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SL12002 - Cell Biology > Lecture 1 - Light microscope > Flashcards

Lecture 1 - Light microscope Flashcards

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1
Q

Why can we see things?

A

Photoreceptor cells in retina activated by photons of light with certain wavelengths

Lens focuses light emitted or bounced off an object

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2
Q

Why are some things not visible

A

Not enough photons
Wrong kind of photons (outside of visible spectrum
Objects too small of photoreceptors to resolve
Objects dont interact with or emit visible light
Objects interact with light the same as surrounding medium
Materials or lenses bend light to cloak object

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3
Q

If an object is not visible due to not enough photons, what can be done to make it visible?

A

Use a condenser

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4
Q

If an object is not visible due to the wrong kind of photons, what can be done to make it visible?

A

Use a detector to detect wavelengths outside of visible spectrum

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5
Q

If an object is not visible due to objects being too small to resolve, what can be done to make it visible?

A

use a compound lens

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6
Q

If an object is not visible due to an object not interacting with light, what can be done to make it visible?

A

Use optics

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7
Q

If an object is not visible due to objects interacting with light the same as the surrounding medium, what can be done to make it visible?

A

Use stains or labels

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8
Q

What do convex lenses do

A

Refract or bend light to increase the angle which spreads out light making an object larger (slide 9)

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9
Q

How does a compound microscope work?

A

Light from specimen hit detector

Eyepiece (ocular) lens magnifies image from objective

Condenser lens focuses incoming light

Virtual image that appears to observer as enlarged specimen
(slide 12)

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10
Q

How is magnification on a microscope calculated

A

Eyepiece (ocular) x objective

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11
Q

What is resolution

A

Smallest distance at which 2 objects can clearly be distinguished from one object

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12
Q

What determines resolution

A

How much light is lost between specimen and lenses
How much light is gathered by lenses
Wavelength used for illumination

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13
Q

What is a refractive index

A

Mismatch between elements resulting in loss of signal between sample, lenses and detector

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14
Q

How can refraction be minimised

A

Properties of lenses used in air can be adjusted to compensate
Special oil which is the same refractive index as glass to prevent loss from refraction

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15
Q

What is angular aperture of a lens

A

The angular aperture of a lens is the angular size of the lens aperture as seen from the focal point
(slide 20)

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16
Q

What affect does angular aperture have on microscopy

A

Higher number = more light = more resolution

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17
Q

What is numerical aperture

A

number that describes light gathering ability and resolving power of an objective lens

18
Q

What wavelengths have the highest energy

A

Shorter wavelengths (also equals higher resolution

19
Q

What is the resolution for light microscopes

A

200nm
500nm without super-res techniques

20
Q

What is the resolution for electron microscopes

A

2nm

21
Q

How are specimen on a microscope slide visible

A

Structures are visible because they interact with light differently from surrounding medium
Many things in cells hard to see without stains or labels

22
Q

How can you boost resolution

A

Phase-contrast boost resulting from differences in refracting between cells and cellular structures
DIC uses polarised light to boost contrast from diffraction of light without the sample

23
Q

What does hematoxylin stain

A

Nuclei

24
Q

What does Eosin stain

A

Eosin

25
Q

What does Oil Red O stain

A

Fat droplets

26
Q

What does Alizann red stain

A

Bone

27
Q

What does alcian blue stain

A

cartilage

28
Q

What does light blue stain

A

Collagen fibres

29
Q

What are fluorophores

A

Molecules that emit energy in the form of photons of a long wavelength, when hit with specific shorter photons
(slide 30)
(emit longer wavelength than they absorb)

30
Q

What is does quinine do

A

Excited by UV light, emits blue light (example fluorophore) (slide 29)

Anti-malarial

31
Q

Describe the mechanics of fluorescence

A

Photon of a particular wavelength, carrying a particular amount of energy excite electrons in a fluorophore
As electrons go back to ground state energy is released as photons
Less energy is released as light than absorbed, so emitted photons have longer wavelengths

32
Q

What are some commonly used fluorescent labels

A

DAPI and Hoescht - molecules bind to DNA - blue
Fluorophore-conjugated molecule - any colour eg. Phalloidin
Fluorescent Protein (GFP) used to incorporate into plasmids and genomes (2008 Nobel prize)
(can localise to cells or organelles)

33
Q

What can Fluorescent proteins be used to study

A

Monitor gene expression

34
Q

How does Fluorescent microscopy work

A

Light passes through objective lens to and from the sample
Goes through excitation and emission filters
Dichroic (two colour) mirror - shorter wavelengths reflected onto the sample, longer wavelengths pass through detector
(slide 32)

35
Q

How can you get better resolution of fluorescent microscope

A

Reduce out of focus fluorophore excitation
Reduce out-of-focus emitted light collection

36
Q

How is a confocal fl. microscope better (2-photon)

A

Removes out of focus light
improves res in z by only exciting fluorophore where beams of light cross paths

37
Q

What is digital deconvolution

A

Improves res using information about light defraxtion

38
Q

What are antibodies

A

Defences against pathogens that bind tightly to target molecules through variable regions

39
Q

What is an antigen

A

What an antibody binds to (on the outside of a pathogen usually)

40
Q

What is an epitope

A

Part of an antigen that antibody recognises

41
Q

What are monoclonal antibodies

A

bind to one epitope

42
Q

What are polyclonal antibodies

A

Mix of antibodies that recognise epitopes on same antigen (slide 42)

SL12002 - Cell Biology (12 decks)

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