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Honours Exam LTC > Imaging > Flashcards

Imaging Flashcards

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)

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

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

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?

A

Phase contrast microscopy

Differential interference microscopy

23
Q

What is phase contrast microscopy

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
Q

What is differential interference microscopy

A

Similar to PCM but uses dif mods to optics
Reveals 3D effect on tissue.

Apps- see good structural detail on thicker samples

25
Q

What is darkfield microscopy

A

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
Q

Apps of fluorescence imaging

A 
Detect autofluorescence 
Cytoskeleton 
Organelle tracking 
Non-living and living cells
Ion channels, receptors
Signal transduction etc etc
27
Q

What is dif between colour pixel and black and white pixel

A

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
Q

What must you ensure when editing images

A

Keep editing to bare minimum
Must not change ‘message’ of image
Must be same between control and sample
Always save original

29
Q

Basic outline of immunohistochemical staining procedure

A 
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
Q

Immunohistochemical detection methods

A

Fluorescent substances/fluorophores

Enzymatic conversion eg. HRP

31
Q

Direct vs indirect immunofluorescence vs enzymatic

A

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
Q

What is ABC system

A

Avidin Biotin Complex
Biotin high affinity for avidin thus good linker
High high sensitivity
Endogenous biotin = background noise

33
Q

Common problems w immunohistochemistry

A

Non specific binding of Abs (ionic/hydrophobic interactions) = background staining
Cross reactivity of Abs to unrelated Ags

34
Q

Effect of exposure time

A

Increase chance of pixel saturation

Less exposure = more noise

35
Q

What is binning

A

Combination of adjacent pixels. Faster. Less resolution. Better signal:noise

36
Q

Role of field diaphragm

A

Diameter of light path on sample

37
Q

Decrease in pinhole diameter causes

A

Reduced light detected
Better Resolution
Decrease signal intensity
More frequent z sections

38
Q

Defn gain

Increase gain =

A

Digital photon detection amplification
More brightness
More noise

39
Q

Ways to increase intensity of fast scan

A

Increase laser

Increase pinhole

40
Q

Problems with fluorescent microscopy

A 
Bleedthrough
Blur
Bleaching
Phototoxicity
Background/ autofluorescence
41
Q

What is bleedthrough

A

With broad peaks and broad collection parameters fluorescence from one channel can appear in another

42
Q

What is photbleaching

A

Irreversible destruction of an excited fluorophore

43
Q

How to counter photobleaching

A 
Reduce scan time
Use high magnification
Use wide emission filters
Reduce excitation intensity
Use 'antifade' reagents (only for dead cells)
44
Q

Dif between fluorescent and confocal microscopes

A

F- arc lamp, emission filter, ocular lens

C- laser, emission pinhole, PMT

45
Q

What is two photon excitation

A

Two photons of half the energy (double wavelength) arrive simultaneously
Will only occur at point of focus therefore, no out of focus emission

46
Q

Adv of two photon confocal

A

Deeper tissue penetration
Less phototoxicity
2nd/3rd harmonic or CARS imaging of unlabeled specimen possible

47
Q

What is 2nd harmonic imaging

A

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
Q

What is PSF

A

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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