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Microscopy. Microscopy methods
1. Microscopy
MSU & SkolTechMicroscopy
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2. Microscopy methods
Light microscopytheoretical resolution
limit 100 nm
Electron microscopy
theoretical resolution
limit 0.1 nm
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3. Microscopy methods
Light microscopyResolution
D = 0.61
N sin
min = 450 nm,
o
N=1 (air) 1.52 (oil), =70
D= 300 nm
objective
specimen
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4. Light microscopy
Bright fieldmicroscopy
Phase contrast
microscopy
Dark field
microscopy
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5. Microscopy methods
Phase contrast microscopy1755
6. Light microscopy
DIC1755
7. Sample preparation
Sample stainingGram staining
Giemsa staining
Antibody
conjugate
staining
Microtome:
preparation of
slices
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8. Fluorescent microscopy
Fluorescent microscope construction1755
9. Confocal microscopy
Principle of confocal microscopyConfocal
Wide field
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10. Fluorescent microscopy
Methods for fluorescent labeling:small molecule fluorophores
DAPI
DNA staining
Fura-2
Calcium sensor
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11. Immunostaning
Method for protein detection in the fixed cells or tissue samplePrimary antibodies
Secondary antibodies
Protein to be detected
fluorophore
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12. Immunostaning
Method for protein detection in the fixed cells or tissue sampleBlue: DAPI (DNA)
Green: FITClabelled antiTubulin antibodies
Red: Alexa 568labelled Phalloidin
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13. Fluorescent protein fusion
Method for protein detection in the living cells or tissueGene of interest
Addition of a fluorescent
protein coding part to the
gene encoding protein of
interest
Expression of the fusion
protein in the living cell
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14. Fluorescent proteins
Green fluorescentprotein (GFP)
...
O
NH
...
O
OH
N
HN
O
NH...
GFP, 27 kD
NN
NH
HO
OH
OH
O
O
...
Chromophore maturation from
natural aminoacids
Osamu Shimomura
Martin Chalfie
Roger Y. Tsien
Nobel Prize 2008
Aequorea victoria
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15. Fluorescent proteins
Further development of fluorescent proteinsFluorophores for
different
wavelengths
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16. Fluorescent proteins
Further development of fluorescent proteins1755
17. Fluorescent proteins
Multiple proteins could be tracked in the single cellGFP, 27 kD
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18. Fluorescent proteins
“Smart” fluorescent proteins with conditionally changed fluorescencepH, H2O2 sensors
Proteolysis and conformation
change sensors
Fluorescent protein timers
Fluorescent timer shows
intracellular locations of
newly made protein
Killer fluorescent protein
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19. RNA imaging in vivo
Sandwich tag could be used to visualize RNA localization in aliving cell
MS2
GFP
coat protein
RNA of interest coat protein
binding site
RNA localization
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20. RNA imaging in fixed cells
FISH - Fluorescent in situ hybridisationgene
nuclease
Fixed cells
o
formamide, 42 C
DNA
polymerase
Denaturation
hybridization
Fluorescent
microscopy
fluorescently labelled
anti-DIG antibodies
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21. DNA imaging in fixed cells
FISH - specific DNA localizationtelomeres
Individual
chromosome
territories
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22. Superresolution microscopy
Resolution limit1755
23. Superresolution microscopy
Structured illumination (SIM)1755
24. Superresolution microscopy
Structured illumination (SIM)Actin and tubulin visualized by
wide field microscopy
and
SIM
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25. Superresolution microscopy
Blinking fluorophores method (STORM)1
2
3
4
5
6
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26. Superresolution microscopy
Blinking fluorophores method (STORM)1755
27. Total internal reflection fluorescence microscopy
Only fluorophores that are close to the surface are visibleWater
Glass
TIRF
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28. Total internal reflection fluorescence microscopy
Cell-surface contacts visualized (vinculin)Usual microscopy
and
TIRF
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29. Förster resonance energy transfer (FRET)
intensitydonor
acceptor
wavelength
k ~ 1/r6
Efficiency of FRET is very sensitive to the distance
between fluorophores
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30. TIRF application for single molecule interaction of biomolecules
FRET between fluorophores visualized by TIRF1755
31. TIRF application for single molecule interaction of biomolecules
FRET between fluorophores visualized by TIRF1755
32. Fluorescently activated cell sorter (FACS)
laserdetector
electric charging
++
+
Red (old)
timer
Blue (new)
timer
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33. Electron microscopy
Principle1755
34. Electron microscopy
Transmission electron microscopy1755
35. Transmission electron microscopy
Metal staining1755
36. Immunostaining in electron microscopy
antibodyProtein of
interest
Gold particles
Gold
Lysosomal localization of
mannose-6-phosphate
Ribosomal protein location within
the ribosome
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37. Scanning electron microscopy
Electron beamSecondary
electrons
Pollen grains
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38. Cryoelecron tomography
Electron beamSupport rotation
HIV
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39. Cryoelectron microscopy
Sample preparationwater solution
graphite support
thin layer of water solution
shock freezing
in liquid ethane
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40. Cryoelectron microscopy
Data acquisitionribosomes
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41. Cryoelectron microscopy
Image processingParticle images
Classification of
protections
Projection set
Computer
reconstitution of 3D
structure
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42. Cryoelectron microscopy
Cryoelectron microscopy approach the resolution of Xray structure analysis6.7 A resolution
structure of
ribosome●EF-Tu
●aa-tRNA complex
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