Microscopy. Microscopy methods

1. Microscopy

MSU & SkolTech
Microscopy
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2. Microscopy methods

Light microscopy
theoretical resolution
limit 100 nm
Electron microscopy
theoretical resolution
limit 0.1 nm
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3. Microscopy methods

Light microscopy
Resolution
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 field
microscopy
Phase contrast
microscopy
Dark field
microscopy
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5. Microscopy methods

Phase contrast microscopy
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6. Light microscopy

DIC
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7. Sample preparation

Sample staining
Gram staining
Giemsa staining
Antibody
conjugate
staining
Microtome:
preparation of
slices
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8. Fluorescent microscopy

Fluorescent microscope construction
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9. Confocal microscopy

Principle of confocal microscopy
Confocal
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 sample
Primary antibodies
Secondary antibodies
Protein to be detected
fluorophore
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12. Immunostaning

Method for protein detection in the fixed cells or tissue sample
Blue: 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 tissue
Gene 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 fluorescent
protein (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 proteins
Fluorophores for
different
wavelengths
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16. Fluorescent proteins

Further development of fluorescent proteins
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17. Fluorescent proteins

Multiple proteins could be tracked in the single cell
GFP, 27 kD
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18. Fluorescent proteins

“Smart” fluorescent proteins with conditionally changed fluorescence
pH, 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 a
living 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 hybridisation
gene
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 localization
telomeres
Individual
chromosome
territories
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22. Superresolution microscopy

Resolution limit
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23. Superresolution microscopy

Structured illumination (SIM)
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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)
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27. Total internal reflection fluorescence microscopy

Only fluorophores that are close to the surface are visible
Water
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)

intensity
donor
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 TIRF
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31. TIRF application for single molecule interaction of biomolecules

FRET between fluorophores visualized by TIRF
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32. Fluorescently activated cell sorter (FACS)

laser
detector
electric charging
++
+
Red (old)
timer
Blue (new)
timer
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33. Electron microscopy

Principle
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34. Electron microscopy

Transmission electron microscopy
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35. Transmission electron microscopy

Metal staining
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36. Immunostaining in electron microscopy

antibody
Protein 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 beam
Secondary
electrons
Pollen grains
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38. Cryoelecron tomography

Electron beam
Support rotation
HIV
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39. Cryoelectron microscopy

Sample preparation
water solution
graphite support
thin layer of water solution
shock freezing
in liquid ethane
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40. Cryoelectron microscopy

Data acquisition
ribosomes
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41. Cryoelectron microscopy

Image processing
Particle 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 analysis
6.7 A resolution
structure of
ribosome●EF-Tu
●aa-tRNA complex
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