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Flashcards in Microscopy Deck (32):

The resolving power of the light microscope is the ...

... the smallest distance between two objects


Abbe's Law

d = (lambda)/2* N.A

where d is the maximum resolution, with values lower not being resolvable


Whether you can see an image depends on ...

... the brightness and not the size


Fluorescence Diagram (3)

  1. Excitation to higher energy level
  2. Decay back to lower energy level
  3. Energy given out as fluorescent light


Fluorescent Microscope (3)

  1. First barrier filter - lets trough only blue ligh with a wavelegth between 450 and 490 nm
  2. Beam spliting mirror - reflects light below 510nm but transmits light above 510nm
  3. Second Barrier Filter - cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm


GFP facilitates ...

... facilitates live cell imaging


What does GFP stand for?

Green Fluorescent Protein from the pacific ocean jelly fish

autofluorescent allows direct visualisation


Why use wide-field microscopy?

Used fixed filter blocks e.g. for specimens but motorised filter wheels and shutter enable more complex time-lapse experiments


What does CLSM stand for?

Confocal Laser Scanning Microscopy


Confocal Microscopy Technique (3)

  1. Fluorescent specimen is illuminated with a focused point of light from a pinhole
  2. Emitted fluorescent light from in-focus point is focused at pinhole and reaches detector
  3. Emitted light from out-of-focus point is out os focus at pinhole and is largely excluded from detector


Pinhole confocal imcroscopy limits ...

... detection to in-focus fluorescence known as "optical Sectioning"


Using sectioning of the cell, the ...

... 3D reconstruction can be built up


What does WFM stand for?

Wide Field Microscope


Deconvolution can signigicantly enhance the ...

... clarity of WFM images depending on the sample properties


How to have a higher resolution? (3)

  • Selective Illumination (TIRF)
  • Beyond the diffraction limit
  • Electron microscopy



What does TIRF Microscopy stand for?

Total Internal Reflection Fluorescence Microscopy


What is a method of Super Resoluton Light Microscopy?

PhotoActivation Localization Microscopy (PALM)

STochastic Optical Reconstruction Microscopy (STORM)


Using fluorophor, we can detect nanometres by ...

... switching on and off fluorophors


Another type of Super Resolution Light Microscopy

STimulated Emission Depletion (STED)


STED Method (2)

Excitation spot created with doughnut spot to create..

spontaneous emission, but gives off very little light and signal.


Confocal can now detect up to ...

... 1 micrometre


3D Light Microscopy (2)

  • Imaging in 3D with less damge
  • Sheet Plane Imaging Microscopy (SPIM) uses a sheet of light rather than a point



How is contrast achieved in the EM?

Specimen is stained with an electron dense material

  • Electrons passing through the specimen are scattered by the structures
  • Others pass through parts of the cell not stained to form an image on a phosphorescent screen.


Scanning EM is analgous to the ...

... Stereo Binocular light microscope because it looks at surfaces rather than through the specimen.


Scanning EM (5)

  1. Electron beam produced
  2. Cross section of EM lenses
  3. Beam passes down the microscope column
  4. Electron beam tends to diverge
  5. Converged by EM lens


Scanning EM is different to Transmission EM because ...

Transmission EM looks through the source rather than Scanning EM which looks at electrons that bounce off source.


Transmission EM Diagram

  1. Condenser Lens
  2. Objective Lens
  3. Eyepiece Lens
  4. Projector Lens
  5. Viewing screen/photographic film


Preparation of Specimens for Tranmission EM (6) 

  • Fixation
    • Glutaraldehyde cross-links proteins
  • Contrast Enhancement
    • Osmium tetroxide binds to lipid bi-layers and proteins (is a heavy metal)
    • Uranyl acetate binds to nucleic acids, and is a heavy metal
  • Dehydration
  • Resin infiltrations (Epoxy resin, Epon)
  • Sectioning (+- 70nm sections)
  • Electron Microscope


What is Vitrification?

Converting a liquid/solid in to a glass like state without a crystal structure.


What is Immuno EM? 


Correlative Light Electron Microscopy (CLEM)

  • Combine the strengths of (live) light microscopy with the high resolution of electron microscopy


Transmission EM (3)

  • Images through samples
  • Correlative Light Electron Microscopy (CLEM) combines the adv of Live Light Microscopy and High Resolution of Electron Microscopy
  • Electron Tomography allows the visualization of 3-D objects in the Transmission Electron Microscope.