Biology (UP) - Microscopes and Graticules

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What is a graticule and how do you calibrate it?

An eyepiece graticule is a little scalebar in the eyepiece of a microscope or on a little disc. It is marked off in graticule units, and to calibrate it, a stage mircometer must be used.

Stage micrometer divisions are usually 0.01mm (10 micrometres) or 0.1mm, and is around 2mm long. The EG is 10mm long, and has 100 subunits. The EG cannot be used for object measurement alone because it magnifies to different degrees for each objective lens, so each lens must be calibrated. Recording the calibration for each microscope saved time later.

  • Place SM on stage
  • Line up divisions on EG with fixed point on SM
  • Count the number of divisions on the eyepiece graticule that correspond with a set measurement on the stage micrometer.
  • Calculate the length of one EG division: divide the SM units by the EG units. 
  • To work out the length of a specimen, you count how many divisions on the EG it is. Then you multiply this with your answer from the previous step
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What is the difference between magnification and r

Magnification = how much bigger the image is than the specimen you're looking at is. Calculated using "magnification = image size/actual size"

Resolution: How well a microscope distinguishes between two points that are close together. If the lens cannot separate two objects then increasing the magnification won't help

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Similarities and differences of TEM and SEM

TEM = Transmission Electron Microscope

SEM = Scanning Electron Microscope

Similarities

  • Both use electrons
  • Same main components: electron source, electro-magnet and electrostatic lenses to control shape/trajectory of electron beam
  • Electron apertures

Differences: 

  • SEM samples are at the bottom of the column and scattered electrons are detected by detectors -> photomultipliers convert into voltage signal
  • TEM samples are in the middle of the column, transmitted electrons pass through and lenses below -> image captured
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How do you work out the length of a scale bar?

  • Calibrate the EG so you know how big each division is e.g. 0.1 micrometre
  • Measure the distance of the drawing e.g. 5cm
  • Draw a 5cm bar and label 0.1 micrometre, for example
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What is a microscope?

Instruments used by scientists to observe specimens too small to be seen with naked day.

Almost all function by magnifying object to make it appear larger

Different microscopes differ in level of magnification, image they produce and degree of resolution provided

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How do light and electron microscopes work?

Light

  • Use light rays to make object visible -> Most shine a beam of light through specimen
  • Two lenses: eyepiece and objective
  • Total magnification = eyepiece mag. x objective mag
  • Specimens placed on glass slide for viewing. Stain may be used to make organelles more visible

Electron

  • First used 1949
  • Beams of high speed electrons instead of light to produce image on fluorescent screen -> greater magnification and resolution
  • Beams are focused by electromagnets
  • Electrons are easily deflected -> need to be in a secure room, and the mircoscope is a vaccum when there is a specimen inside
  • Two types
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How do TEM and SEM work?

TEM

  • Shine electron beam through thin sections of specimen, like cells and tissues
  • Cells are stained with heavy/dense metals, e.g. osmium, to block transmission of the electrons, the ones that pass through strike a detector producing an image
  • Provide 2D detailed image 

SEM

  • Shine beam of electron onto surface of specimen and are reflected back onto a detector to produce image. Provides detailed 3D image of surface e.g. tissues. Specimen needs to be coated in metals like gold to reflect electrons
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What are the different light microscope parts and

Stage: Place your slide on here

Objective lens: Optical element closest to the specimen. Gathers light from the specimen, which is focused to produce the real image that is seen on the eyepiece lens

Eyepiece lens: AKA ocular lens. The lens the viewer looks through to see the specimen. Uusually contains a 10X or 15X power lens

Stage clips: Holds slide in place for viewing

Base: Provides support and stability

Arms: The arm connects the body tube to the base of the microscope

Coarse adjustment knob: moves the stage up and down to bring the specimen into focus

Fine adjustment knob: used to bring the specimen into sharp focus under low power and is used for all focusing when using high power lenses

Light source is usually a light underneath the stage

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