HC10 - Visualizing cells Flashcards
electron microscopy in
dead, fixed cells
transmission light microscopy
brightfield microscopy
due to low contrast
organelles are hardly visble
histological stains
to visualize proteins, fats, DNA, organelles
dyes for histologicals stains can
absorb a certain colour but don’t emit
light (3)
- electromagnatic radiation
- quantified in protons
- can act as both a wave and particle
enhancing contrast
in phase = bright, out of phase= dim
dark field microscopy
light enters at an angle > only scattered light rays will enter the objective > thicker pieces will light up
phase contrast/DIC microscopy
waves out of phase create contrast when combined > difference in density/thickness detected
resolution (r)
minimal distance where one can still distinguish 2 points of equal intensity from each other
smaller r
more details observed
objective lens
collects a cone of light rays to create an image
condensor lens
focuses a cone of light onto each point of the specimen
a higher numerical aperture leads to
greater resolution
microtome
creates thin sample slices (10-50 um) of specimen embedded in wax or resin
hematoxylin
ECM
eosin
nucleus
dast green
cellulose of cell wall
safarin
xylem
RNA in sity hybridization
tracking activities of genes
electron microscopy
highly detail in dead cells
dead cells electron microscopy
high energy electron beam
fluorescence microscopy can occur
in living cells
fluorescence
a dye molecule absorbs energy and can send out light afterwards
red-shifted colour
emission light has a lower energy and higher wavelength
altering of GFP DNA sequence creates
different variants fluophores
beam-splitting mirror
selects the right wavelength for fluorescence
emission filter
filters out unwanted fluorescent signals
quantum dots (selenium, cadnium) charachteristics (3)
- have very sharp emission bands > only emits one colour
- photostable > take images without losing colour
- disadvantage = coating needed > hydrophilic and big in size (10 nm)
immunofluorescence (in fixed cells)
fluorescent antibodies for specific proteins
diffraction limitations > blur
Point Spread Function (PSF)
improving resolution (2)
- smaller lambda
- larger theta and n
deconvoluting images
with the use of computer software
wide field fluorescence microscopy
in-focus and out-of-focus plane
multi-point detector
camera
confocal microscopy uses a
laser
objective confocal microscopy
focused light = point of focus > molecules in out-of-focus will also be excited
confocal pinholes
emitted fluorescent ligh from in focus point is focused at the pinhole and reaches detector > more contrast in z-direction
obtaining 3D images
moving the illumination spot over the sample using scanners > Galvanometer mirror
for 3D imaging
multiple images are obtained at different heights = slower
calcium waves fertilized egg cel
will activate the injected Aequorin protein that luminesces in blue light
time dynamics of spliceosome proteins
moving Cajal bodies in plant nucleus
Annexin A4 > repair of plasma membrane
clustering of Annexin A4-sGFP in HeLa cells induced by adding calcium usin 1uM ionomycin
breakage of plasma membrane
influx calcium > positive protein bound
Fröster Resonance Energy Transfer (FRET)
for visualizing interactions > close proximity = interaction
FRET calcium biosensor
FRET between CFP and YFP > cadmodulin calcium binding sites > conformational change> shortens distance > FRET
- low CFP and high YFP
visualization of movement
photoactivation of dark GFPs
photoactivation
fluorescence in selected region > diffusion of proteins
Fluorescence Recovery After Photobleaching (FRAP)
visualizing movement by destroying fluorescent dyes > replenishment from other regions
example FRAP
galactosyl transferase in Golgi and ER
Total Interal Reflection Microscopy (TIRM)
visualization of structures at/near membrane
critical angle for total internal reflection
no light through the sample > only molecules in evanescent field fluoresce
fixing cells EM
glutaraldehude
contrast dye EM
osmium tetoxide = metals
copper grid EM
covered with carbon and/or plastic film
Transmission EM (TEM)
electron bundle through sample
Scanning EM (SEM)
3D imaging and reconstruction > different angles and movement of detector
Super Resolution Microscopy (SRM)
to overcome the resolution limitation of ~200 nm
types of SRM (4)
- Structered Illumination Microscopy (SIM)
- Expansion Microscopy
- Stimulated Emission by depletion (STED)
- Stochastic localization techniques
Stimulated Emission by Depletion (STED)
SRM for live cells > 5-7 nm
STED beam (red light)
causes excited molecules to return to resting state > only molecules not in STED beam emit light = effective fluorescent spot
Stochastic localization techniques
PALM, STORM, GSDIM
mechanism stochastic localization techniques
the position of a fluorphore can be determined with higher accuracy than the resolution of a confocal microscope > determination of center blurred spot
succesive cycles of activation and bleaching for
well-separated single fluorescent molecules
