1000μm
• Calibration Parts

  Eyepiece Graticule

  • Scale inside eyepiece lens that fits into microscope top
  • You can use it to measure sample and calibrate to calculate object size
  • Objective magnification 4x = each division 0.025mm/25 microns
  • Objective magnification 10x = each division 0.01mm/10 microns
  • Objective magnification 20x = each division 0.005mm/5 microns
  • Objective magnification 40x = each division 0.0025mm/2.5 microns
  • Objective magnification 100x = each division 0.001mm/1 micron

  Stage micrometer

  • Scale on microscope slide, divided into 100 small divisions (1-10/10-100)
  • Each division = 0.1mm (10mm long)
• Calibrating Microscope

  • Set object lens to x4
  • Place stage micrometre on microscope stage + line graticule up (line 0’s)
  • Calculate how many stage micrometre divisions eyepiece graticule take up
  • 100 eyepiece divisions = 25 stage divisions
  • 1 SD = 0.1mm
  • 25 SD = 2.5mm = 100 ED
  • We want to know how many mm, eyepiece graticule measures, we need to find out 1 length
  • 1 ED = 2.5/100 = 0.025mm
  • Convert to μm as it’s more useful when measuring cells
  • 0.025mm x 1000 = 25μm
  • This is final conversion value for our eyepiece graticule at x4