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Neuroscience > Models and Microscopy > Flashcards

Models and Microscopy Flashcards

1
Q

model organism

A

species that has been widely studies beacause It is easy to maintain and breed in a lab and has experimental advantages

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2
Q

why use model organisms

A

help understand fundamental mechanisms applicable to more complex systems - human

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3
Q

what should an ideal model provide the researcher

A
  1. accurately mimic the desired function or disease
  2. species availability
  3. data extrapolatable
  4. be available to multiple investigators
  5. be handled easily
  6. survive long enough to be functional
  7. fit available animal housing facilities
  8. be of sufficient size to provide multiple samples
  9. be polytococcous so that multiple offspring are produced for each gestation
  10. ethical approved for use.
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4
Q

extrapolatable

A

the ability to infer the unknown from the known
predict human data by replying on animal data

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5
Q

2 main characteristics of data extrapolatable to man

A

fidelity and discrimination

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6
Q

fidelity

A

how close a model is to the organism or condition we are studying in our target species

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7
Q

discrimination

A

means the extent to which the model reproduces one particular property of the original in which we happen to be interested

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8
Q

high fidelity

A

no extrapolation
(HIV study)

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9
Q

low fidelity

A

high level of discrimination for oestrogen receptors study =extrapolation possible

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10
Q

why is yeast a good model

A

exhibits high degree of evolutionary conservation humans - but can be extrapolated to humans.

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11
Q

why is the mus musculus a good model

A

permits the most diverse strategies of assessing the role of specific genes and the phenotypic manifestation of genetic variation in mammals

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12
Q

when are genetic modifications performed in rates

A

to study complex behaviours - stress, anxiety, depression, aggressively, learning

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13
Q

advantages of mus musculus model

A

complex behaviours
organs homologous to humans
genetic similarity to humans

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14
Q

limitations of mus musculus model

A

very expensive husbandry costs
experimental cycle long
ethical constraint

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15
Q

why are zebra fish used in research (Danio rerio)

A

Zebrafish embryos are transparent and they develop outside of the uterus
allows scientists to study details of development = from fertilisation and continuing throughout development

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16
Q

advantages of using zebrafish

A

high reproductive rate
development is external
genetic similarity to humans
embryos and larvae are transparent
possibility to study complex behaviours

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17
Q

limitations of using zebra fish

A

moderate perdictivity
moderate translational value

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18
Q

why use drosophila melanogaster

A

to study diverse range of biological processes - genetics and inheritance, embryonic development, learning, behaviour, aging

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19
Q

advantages of drosophila

A

easy to work with
short generation time (10 days for egg to adult)
low cost of maintenance
small genome - 4 chromosomes
useful model to study behaviours - aggression, sex drive, motivation and insomnia

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20
Q

limitations of drosophila

A

genetically distant from humans
relatively simple anatomy (100000 neurons)
no adaptive immune system

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21
Q

3 Rs in research

A

replacement - methods to avoid/replace use of animals
reduction - methods to minimise the number of animals used per experiment
refinement - methods which minimise suffering and improve animal welfare

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22
Q

advantages of in vitro models

A

easy to work with
lost cost of maintances
short experimental cycle

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23
Q

limitations of in vitro models

A

simplified system
highly controlled
poor correlation with in vivo mechanisms

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24
Q

difference between cells in a dish and in a body

A

2D vs 3D - matrix studies
cell/cell contacts - co-culture
complex signals - fancy media/sera
matrix rigidity - bendy matrix
pO2 - normoxic conditions

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25
Q

organdies

A

in vitro 3D cellular clusters
synthetic organs

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26
Q

what are organdies derived from

A

primary tissue, embryonic stem cells or induce pluripotent stem cells

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27
Q

function of organoids

A

capable of self renewal
self organisation
exhibiting similar organ functionality as the tissue of origin

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28
Q

how to produce organoids

A

fibroblast + blastocyst
= ESCs aggregation
neuroectoderm
matrix embedding
spinning bioreactor
whole brain organoids

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29
Q

4 light microscopy techniques based on scattering reflection and absorption

A

bright field
dark field
phase contrast
differential interference contrast

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30
Q

light microscopy technique based on fluorescence

A

epifluorescence - widefield

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31
Q

types of epifluorescence - widefield

A

confocal
2 photon
light sheet
microscopy in behaving animals

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32
Q

brighfield microscopy

A
  • light from light source
  • to specimen through eyepiece or camera
  • light is transmitter through specimen
  • specimen scatters light passing through it
  • makes it appear dark against illuminated white background
33
Q

difference between stained and unstained specimens in brighfield microscopy

A

unstained - scattering is weak = low contrast
stained = scattering it high = high contrast

34
Q

dark field microscopy

A

direct light is blocked by an opaque disk in condense
- light passing through specimens from oblique angles - diffracted, refracted, reflected int microscope objective
- white image on dark background

35
Q

when to use dark field microscopy

A

very thin bacteria not visible normally since the reflection of light makes them appear larger

36
Q

phase contrast microscopy

A

converts differences in phases into differences in intensity of light
produces light and dark contract in the image

37
Q

differential interference contrast microscopy

A

phase contrast microscopy technique transforms small spatial variations in phase into corresponding changes in the intensity of transmitted light
similar to phase contrast without the halo
3D effect
very useful for electrophysiologists

38
Q

what is flourescence

A

the property of absorbing light of short wavelength and emitting light of longer wavelength

39
Q

what is higher emission of excitation wavelength

A

emission wavelength is always higher than the excitation wavelength

40
Q

where did Green Flourescent protein originate from

A

isolted from Aequorea Victoria jellfish
closed in 1992
crystal structure 1996

41
Q

who won Nobel prize for GFP

A

2008 = nobel prize - discovery and use of GFP
Martin Shelvey, Osamu Shimomura, Roger Tsien

42
Q

how does GFP work

A

blue light in
green light out

43
Q

DAPI (4,6 diamidino-2-phenylindole)

A

A blue-emitting fluorescent molecule which specifically binds DNA and is used for the localization of nuclei

44
Q

what neurons express GFP

A

all excitatory neurons = express GFP
some fluorescent molecules = linked to antibodies and immunofluorescence = reveal the presence of certain proteins/targets

45
Q

4 main types of light source used in an epifluorescence microscope

A

xenon arc lamps
mercury vapour lamps
with excitation filter, lasers, high power LEDs

46
Q

the excitation filter in epifluorescence microscope

A

A bandpass filter that passes only the wavelengths absorbed by the fluorophore

47
Q

the dichroic matter in epifluorescence microscope

A

very accurate colour filter used to selectively pass light of a small range of colours while reflecting other colours

48
Q

the emission filter in epifluorescence microscope

A

the emitter is a bandpass filter = passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band (excitation light)
by blocking unwanted excitation energy or sample autoflorescence - optic filters ensure the darkest background

49
Q

advantages and disadvantages of brightfield

A

+ = low illumination, no labelling required, cheap, useful for fixed stain specimens
- = impossible to automate image analysis of unstained samples

50
Q

advantages and disadvantages of dark field

A

+ = increase contrast without staining
useful for very small specimen
- = impossible to automate image analysis of unstained samples
dust can be misinterpreted for specimen

51
Q

advantages and disadvantages of phase contrast DIC

A

+ = use interference patterns to enhance contrast, low illumination, no labelling required, cheap
- = almost impossible to automate image analysis, can only differentiate structures with high contrast

52
Q

advantages and disadvantages of fluorescent widefield

A

+ discrimination of up to 4 fluorophores
- contribution of out of focus light, high illumination, limited depth information

53
Q

4 advanced microscopy techniques

A

confocal
multi photon
light sheet
fluorescence microscopy in behaving animals

54
Q

drawback of epifluorescence microscopy

A

unless the specimen I very thin = areas of the specimen above and below the focal plane still contribute to the image as out of focus blur

55
Q

confocal microscopy principle

A

pinhole between specimen and detector is used to select information from a single focal plan = producing a sharply focussed optical slice through the specimen

56
Q

how to get 3d image from confocal microscopy

A

take series of optical slices from different focus levels in the specimen generates a 3d data set
3d data set can be visuals as a Z stack or as maximum projection

57
Q

advantages of confocal microscopy

A

accurate res in 3D
discrimination of multiple fluorophores
possibility of automates image analysis

58
Q

disadvantages of confocal microscopy

A

expensive
high illumination and long acquisition time =
can lead to photobleaching

59
Q

what is photobleaching

A

photochemical alteration of a dye or fluorophore molecule such that it is permanently unable to fluoresce

60
Q

how is photobleaching caused

A

caused by cleaving a covalent bonds or non specific reactions between fluorophore and surrounding molecules

61
Q

how to avoid photobleaching

A

mounting medium
choir of fluorophores
slide storage
lower light power
short exposure time
other methods

62
Q

what is 2 photon microscopy

A

allows visualisation of living tissue at depths unachievable with conventional (one photon) fluorescence or confocal microscopy

63
Q

how can fluorescent be induced

A

absorption of one photon of a given energy
stimultaneous absorption of 2 photons of half the energy (twice the wavelength)

64
Q

beams in 2 photon microscopes

A

a near infrared laser beam (800nm) with 100fs long pulses at a repetition rate of 80 MHz is focused through focusing objective

65
Q

2 photon microscopy compared to single phon confocal microscopy

A

IF lasers used for 2 photon microscopy scatter much less
IF lasers - excite fluorophores up to around 1mm in living tissues
single photon confocal = penetrate 200um

66
Q

what is light sheet fluorescent microscopy

A

good optical sectioning capabilities
high speed
only a thin slice of sample is illuminated perpendicularly to the direction of observation

67
Q

main advantages of 2 photon microscopy

A

imaging of deeper structures
less photobleaching

68
Q

main advantages of 2 photon microscopy

A

imaging of deeper structures
less photobleaching

69
Q

how does light sheet fluorescent microscopy reduce photobleaching

A

as only the observed section is illuminated
reduces photo damage and stress induced on living sample

70
Q

advantages of light sheet fluorescent microscopy

A

combines the speed of wide field imaging with optical sectioning and low photobleaching

71
Q

disadvantages of light sheet fluorescent microscopy

A

expensive
unsuitable for tissue with strong light scattering property

72
Q

volumetric imaging using light sheet fluorescent microscopy - tissue clearing

A

making larger fixed biological samples transparent
creating consistent refractive index through the tissue = clearer image

73
Q

microscopy techniques in behaving animals

A

visualise dynamic signals (neuronal activity) in live animals
fluorescent probes can be used to visualised those dynamic signals

74
Q

GCaMP

A

encoded calcium indicator used as a proxy for electrical activity

75
Q

how does GCaMP work

A

calcium binds to the calmodulin domain and causes a conformational change that causes GFP fluorescence

76
Q

when is GCaMP used

A

to visualise dynamic signals in live animals - shows neuronal activity

77
Q

what is GRIN - gradient index lens

A

optical lenses that an be chronically implanted into the brain to provide optical access to the neurons of interest

78
Q

miniature epifluorescence microscope

A

similar to bench top microscope by weights 2g!!
fix onto mouse

79
Q

summarise confocal microscopy, 2 photon microscopy, light sheet fluorescence microscopy and in vivo fluorescence microscopy

A

confocal microscopy = 3D images - very useful for colocalisation studies
2 photon microscopy = excellent spatial resolution
light sheet fluorescence microscopy = volumetric imaging of transparent samples
in vivo fluorescence microscopy - GCaMP as a proxy gor neuronal activity and miniature microscopes

Neuroscience (14 decks)

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