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Microscopy Flashcards

1
Q

The resolving power of the light microscope is the …

A

… the smallest distance between two objects

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2
Q

Abbe’s Law

A

d = (lambda)/2* N.A

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

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3
Q

Whether you can see an image depends on …

A

… the brightness and not the size

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4
Q

Fluorescence Diagram (3)

A
  1. Excitation to higher energy level
  2. Decay back to lower energy level
  3. Energy given out as fluorescent light
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5
Q

Fluorescent Microscope (3)

A
  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
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6
Q

GFP facilitates …

A

… facilitates live cell imaging

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7
Q

What does GFP stand for?

A

Green Fluorescent Protein from the pacific ocean jelly fish

autofluorescent allows direct visualisation

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8
Q

Why use wide-field microscopy?

A

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

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9
Q

What does CLSM stand for?

A

Confocal Laser Scanning Microscopy

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10
Q

Confocal Microscopy Technique (3)

A
  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
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11
Q

Pinhole confocal imcroscopy limits …

A

… detection to in-focus fluorescence known as “optical Sectioning”

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12
Q

Using sectioning of the cell, the …

A

… 3D reconstruction can be built up

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13
Q

What does WFM stand for?

A

Wide Field Microscope

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14
Q

Deconvolution can signigicantly enhance the …

A

… clarity of WFM images depending on the sample properties

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15
Q

How to have a higher resolution? (3)

A
  • Selective Illumination (TIRF)
  • Beyond the diffraction limit
  • Electron microscopy
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16
Q

What does TIRF Microscopy stand for?

A

Total Internal Reflection Fluorescence Microscopy

17
Q

What is a method of Super Resoluton Light Microscopy?

A

PhotoActivation Localization Microscopy (PALM)

STochastic Optical Reconstruction Microscopy (STORM)

18
Q

Using fluorophor, we can detect nanometres by …

A

… switching on and off fluorophors

19
Q

Another type of Super Resolution Light Microscopy

A

STimulated Emission Depletion (STED)

20
Q

STED Method (2)

A

Excitation spot created with doughnut spot to create..

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

21
Q

Confocal can now detect up to …

A

… 1 micrometre

22
Q

3D Light Microscopy (2)

A
  • Imaging in 3D with less damge
  • Sheet Plane Imaging Microscopy (SPIM) uses a sheet of light rather than a point
23
Q

How is contrast achieved in the EM?

A

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.
24
Q

Scanning EM is analgous to the …

A

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

25
Q

Scanning EM (5)

A
  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
26
Q

Scanning EM is different to Transmission EM because …

A

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

27
Q

Transmission EM Diagram

A
  1. Condenser Lens
  2. Objective Lens
  3. Eyepiece Lens
  4. Projector Lens
  5. Viewing screen/photographic film
28
Q

Preparation of Specimens for Tranmission EM (6)

A
  • 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
29
Q

What is Vitrification?

A

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

30
Q

What is Immuno EM?

(CLEM)

A

Correlative Light Electron Microscopy (CLEM)

  • Combine the strengths of (live) light microscopy with the high resolution of electron microscopy
31
Q

Transmission EM (3)

A
  • 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.

Bio-Med (9 decks)

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