L3 - microscopes and graticules

• Electron microscopy: microscopy using a microscope that employs a beam of electrons to illuminate the specimen. As electrons have a much smaller wavelength than light they produce images with higher resolution than light microscopes
• Compound light microscope: a light microscope which uses two lenses to magnify an object; the objective lens, which is placed near to the specimen and an eyepiece lens, through which the specimen is viewed
• Eyepiece graticule: a disc in the microscope with a printed scale from 0 to 100. The absolute size of the scale is not important as the graticule calibration will account for this
• Gram negative bacteria: bacteria with cell walls that stain red with Gram stain
• Contrast: staining or treating specific cell components so that they are visible compared to untreated components
• Differential staining: using specific stains to distinguish different types of cell
• Resolution: the shortest distance between two objects that are still seen as separate objects
• Scanning electron microscope (SEM): an electron microscope on which a beam of electrons is sent across the surface of a specimen and the reflected electrons are focused to produce a 3D image of the specimen surface
• Light microscope: an instrument that uses visible light and glass lenses to enable the user to see objects magnified many times
• Transmission electron microscopy (TEM): an electron microscope on which a beam of electrons is transmitted through a specimen and focused to produce an image
• Stage micrometer: a slide with a scale in micrometres (um) etched into it. Used to measure the size of a sample under a light microscope
• Laser scanning confocal microscope: a microscope that employs a bean of fluorescence and a pin-hole aperture to produce an image with a very high resolution
• Gram positive bacteria: bacteria with cell walls that stain purple-blue with Gram stain
• Stains (staining): dyes used in microscopy sample preparation to increase contrast or identify specific components
• Counterstain: application of a second stain with a contrasting colour to sample for microscopy
• Microscopes:

  -instruments that magnify the image of an object

  -scientists use two types; compound light and electron
• Using an eyepiece graticule:

  -an eyepiece graticule is a glass disc fitted into the eyepiece of the microscope

  -the disc is marked with a fine scale of 100 divisions

  -the absolute size of this scale is not important; it is arbitrary and needs to be calibrated
• The stage micrometer:

  -used to calibrate the eyepiece graticule

  -consists of a microscope slide on which a fine and accurate scale is etched onto the glass

  -the stage micrometer is 1mm in length, split into 100 divisions
• Calibrating a microscope:

  -place stage micrometer onto stage

  -focus microscope on the lowest powered lens first so SM and EPG are in clear view

  -line up SMU and EPU scales and find two points of coincidence

  -count number of EPG divisions that represent total length of stage micrometer (1000um)

  -calculate actual length of one division of EPG

  -repeat for all objective lenses
• Resolution:

  -the ability to distinguish between two separate points

  -if two objects cannot be resolved they will be seen as one object

  -two objects can only be distinguished if light waves can pass between them
• Resolution depends on:

  -wavelength (light is made of longer wavelengths but shorter ones give better resolution)

  -type of radiation being used
• Light microscope resolution:

  -max resolution of a light microscope is 200nm (0.2um)

  -if two points are closer than 200nm they cannot be distinguished as separate
• General resolution rule: the limit of resolution is about half the wavelength of radiation used to view the object
• Magnification:

  -the number of times larger an image is compared with the real size

  -to work out how much an object is magnified under a light microscope; $total\ magnification=$$objective\ lens\times eyepiece\ lens$
• $magnification=$$\frac{image\ size}{actual\ size}$
• 2 types of electron microscopes:

  -transmission electron microscope (TEM)

  -scanning electron microscope (SEM)
• Light vs electron microscope:

  -radiation; light rays vs electron beams

  -magnification; x2000 vs x500000

  -resolving power; 200nm vs 0.2nm

  -focused by; glass lenses vs electromagnets

  -bio material; living/dead vs dead

  -size; small&portable vs large&static

  -material prep; quick&easy vs long&complex

  -cost; cheap vs very expensive
• Light microscope advantages:

  -relatively cheap to buy

  -cheap to run

  -specimen prep is simple

  -living and dead specimen may be observed

  -coloured images are obtained
• Light microscope disadvantages:

  -resolution is limited by the wavelength of light

  -limited magnification (x2000)

  -depth of field is restricted
• Electron microscope disadvantages:

  -expensive to buy

  -expensive to run when generating electron beam

  -specimen prep is complex

  -living cells can’t be observed

  -images in black and white
• Electron microscope advantages:

  -high resolution

  -high magnification (500000)

  -greater depth of field may be observed
• Laser scanning confocal microscopy:

  -developed light microscope

  -also called ‘confocal microscopes’

  -uses a laser light to scan an image point to point

  -information collected by a computer to generate an image on a screen

  -high resolution and contrast

  -can focus on different depths so can look at whole living specimens as well as cells

  -used medically (eg. looking at the retina of the eye)
• Staining in electron microscopy:

  -achieved using metal particles or metal salts

  -purpose; to distinguish or reveal different features

  -images are always black, white and grey

  -colours can be added using computer software giving false colour electron micrographs
• Stains are chemicals that bind to chemicals or structures on or in the specimen
• Stains are used to:

  -make transparent material visible

  -allows specific structures to be seen
• Wide variety of stains that colour different tissues and organelles:

  -eg. Acetic Orcein stains dna dark red

  -eg. Hoechst stains dna dark blue