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Cell Biology, Light microscopy, Image analysis, Chromosome organisation in…
Cell Biology
QA:
- Как можно изучать отдельный уровень в контексте всего?
- Какие проблемы появляются при фиксации клеток и органов
- Почему на ранних этапах при развитии смертей больше и ошибок больше чем потом?
- Почему эмбрионы чувствительны к свету?
- Что контролирует длину микротрубочек? Важен ли их размер?
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Клетка - наименьшая единица жизни,
суммирует омиксную информацию,
применимы физико-химические методы,
понимание как возникла жизнь
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Деление клеток:
- Как ядро собирается в клетках
- Как собираются и сворачиваются хромосомы
- Как клетки делятся на ранних этапах развития. Митотическая анеуплоидия.
- Inverted life sheet microscope - только часть света попадает на образец, чтобы его не повреждать.
- В раннем митозе два митотических центриоли две системы сегрегации (только у мышей?)
Какова роль актина при делении клеток?
- 4 -> 8 с наибольшим количеством ошибок
Киназа отвечает за стыковку микротрубочек и кинетохора
Light microscopy
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optical abberation:
- chromatic - index of refraction for dif lenght is slitly different - How to fix?
-corraction using achromatic doublet
- spherical - lense - coma
-use single white dot, single object
- Field culveture - edges or center forcus
-if very important look outside the center
- Geometric distortions - computer chip - distances between the objects!!!
two lenses and magnification = f1/f0
+uncritical space for filtares
to look with eys you need + 2 lenses - project magnified image to our eyes lenses
Illuminate evenly - Kohler illumination
add lenses in spatial conformation
How do you order microscopy lenses? Why micro is so expensive?Different objective qualities
- Achromat - simple, 1 color
- Plan Achromat - variety of colors + correct culveture
- Plan fluorita
- Plan apochromat
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Break diffraction limit
increase NA:
immersion
decrease wave lengthsuper-resolution techniques - restrict excitation
turn of or on to localise dots of the imageMulticolor image:
excitation/emmision bandsProblems with fluorescence:
- Photobleaching
- lower intensity = longer imaging
organic dyes for time lapse
- Photo-toxicity
Loght sheet microscopyc
FLIM - fleorescence lifetime imaging
single pulse - hight resolution + decay curve to even fluoroforse
FRET
between light sensitive molecules
scales with d^6 - cjlo with nm
scattering:
light interacts with tissues, and waves are perturbed = blurd image
Non-linear mucr, two-photn mic
Image analysis
Изображение:
- Set of values at coordinates
- Blur - photones have size and noise
- microscopy is physic
- Draw what you image - edge might be brighter than the whole cell
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Filters:
- topchat - arrogeon, get rid of dots + nucleor shrinks + dialation = cell background
- Substract the background
- get your image
QA:
- How to make sure that you are doing the right analysys, the correct
- Trouble shooting techniques when it comes to morphology?
Convolution - compute average and replace the pixel with average
label mask image - for computer how big the image is
threshold- connected components
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Calibration
get from just intensities to the number of molecules!! Jan Ellenberg group (Antonio Pollity Nature)
Quality control:
- scatter plot - dot are cells - click on the dot and see the cell
Precision: TP/(TP + FP) true + false positives
Recall: TP/(TP+FN) - how many true positives did ypu get
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Fluorescence
History:
- Quinine - antimalerial compound, was used in tonic water
- Stokes observed the change waves in light
- Synthetise quinine, but obtained mauveine = purple dye
- Frorescein and oregon green, rhodamines - to visible range
Properties:
- Rotation, moving - destroy fluorescence??
Do you have dyes, which change more than 2 colours?
Problems:
- Achieving specific labelling
-synthetic dyes
cellular stains - DAPI into DNA, mitotracker, lysotracker, nile red - stability? - easy to use, only organelle specificity
Maleimide - to the cystein react for protein and nucleic asides, not specificic, can use for hybrid/FISH
AlexaFluor
Cy dyes
-fluorescent proteins
GFP
transgenic animals
genetically encoded, maturation time, oxigen, pH sensitive, suffer of brightness
-hybrid = protein + dye
label specific proteins
idea from protein tags + ligand
SNAP-tag, Halo-tag
Rhodamine as a tag
-can exist in equilibrium between no fluorescence and fluorescence
-protein shifts equilibrium to the open statues, increase fluorescence
Janelia Fluor, MaP555, MaP618
rhodamine and halo-tag
Deo group:
dynamical dyes
engineering new Calcium indicators
GCaMP - GFP+calmodulin
Deo created HaloCaMP
What is an intermediate region between domains - hard to pick up??
+Photoacoustic
LOV2 domein
use sound for visualisation - release excitment by vibration = temperature and pressure
need to work in far red
Are there dyes, which react to mechanical signals?
Cell mechanics
Are there dyes or fluorofores, which react to mechanical signals? measure tension with them or elastisity - side project for me
Cell properties affect cascades
-membrain to cortex attachment
-arcitexture of the cytoskeleton
-bending regidity
How cell shape emergies?
Cryelectron tomography
Membrane topography?
ECM mechanics
iMC linker - mCherry = membraine to cortex attachment
keep cells with hight MCA dark green to light green
RECS1 - canonical naive gene how much do you express = how naive you are
What is the mechanism for the exite from the pluripotency??
Migrating cells, can't turn?!
Anna Erzbenger
negative coupling between actin and rufles membrane
BAR domain proteins are curvature-sensitive
Snx33
Shallove negative curveture binding and localise in ruffles
nockout spread further and elongated
resque GFP
CRISPR - right control and specific make sure
WAVE protein for the actin polimerisation
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