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Honours Exam LTC > Imaging > Flashcards

Imaging Flashcards

(49 cards)

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0
Q

What determines colour of light

A

Wavelength

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1
Q

How to make invisible objects visible

A

Stain with dyes
Use phase altering properties of object to create contrast
Manipulating optical pathway of microscope

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2
Q

What determines brightness of light

A

Amplitude

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3
Q

What has caused a revival in microscopy

A 
Better optics
Computer interfacing
User friendly
Multi apps
Molecular probes
Genetic engineering
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4
Q

Purpose of objective lens

A

Critical for resolution

Focusing on image

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5
Q

What does PLAN on objective mean

A

Flat field - whole field is flat and in focus (no blurry edges)

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6
Q

What does apochromat mean

A

Lens is colour corrected so colours are transmitted accurately. (No chromatic aberration)

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7
Q

Higher NA equals

A

Better resolution

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8
Q

Why is thin cover glass better

A

Less refraction

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9
Q

Purpose of immersion lenses in microscopy

A

Reduce refraction improving resolution

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10
Q

What is the purpose of the condenser

A

Focuses light onto specimen

Fills objective lens with light (except in dark field)

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11
Q

What is Abbe’s Law

A

Minimum resolving distance (d) is related to wavelength of light divided by the numerical aperture, which is proportional to the angle of the light cone formed by a point on the object, to the objective

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12
Q

What is kohler illumination

A

Provides an evenly illuminated field of view whilst illuminating the specimen with a wide cone of light

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13
Q

How does kohler illumination work

A

Forms two conjugate planes of light:
One contains specimen image
Other the filament from the light

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14
Q

Why NB to have kohler illumination

A

Best for recording data
Best illumination, resolution
Standardized viewing and imaging conditions

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15
Q

Why don’t we visualize fresh tissue

A

Soft
Decays quickly
Can’t get thin enough samples
Living cells too thin to see when unstained

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16
Q

Methods of fixing tissue

A

Chemical - aldehydes, oxidizing agents, protein denaturation

Freezing (physical) - liquid nitrogen, propane

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17
Q

What molecule forms backbone of dyes

A

Benzene

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18
Q

How do dyes stain

A

Acid-base interactions
Permeability and displacement
Deposition, impregnation, precipitation
Chemical reaction with colourless dye to form colorful compound

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19
Q

Disadvantages of acid-base (h&e)

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A

Different tissue components with similar charge stain the same.
Cytoplasm and ECM show similar staining
Fine extracellular fibres not visible
Lipids are unstained

20
Q

How to overcome acid-base shortfalls

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A 
Trichromatic stains (all acid dyes. Permeability and displacement. Shows densities of tissue rather than charge)
Metal/salt deposition (enhance size and contrast of fine structures)
PAS (histochemical reaction. Stains mucopolysaccharides)
21
Q

How does enzyme histochemical staining work

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A

Active enzymes in tissue react with substrate to form a primary reaction product (invisible) . Combines with dye salt to form colorful insoluble precipitate.

22
Q

How does one view unstained living cells?

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A

Phase contrast microscopy

Differential interference microscopy

23
Q

What is phase contrast microscopy

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A

light background, uses inherent phase altering properties (modify optics) image is formed by diffraction and wave recombination increases contrast

Apps- living cells/tissues, thin unstained sections

24
What is differential interference microscopy
Similar to PCM but uses dif mods to optics Reveals 3D effect on tissue. Apps- see good structural detail on thicker samples
25
What is darkfield microscopy
Viewed on dark bkground (DF condenser) Principle of deflection Only dense objects detected Higher resolution than BF Apps- in situ hybridization, metal staining, live cell imaging.
26
Apps of fluorescence imaging
``` Detect autofluorescence Cytoskeleton Organelle tracking Non-living and living cells Ion channels, receptors Signal transduction etc etc ```
27
What is dif between colour pixel and black and white pixel
Black and white - each pixel has one binary unit eg. On or off Colour - has one binary unit for each primary colour therefore, three times larger.
28
What must you ensure when editing images
Keep editing to bare minimum Must not change 'message' of image Must be same between control and sample Always save original
29
Basic outline of immunohistochemical staining procedure
``` Fix, embed, section Wash with PBS Incubate with BSA Incubate w primary Ab (NB positive and negative controls) Wash with PBS Apply detection system Mount and analyze ```
30
Immunohistochemical detection methods
Fluorescent substances/fluorophores Enzymatic conversion eg. HRP
31
Direct vs indirect immunofluorescence vs enzymatic
Direct - easy, not v sensitive, primary Ab need to be labelled. Indirect - more steps, sensitive, more versatile as 2ndry Ab labelled (combos) Enzymatic - BF microscope fine, resolution not as good but can use EM, long shelf life, toxic substrates used
32
What is ABC system
Avidin Biotin Complex Biotin high affinity for avidin thus good linker High high sensitivity Endogenous biotin = background noise
33
Common problems w immunohistochemistry
Non specific binding of Abs (ionic/hydrophobic interactions) = background staining Cross reactivity of Abs to unrelated Ags
34
Effect of exposure time
Increase chance of pixel saturation | Less exposure = more noise
35
What is binning
Combination of adjacent pixels. Faster. Less resolution. Better signal:noise
36
Role of field diaphragm
Diameter of light path on sample
37
Decrease in pinhole diameter causes
Reduced light detected Better Resolution Decrease signal intensity More frequent z sections
38
Defn gain | Increase gain =
Digital photon detection amplification More brightness More noise
39
Ways to increase intensity of fast scan
Increase laser | Increase pinhole
40
Problems with fluorescent microscopy
``` Bleedthrough Blur Bleaching Phototoxicity Background/ autofluorescence ```
41
What is bleedthrough
With broad peaks and broad collection parameters fluorescence from one channel can appear in another
42
What is photbleaching
Irreversible destruction of an excited fluorophore
43
How to counter photobleaching
``` Reduce scan time Use high magnification Use wide emission filters Reduce excitation intensity Use 'antifade' reagents (only for dead cells) ```
44
Dif between fluorescent and confocal microscopes
F- arc lamp, emission filter, ocular lens C- laser, emission pinhole, PMT
45
What is two photon excitation
Two photons of half the energy (double wavelength) arrive simultaneously Will only occur at point of focus therefore, no out of focus emission
46
Adv of two photon confocal
Deeper tissue penetration Less phototoxicity 2nd/3rd harmonic or CARS imaging of unlabeled specimen possible
47
What is 2nd harmonic imaging
Irradiation with femtosecond laser results in forward scatter of light which is double frequency. In vivo and in situ wout staining. High res: collagen, micro tubules, skeletal muscle.
48
What is PSF
Point spread function | Describes how light of point object is distorted by optical system. Can be fixed using de convolution software

Honours Exam LTC (10 decks)

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