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Cell Function and Structure > Microscopy > Flashcards

Microscopy Flashcards

1
Q

Light Microscopy

A
  • Uses visible light and system of lenses to magnify images
  • Commonly used to examine tissues
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2
Q

Super-Resolution Light Microscopy

A
  • Encompasses fluorescence techniques with capabilities to resolve objects below the classical diffraction limit of optical resolution
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3
Q

Electron Microscopy

A
  • Uses a beam of concentrated electron against the sample at a greater resolution
  • Does not work on living cells because it will fry them
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4
Q

Atomic Force Microscopy

A
  • Scanning probe microscope which primary roles include measuring properties such as magnetism, height, and friction
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5
Q

Fluorescent Microscopy

A
  • Revolutionized the field of cell biology
  • Provides an enormous contrast (black, white, etc.)
  • Linked to specific probes, such as antibodies or small molecules to localize discrete cell types or cellular compartments
  • Possible by virtue of the principle of fluorescence
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6
Q

Principle of Fluorescence

A

Increased absorption of photons -→ reaches an excitatory state

When released from the excitatory state, emission of photons at a longer wavelength

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7
Q

First Barrier Filter

A

Lets through only blue light with a wavelength between 450 and 490nm

Restrict; Allows light that excites

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8
Q

Beam Splitting Mirror

A

Reflects light below 510nm and transmits light above 510nm

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9
Q

Second Barrier Filter

A

Cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560nm

Restricts; Allows you to view the range you’re expecting

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10
Q

Phase Contrast Microscopy

A
  • If staining is not possible
  • Takes advantage of the change in phase caused by regions of the sample have differential refractive index to introduce context into the image
  • Look at occlusion within cells
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11
Q

Refractive Index

A

Measurement of a bending of light ray when passing through one medium and into another

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12
Q

Differential Interference Contrast Microscopy

A
  • If staining is not possible
  • Another method of generating contrast is based on the interference pattern generated by light passing through adjacent regions of a sample
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13
Q

Fluorescent Protein Technologies and in vivo Imaging

A
  • The creation of fusion proteins between a protein like GFP and a cellular gene in yeast or any other organism permits us to light that cellular product in a living organism
  • The presence of GFP does not interfere with the function of the cellular protein
  • No antibody or fixation required
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14
Q

Jellyfish Aequorea Victoria - Green Fluorescent Protein (GFP)

A
  • System to TURN ON GFP: Aequorin + Coelenterazine  (Ca2+)  Aequorin (Blue emission energy transfer to GFP)  GFP
  • Light around 480nm will image a longer wavelength
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15
Q

GFP and DsRed

A
  • Two families of fluorescent proteins
  • Both tinkered and engineered to emit most of the visible spectrum
  • DsRed and GFP functions are similar but DsRed can emit different wavelengths
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16
Q

Photoactivable Fluorescent Proteins

A
  • Useful for looking at the dynamics of specific molecules
  • Can be turned on with a laser of specific wavelength and turned off by an intense laser, photobleached (bright light)
17
Q

PhotoActivable Localization Microscopy (PALM)

A
  • Only activates specific molecules at a time
  • Current iterations of PALM technology enable 20nm resolution
18
Q

Eukaryotic Cell

A

The size of an organelle is just at the limit of resolution for a conventional microscope

19
Q

Factors that affect the Quality of Data

A
  1. Contrast
  2. Microscope Design
  3. Resolution
20
Q

Contrast

A
  • The use of stain to selectively mark cellular components
  • To observe cellular structure stains or dyes were necessarily introduced into samples to increase contrast between different cellular constituent
21
Q

Stains

A

Stains impart differential absorption of light as it passes thru samples, thus providing contrast to an otherwise uniformly translucent sample

  • Different stains will bind to different regions
  • “Differential absorption of light”
  • Common stain: Hematoxylin/Eosin (H&E)
22
Q

Hematoxylin (BLUE)

A

Binds arginine and lysine AA side chains enriched in nuclei (Basophillic)

23
Q

Eosin (PINK)

A
  • Stains protein the cytoplasm
  • Staining is done on fixed tissue (dead)
24
Q

Benefits of Fixed Tissues

A
  • Fixation procedures allow for stains to enter the sample (more stable)
  • Helps in catalog and archiving samples
25
Q

Antibody Probes

A

Antibodies can be linked to fluorescent molecules and become stains themselves

  • Antibodies to many cellular antigens exists to recognize many cellular constituents
26
Q

Covalently Coupled Markers

A

Enhance contrast in the cell or sub-cellular region in which the antigen was contained

27
Q

Cell Impermanent

A

Opens hole using alcohol to allow the stain to enter

28
Q

Digitization

A
  • Of image data using digital cameras extend the ability to accurately detect very weak signals and increase contrast
  • Digitization permits processing to enhance image quality or presentation
29
Q

Digitization Examples

A
  • In Calcium imaging, where the sperm enters. Color coding identifies different concentrations of calcium
  • 3D structures of organelles can be determined through digitization and processing
30
Q

Microscope Design - 3D Reconstruction

A
  • Removes out of focus light
  • Amending the microscope design
31
Q

Standard Fluorescence Microscope

A
  • Out of focus light
  • As you image a particular focal plane (optical section) light from above and below that plane is illuminated transmitted to the detector
  • Fuzzy image that is a composite of the plane of focus and the out of plane light
32
Q

Cortical Microscope

A

Producers an image of all the light coming off the sample both above and below the point at which you are focused

  • “ Just look where you are focused”
33
Q

Deconvolution

A

Algorithm is used to remove out of focus based on the behavior of light in the system

34
Q

Laser Scanning Confocal Microscope

A
  • Removal of out of focus light through fine engineering
  • Confocal pinholes are controlled precisely by and relative to one another where light illuminates to the point of focus and what emitted light makes it to the detector
    • Only fluorescence emitted from the point of focus makes it through the second pinhole to the detector
    • Coupled with digitization of data permits us to view the cell in 3D
35
Q

Resolution

A

Development in super-resolution technology

36
Q

Diffraction Limited Resolution

A

The Raleigh criterion defines the lower limit of resolution

37
Q

The Raleigh Criterion

A

two images are just resolvable when the center of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other

  • The size of the disc, its interference rings, and the overall resolution of the optical system is a function of the following: (Resolution eq: wavelength/numerical aperture)
  • Lower value for resolution is desirable: Decreased Resolution = Increased ­ Light

Cell Function and Structure (7 decks)

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