Chapter 2.3 More Microscopy Flashcards
Different types of microscopy
Describe electron microscopy
A beam of electrons with a wavelength of less than 1nm used to illuminate a specimen. More detail of cell ultrastructure can be seen. Magnification up to x500 000
Disadvantage of electron microscopes
Expensive
Can only be used in carefully controlled environments
Specimens can be damaged by electron beam as preparation is complex
Artefacts (structures that are produced due to preparation process)
Types of electron microscope
Transmission electron microscope (TEM) beam of electrons transmitted through specimen focused to produce an image similar to light microscopy best resolution (0.5nm)
Scanning electron microscope (SEM)
beam of electrons sent across the surface of specimen
reflected electrons are collected
resolving power of 3-10nm
3-d images of surfaces produce
gives information on appearance of different organisms
Sample preparation for electron microscope
Vacuum to ensure electron beams travel in straight lines
Fixation - chemicals or freezing
Staining with heavy metals
Dehydration with solvents
TEM - set in resin, maybe stained again
SEM - may be fractures to expose inside, coated with heavy metals
What are artefacts
Visible structural detail caused by processing the specimen.
They are not part of the specimen.
Appear in light and electron microscopy
Inevitable in electron microscopy
Laser scanning confocal microscopy
Uses flouresence and flourescent microscope.
A single spot of focused light is moved across the specimen (point illumination)
Specimen has been treated with flourescent ‘dye’
High light intesity from flourescent microscope is used to illuminate treated specimen
Flourescence is caused from components labelled with ‘dye’
Emitted light is filtered through pinhole aperture
Only light radiated from very close to focal plane is detected.
Laser used as it has a higher intensity than light improving illumination
Spot illuminating specimens is moved across it - 2D images
3D can be obtained by creating images from different focal planes
Explain resolution in laser scanning confocal microscopy
Only light from focal plane is detected as other light would reduce resolution
Unwanted radiation doesn’t pass through pinhole aperture as it is undetected
Thin sections of specimens examined and light from elsewhere is removed
Uses of laser confocal microscopy
Non invasive
Cam be used in diagnosis of eye diseases
Use in endoscopic procedures
Development of drugs (see the distribution of molecules within cells)
Uses for advanced optical microscopy
Virtual biopsies (suspected skin cancer)
What is a beamsplitter
Dichroic mirror
Reflects only one wavelength
Allows other to pass through
Purpose of the term confocal
Positions of two pinhole means light waves from laser follow same path as light waves radiated when sample flouresces.
Both have same focal plane
Use of antibodies with Fluorescent tags
Specific features can be targeted and studied by confocal microscopy with more precision than when using staining and light microscopy
Example of a fluorescent tag
Green fluorescent protein
Produced by jellyfish Aequorea Victoria
Protein emits green light when illuminated by UV light
Molecules engineered to flouresce different colours
Different components of specimen studied at same time
Gene for protein isolated and attached to genes coding for proteins that are under investigation
Attached through genetic engineering
Fluorescence indicates protein is being made
Used to see what goes on in cell/organism
Purpose of fluorescing proteins
Non invasive technique to study production and distribution of proteins in cells and organisms
Cells that have been modified to express GFP and flouresce
Bacterial
Fungal
Plant
human
Atomic Force Microscopy
Gathers information about specimen by feeling surface with mechanical probe
Scanning microscope generates 3D images of surfaces
Consists of sharp probe on a cantilever
When brought close to surface, force between probe and specimen cause reflections of cantilever
Reflections measured using laser beam reflected from top of cantilever to detector
Why is fixation and staining not required in AFM
Specimen can be viewed in almost normal cell conditions without damage caused during preparation of specimens for electron microscopy.
Even living systems can be examined.
Resolution of AFM
Very high. 0.1nm
Information at atomic level can be gathered
Uses of AFM
Identify potential drug targets on cellular proteins and DNA
lead to better understanding of how drugs interact with their target cell
Identify new drugs - molecular structures need to be understood. AFM speeds up process saving money and lives
Case study of AFM
2010 scientists working on species of bacteria from sample of mud
Mariana trench
Bacteria produced unknown chemical compound
Chemical composition determined but molecular structure difficult to figure out
AFM used
Molecular structure found within a week
Super resolved fluorescence microscopy
2 principles -
1.build up high resolution using many small images
2.superimpose images with normal resolution to create one high resolution image
STED - two lasers slightly offset. First laser scans specimen causing fluorescence. Second laser negates fluorescence from molecular sized area
Picture is built up with resolution much greater than produced in light microscopy
Individual strands of DNA are visible
Specimens scanned multiple times
Each time different molecules flouresce
Images are superimposed
Uses of SRFM
Follow individual molecules during cell processes
Proteins in Parkinson’s and Alzheimer’s can be observed interacting
Fertilized eggs dividing into embryos can be studied at a molecular level
