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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
organdies
in vitro 3D cellular clusters synthetic organs
26
what are organdies derived from
primary tissue, embryonic stem cells or induce pluripotent stem cells
27
function of organoids
capable of self renewal self organisation exhibiting similar organ functionality as the tissue of origin
28
how to produce organoids
fibroblast + blastocyst = ESCs aggregation neuroectoderm matrix embedding spinning bioreactor whole brain organoids
29
4 light microscopy techniques based on scattering reflection and absorption
bright field dark field phase contrast differential interference contrast
30
light microscopy technique based on fluorescence
epifluorescence - widefield
31
types of epifluorescence - widefield
confocal 2 photon light sheet microscopy in behaving animals
32
brighfield microscopy
- 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
difference between stained and unstained specimens in brighfield microscopy
unstained - scattering is weak = low contrast stained = scattering it high = high contrast
34
dark field microscopy
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
when to use dark field microscopy
very thin bacteria not visible normally since the reflection of light makes them appear larger
36
phase contrast microscopy
converts differences in phases into differences in intensity of light produces light and dark contract in the image
37
differential interference contrast microscopy
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
what is flourescence
the property of absorbing light of short wavelength and emitting light of longer wavelength
39
what is higher emission of excitation wavelength
emission wavelength is always higher than the excitation wavelength
40
where did Green Flourescent protein originate from
isolted from Aequorea Victoria jellfish closed in 1992 crystal structure 1996
41
who won Nobel prize for GFP
2008 = nobel prize - discovery and use of GFP Martin Shelvey, Osamu Shimomura, Roger Tsien
42
how does GFP work
blue light in green light out
43
DAPI (4,6 diamidino-2-phenylindole)
A blue-emitting fluorescent molecule which specifically binds DNA and is used for the localization of nuclei
44
what neurons express GFP
all excitatory neurons = express GFP some fluorescent molecules = linked to antibodies and immunofluorescence = reveal the presence of certain proteins/targets
45
4 main types of light source used in an epifluorescence microscope
xenon arc lamps mercury vapour lamps with excitation filter, lasers, high power LEDs
46
the excitation filter in epifluorescence microscope
A bandpass filter that passes only the wavelengths absorbed by the fluorophore
47
the dichroic matter in epifluorescence microscope
very accurate colour filter used to selectively pass light of a small range of colours while reflecting other colours
48
the emission filter in epifluorescence microscope
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
advantages and disadvantages of brightfield
+ = low illumination, no labelling required, cheap, useful for fixed stain specimens - = impossible to automate image analysis of unstained samples
50
advantages and disadvantages of dark field
+ = increase contrast without staining useful for very small specimen - = impossible to automate image analysis of unstained samples dust can be misinterpreted for specimen
51
advantages and disadvantages of phase contrast DIC
+ = 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
advantages and disadvantages of fluorescent widefield
+ discrimination of up to 4 fluorophores - contribution of out of focus light, high illumination, limited depth information
53
4 advanced microscopy techniques
confocal multi photon light sheet fluorescence microscopy in behaving animals
54
drawback of epifluorescence microscopy
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
confocal microscopy principle
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
how to get 3d image from confocal microscopy
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
advantages of confocal microscopy
accurate res in 3D discrimination of multiple fluorophores possibility of automates image analysis
58
disadvantages of confocal microscopy
expensive high illumination and long acquisition time = can lead to photobleaching
59
what is photobleaching
photochemical alteration of a dye or fluorophore molecule such that it is permanently unable to fluoresce
60
how is photobleaching caused
caused by cleaving a covalent bonds or non specific reactions between fluorophore and surrounding molecules
61
how to avoid photobleaching
mounting medium choir of fluorophores slide storage lower light power short exposure time other methods
62
what is 2 photon microscopy
allows visualisation of living tissue at depths unachievable with conventional (one photon) fluorescence or confocal microscopy
63
how can fluorescent be induced
absorption of one photon of a given energy stimultaneous absorption of 2 photons of half the energy (twice the wavelength)
64
beams in 2 photon microscopes
a near infrared laser beam (800nm) with 100fs long pulses at a repetition rate of 80 MHz is focused through focusing objective
65
2 photon microscopy compared to single phon confocal microscopy
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
what is light sheet fluorescent microscopy
good optical sectioning capabilities high speed only a thin slice of sample is illuminated perpendicularly to the direction of observation
67
main advantages of 2 photon microscopy
imaging of deeper structures less photobleaching
68
main advantages of 2 photon microscopy
imaging of deeper structures less photobleaching
69
how does light sheet fluorescent microscopy reduce photobleaching
as only the observed section is illuminated reduces photo damage and stress induced on living sample
70
advantages of light sheet fluorescent microscopy
combines the speed of wide field imaging with optical sectioning and low photobleaching
71
disadvantages of light sheet fluorescent microscopy
expensive unsuitable for tissue with strong light scattering property
72
volumetric imaging using light sheet fluorescent microscopy - tissue clearing
making larger fixed biological samples transparent creating consistent refractive index through the tissue = clearer image
73
microscopy techniques in behaving animals
visualise dynamic signals (neuronal activity) in live animals fluorescent probes can be used to visualised those dynamic signals
74
GCaMP
encoded calcium indicator used as a proxy for electrical activity
75
how does GCaMP work
calcium binds to the calmodulin domain and causes a conformational change that causes GFP fluorescence
76
when is GCaMP used
to visualise dynamic signals in live animals - shows neuronal activity
77
what is GRIN - gradient index lens
optical lenses that an be chronically implanted into the brain to provide optical access to the neurons of interest
78
miniature epifluorescence microscope
similar to bench top microscope by weights 2g!! fix onto mouse
79
summarise confocal microscopy, 2 photon microscopy, light sheet fluorescence microscopy and in vivo fluorescence microscopy
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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