Models and Microscopy Flashcards
model organism
species that has been widely studies beacause It is easy to maintain and breed in a lab and has experimental advantages
why use model organisms
help understand fundamental mechanisms applicable to more complex systems - human
what should an ideal model provide the researcher
- accurately mimic the desired function or disease
- species availability
- data extrapolatable
- be available to multiple investigators
- be handled easily
- survive long enough to be functional
- fit available animal housing facilities
- be of sufficient size to provide multiple samples
- be polytococcous so that multiple offspring are produced for each gestation
- ethical approved for use.
extrapolatable
the ability to infer the unknown from the known
predict human data by replying on animal data
2 main characteristics of data extrapolatable to man
fidelity and discrimination
fidelity
how close a model is to the organism or condition we are studying in our target species
discrimination
means the extent to which the model reproduces one particular property of the original in which we happen to be interested
high fidelity
no extrapolation
(HIV study)
low fidelity
high level of discrimination for oestrogen receptors study =extrapolation possible
why is yeast a good model
exhibits high degree of evolutionary conservation humans - but can be extrapolated to humans.
why is the mus musculus a good model
permits the most diverse strategies of assessing the role of specific genes and the phenotypic manifestation of genetic variation in mammals
when are genetic modifications performed in rates
to study complex behaviours - stress, anxiety, depression, aggressively, learning
advantages of mus musculus model
complex behaviours
organs homologous to humans
genetic similarity to humans
limitations of mus musculus model
very expensive husbandry costs
experimental cycle long
ethical constraint
why are zebra fish used in research (Danio rerio)
Zebrafish embryos are transparent and they develop outside of the uterus
allows scientists to study details of development = from fertilisation and continuing throughout development
advantages of using zebrafish
high reproductive rate
development is external
genetic similarity to humans
embryos and larvae are transparent
possibility to study complex behaviours
limitations of using zebra fish
moderate perdictivity
moderate translational value
why use drosophila melanogaster
to study diverse range of biological processes - genetics and inheritance, embryonic development, learning, behaviour, aging
advantages of drosophila
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
limitations of drosophila
genetically distant from humans
relatively simple anatomy (100000 neurons)
no adaptive immune system
3 Rs in research
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
advantages of in vitro models
easy to work with
lost cost of maintances
short experimental cycle
limitations of in vitro models
simplified system
highly controlled
poor correlation with in vivo mechanisms
difference between cells in a dish and in a body
2D vs 3D - matrix studies
cell/cell contacts - co-culture
complex signals - fancy media/sera
matrix rigidity - bendy matrix
pO2 - normoxic conditions
organdies
in vitro 3D cellular clusters
synthetic organs
what are organdies derived from
primary tissue, embryonic stem cells or induce pluripotent stem cells
function of organoids
capable of self renewal
self organisation
exhibiting similar organ functionality as the tissue of origin
how to produce organoids
fibroblast + blastocyst
= ESCs aggregation
neuroectoderm
matrix embedding
spinning bioreactor
whole brain organoids
4 light microscopy techniques based on scattering reflection and absorption
bright field
dark field
phase contrast
differential interference contrast
light microscopy technique based on fluorescence
epifluorescence - widefield
types of epifluorescence - widefield
confocal
2 photon
light sheet
microscopy in behaving animals
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
difference between stained and unstained specimens in brighfield microscopy
unstained - scattering is weak = low contrast
stained = scattering it high = high contrast
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
when to use dark field microscopy
very thin bacteria not visible normally since the reflection of light makes them appear larger
phase contrast microscopy
converts differences in phases into differences in intensity of light
produces light and dark contract in the image
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
what is flourescence
the property of absorbing light of short wavelength and emitting light of longer wavelength
what is higher emission of excitation wavelength
emission wavelength is always higher than the excitation wavelength
where did Green Flourescent protein originate from
isolted from Aequorea Victoria jellfish
closed in 1992
crystal structure 1996
who won Nobel prize for GFP
2008 = nobel prize - discovery and use of GFP
Martin Shelvey, Osamu Shimomura, Roger Tsien
how does GFP work
blue light in
green light out
DAPI (4,6 diamidino-2-phenylindole)
A blue-emitting fluorescent molecule which specifically binds DNA and is used for the localization of nuclei
what neurons express GFP
all excitatory neurons = express GFP
some fluorescent molecules = linked to antibodies and immunofluorescence = reveal the presence of certain proteins/targets
4 main types of light source used in an epifluorescence microscope
xenon arc lamps
mercury vapour lamps
with excitation filter, lasers, high power LEDs
the excitation filter in epifluorescence microscope
A bandpass filter that passes only the wavelengths absorbed by the fluorophore
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
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
advantages and disadvantages of brightfield
+ = low illumination, no labelling required, cheap, useful for fixed stain specimens
- = impossible to automate image analysis of unstained samples
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
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
advantages and disadvantages of fluorescent widefield
+ discrimination of up to 4 fluorophores
- contribution of out of focus light, high illumination, limited depth information
4 advanced microscopy techniques
confocal
multi photon
light sheet
fluorescence microscopy in behaving animals
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
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
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
advantages of confocal microscopy
accurate res in 3D
discrimination of multiple fluorophores
possibility of automates image analysis
disadvantages of confocal microscopy
expensive
high illumination and long acquisition time =
can lead to photobleaching
what is photobleaching
photochemical alteration of a dye or fluorophore molecule such that it is permanently unable to fluoresce
how is photobleaching caused
caused by cleaving a covalent bonds or non specific reactions between fluorophore and surrounding molecules
how to avoid photobleaching
mounting medium
choir of fluorophores
slide storage
lower light power
short exposure time
other methods
what is 2 photon microscopy
allows visualisation of living tissue at depths unachievable with conventional (one photon) fluorescence or confocal microscopy
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)
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
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
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
main advantages of 2 photon microscopy
imaging of deeper structures
less photobleaching
main advantages of 2 photon microscopy
imaging of deeper structures
less photobleaching
how does light sheet fluorescent microscopy reduce photobleaching
as only the observed section is illuminated
reduces photo damage and stress induced on living sample
advantages of light sheet fluorescent microscopy
combines the speed of wide field imaging with optical sectioning and low photobleaching
disadvantages of light sheet fluorescent microscopy
expensive
unsuitable for tissue with strong light scattering property
volumetric imaging using light sheet fluorescent microscopy - tissue clearing
making larger fixed biological samples transparent
creating consistent refractive index through the tissue = clearer image
microscopy techniques in behaving animals
visualise dynamic signals (neuronal activity) in live animals
fluorescent probes can be used to visualised those dynamic signals
GCaMP
encoded calcium indicator used as a proxy for electrical activity
how does GCaMP work
calcium binds to the calmodulin domain and causes a conformational change that causes GFP fluorescence
when is GCaMP used
to visualise dynamic signals in live animals - shows neuronal activity
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
miniature epifluorescence microscope
similar to bench top microscope by weights 2g!!
fix onto mouse
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
