Microscopy Flashcards
Definition
Optical instruments which use a combination of lenses to produce a magnified image of small objects
- Magnify an object
- increase the resolution and contrast of the image
Types of Microscope
Hand magnifiers
Stereo (dissecting)
Compound (Lab)
- Polarising
- Phase contrast
- Infra-red
- Visible/ultraviolet
- Fluorescence
Comparison
Transmission and Scanning electron microscopes
Microscopy Features
Resolution
- Ability to distinguish between two points on the specimen
- Increasing the resolution makes the image sharper
Contrast
- Difference in intensity between parts of the image
- Enhanced by use of stains
Measurement on slides
- Place slide on the microscope stage and adjust the focus so that the image is clear
- Insert the graticule into the eyepiece
- Remove the slide and replace it with the stage micrometer
- Calibrate the graticule
- Replace the micrometer with the slide and measure the image using the graticule
stage divisions/eyepiece divisions x 0.01mm
Explain staining and the different stain types used.
Staining
- Adds contrast to the image
- Identifies components of interest
- Locates tissues and organelles
Stain types
- Metachromic - Stain different structures different colours
- Orthochrimic - Never change colour - whatever they stain
- Counterstain - Stains background to give contrast to stained structures
- Negative stain - Background stained rather than structure of interest
What are the two main stains used in forensic science?
Haematoxylin
- Basic (cationic) dye
- Blue-violet colour
Stains structures that are
- Acidic
- Anionic
- Basophilic
Used for nucleic acids and cell nuclei
Eosin
- Acidic (anionic) dye
- Shades of red
Stains structures that are
- Basic
-Cationic
-Acidophilic
Tends to stain all components of the cell
Progressive - Dyes substances preferentially
Regressive - Initial over-staining followed by decolourisation
How do you mount a sample?
- Seal sample on a slide under a coverslip using a mountant
- Mountants are clear resin that harden quickly
Sample mounting protects (sections of) sample and allows storage without deterioration - allow rays to enter objective lens
What is Refractive Index?
- Change of direction of a beam of light as it passes through another medium
- RI = sin i/sin r
- i = incidence angle
- r = refractive angle
Explain the Becke Line and its principles.
Becke Line - a bright halo of light appearing near the perimeter of a fragment
- Observed when the RI of a fragment is different to that of its surrounding medium
- Used to determine RI of fragments of glass
- Becke line is inside particle if glass has the higher RI
- Becke line is outside particle if glass has a lower RI
What is GRIM?
Glass Refractive Index Measurement
- Glass fragment is immersed in oil which has a higher RI
- Temperature of oil is increased, lowering RI
- Temperature is changed until Becke line disappears
- At this point - RI of the sample = RI of the liquid
What is the conclusion that a forensic scientist can make?
If two pieces of glass have the same refractive index, the forensic scientist can conclude that there is strong support that the glass originated from the same (type of) source
What is polarised microscopy and how does it work?
Most light consists of a random distribution of orientations where a polarised allows only one of these orientations to pass through, resultant light vibrates in a single plane whereas polarised light interacts with the sample
What is polarised microscopy used for?
Trace evidence analysis
Fibre analysis
Repeating monomer units
Variation in density in two directions
Light is separated into two rays inside the fibre
- Parallel
- Perpendicular
Birefringence
- Difference between two refractive indices
What is pleochroism in fibre analysis?
Sample displays different colours depending on its orientation to the polarised light
What is microspectrophotometry?
A combination of an optical microscope with a highly sensitive spectrophotometer.
Designed to measure the absorbance of visible and UV light by microscopic samples
Operates in Visible (380-800nm) and UV (190-380nm)
Spectra are collected over small measurement steps or increments of 1/2 to a few nanometers each
A standard, control or blank is analysed and the response of the sample relative to this is then reported
