Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion...
Transcript of Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion...
![Page 1: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/1.jpg)
Rädler SS08 Biophysik der Zelle 1
Microscopy in Cell Biophysics
• Light Microscopy (200 nm)– Brightfield– Phasekontrast– Fluorescence– Advanced
• Electron Microscopy (1 nm)– Scanning– Transmission
• Scanning Probe Microscopy (AFM, STM)
• Contrast • Spatial and temporal resolution• Environmental conditions
![Page 2: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/2.jpg)
Rädler SS08 Biophysik der Zelle 2
1901W. C. Röntgen (Germany, 1845-1923)Discovery of X rays
1953F. Zernike (Netherlands)Phase-Contrast Microscopy
1986Ernst Ruska (Germany, 1906 - 1988)Gerd Binnig (Germany)Heinrich Rohrer (Switzerland)Electron microscope and raster tunnel microscope, respectively
2003 Paul C. Lauterbur (USA)Sir Peter Mansfield (United Kingdom)for their discoveries concerning magnetic resonance imaging
Nobel prizes directly related to „bioimaging“
phasencontrast (Zernike)
bright field
![Page 3: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/3.jpg)
Rädler SS08 Biophysik der Zelle 3
Grundlagen: optisches Mikroskop
Δ: Tubuslänge
Vergrößerung des Sehwinkels
![Page 4: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/4.jpg)
Optisches Mikroskop
OkularObjektiv
2-100x10-20x
![Page 5: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/5.jpg)
Rädler SS08 Biophysik der Zelle 5
![Page 6: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/6.jpg)
Rädler SS08 Biophysik der Zelle 6
![Page 7: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/7.jpg)
Rädler SS08 Biophysik der Zelle 7
Immunhistochemie
Anfärbung durch „Elektroadsorption“Schichtdicken ca. 10µm
![Page 8: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/8.jpg)
Rädler SS08 Biophysik der Zelle 8
bright field phase contrast
Normaski DIC dark field
Phase Contrast Microscopy
![Page 9: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/9.jpg)
Rädler SS08 Biophysik der Zelle 9
Abbesche Abbildungstheorie
Entstehung einesFourierbildes in derBrennebene einer Linse
Darstellung der Hin-und Rücktransformationdurch Anordnung zweierLinsen
![Page 10: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/10.jpg)
Rädler SS08 Biophysik der Zelle 10
OptischeDatenverarbeitungim Fourier-Raum
Originalbild
Bild mit Rasterpunkten
Entfernung der periodischenRasterpunkten durch Blendeim Fourierbild
![Page 11: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/11.jpg)
Rädler SS08 Biophysik der Zelle 11
Das Phasenkontrastmikroskop
Kondensor
Ringblende
Probe
Objektiv
ungebeugtes Lichtgebeugtes Licht
Phasenring
Der Phasenring verschiebt dasungebeugte Licht um 90 Grad.Zusätzlich wird das ungebeugte Lichtauch noch abgeschwächt, damit dieIntensitäten der beideninterferierenden Komponentenähnlich sind.
90 Grad verschoben
![Page 12: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/12.jpg)
Rädler SS08 Biophysik der Zelle 12
http://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html
![Page 13: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/13.jpg)
Rädler SS08 Biophysik der Zelle 13
![Page 14: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/14.jpg)
Rädler SS08 Biophysik der Zelle 14
Fluoreszenz
495 nm 520 nm
Stokes Shift is 25 nmFluorescein
Fluo
resc
nece
Inte
nsity
Wavelength
Chromophore (Farbstoffe) enthalten große delokalisierte π-Elektronensysteme. Oft sind dies aromatische Ringe.
![Page 15: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/15.jpg)
Rädler SS08 Biophysik der Zelle 15
Fluoreszenz
ENER
GY
S0
S1
S2
T2
T1ABS FL I.C.
ABS - Absorbance S 0.1.2 - Singlet Electronic Energy LevelsFL - Fluorescence T 1,2 - Corresponding Triplet StatesI.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence
IsC
IsCPH
[Vibrational sublevels]
Jablonski Termschema
Vibrational energy levelsRotational energy levelsElectronic energy levels
Singlet States Triplet States
![Page 16: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/16.jpg)
Rädler SS08 Biophysik der Zelle 16
Dichroitischer Spiegel
Objektiv
Bogenlampe
Emissionsfilter
Anregungsblende
Okular
Anregungsfilter
EPI-Beleuchtung
Fluoreszenzmikroskop
![Page 17: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/17.jpg)
Rädler SS08 Biophysik der Zelle 17
![Page 18: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/18.jpg)
Rädler SS08 Biophysik der Zelle 18
!D
D
!" #= 22,1min
Lage des ersten Beugungsminimums
Beugung an einer kreisrunden Öffnung
![Page 19: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/19.jpg)
Rädler SS08 Biophysik der Zelle 19
Auflösungsbegrenzung durch Beugung:
Für zwei selbstleuchtende Objekte istder kleinste auflösbare Abstand Δx :
!
"
sin
61.0min
#
#=$n
x
Wobei λ die Wellenlänge undn sinα die numerische Apertur ist.(α: Öffnungswinkel der Linse)
![Page 20: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/20.jpg)
Rädler SS08 Biophysik der Zelle 20
Objective
n=1.52
n = 1.52
n = 1.52
Specimen
Coverslip
Oil
n=1.33
n = 1.52
n = 1.0
n = 1.5
Water
Air
Ölimmersion erhöht die numerische Apertur
![Page 21: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/21.jpg)
Rädler SS08 Biophysik der Zelle 21
![Page 22: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/22.jpg)
Rädler SS08 Biophysik der Zelle 22
Wellencharakter massiver Teilchen:
![Page 23: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/23.jpg)
Rädler SS08 Biophysik der Zelle 23
Elektronen Mikroskop
vm
h
p
h==!
(de Broglie Beziehung)
em
Ue !=2
v
Uem !=2
2
1vElektronenbeschleunigung
!
" nm[ ] =h
2mee #U
=1.23
U V[ ]
z.B. 100keV λ~3pm
![Page 24: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/24.jpg)
Rädler SS08 Biophysik der Zelle 24
Transmission electron microscopy (TEM)
• Operates in vacuum• Specimen usually fixed, embedded, sectioned, and stained with
an electron-dense material• typical specimen thickness 50nm
Special techniques:• Metal shadowing: visualize surface structures, cell components• Cryoelectron: visualize unfixed, unstained samples• Freeze fracture, freeze etch: visualize membrane interior• Freeze etch: visualize cell interior
![Page 25: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/25.jpg)
Rädler SS08 Biophysik der Zelle 25
Transmission Electron Microscope (TEM)
![Page 26: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/26.jpg)
Rädler SS08 Biophysik der Zelle 26
![Page 27: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/27.jpg)
Rädler SS08 Biophysik der Zelle 27
Scanning Electron Microscopy (SEM)• Can visualize surfaces of tissues, cells, isolated cell parts• Specimen is fixed and coated with thin layer of heavy metal• Images secondary electrons, resolution = 10 nm
![Page 28: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/28.jpg)
Rädler SS08 Biophysik der Zelle 28
STEM
![Page 29: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/29.jpg)
Rädler SS08 Biophysik der Zelle 29
A) REMB) opt. Mic. C) TEM
Drei Bildgebende Techniken im Vergleich
![Page 30: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/30.jpg)
Rädler SS08 Biophysik der Zelle 30
Negative staining and freeze fracture
actin filaments
Myosin molecules
![Page 31: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/31.jpg)
Rädler SS08 Biophysik der Zelle 31
![Page 32: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/32.jpg)
Rädler SS08 Biophysik der Zelle 32
Helium-Ion Microscope
The helium ion beam has a DeBrogliewavelength that is approximately 300times smaller than an electron beamresulting in much less diffraction.
See low-Z materials, like carbonnanotubes, with resolution andsurface information unavailablefrom a typical SEM.Orion He-Ion Microscope (Zeiss)
Quelle: Zeiss
He++: ion beam energie : meV
![Page 33: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/33.jpg)
Rädler SS08 Biophysik der Zelle 33
TEM Bilder sind Projektionen !
![Page 34: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/34.jpg)
Rädler SS08 Biophysik der Zelle 34
A deconvolution algorithm subtractsout-of plane image information usingstacks of image data
![Page 35: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/35.jpg)
Rädler SS08 Biophysik der Zelle 35
DasKonfokaleMikroskop
Objektiv
Laser
Emissionsblende
Anregungsblende
PMT
Emissions-filter
Anregunsfilter
![Page 36: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/36.jpg)
Rädler SS08 Biophysik der Zelle 36
KonfokalKonventionellesFluoreszenzmikroskop
![Page 37: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/37.jpg)
Rädler SS08 Biophysik der Zelle 37
Two-Photon Excitation
<P> ≈ mW P > MW => ps Pulse
Virtuallevel
Point-spreadfunction!
1.5µm
![Page 38: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/38.jpg)
Single-photonexcitation(488 nm)
Two-photonexcitation(900 nm)
Because simultaneousabsorption of two photonsis required for excitation,the emission depends onthe square of the intensity,rather than being linearlyproportional.
Significant excitation occursonly in the focal volume(at “normal” imagingintensities - mW range).
F
Ix
Femtosecond pulsetrain @ 80 MHz
Two photon excitation is spatially localized
0 5 10 15 20 25
![Page 39: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/39.jpg)
Rädler SS08 Biophysik der Zelle 39
![Page 40: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/40.jpg)
Rädler SS08 Biophysik der Zelle 40
STED-Mikroskopie schlägt das BeugungslimitSTED:Stimulated Emission Depletion
Der Anregungsfokus wird „maßgeschneidert“.
![Page 41: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/41.jpg)
Rädler SS08 Biophysik der Zelle 41
![Page 42: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/42.jpg)
Rädler SS08 Biophysik der Zelle 42
Electron tomographyof vitrified cells is a noninvasive three-dimensional imaging technique that opensup new vistas for exploring the supramolecular organization of the cytoplasm.
Further reading:Macromolecular Architecture in Eukaryotic CellsVisualized by Cryoelectron TomographySCIENCE VOL 298 (2002) p.1209
W. Baumeister (MPI Biochemie)
![Page 43: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/43.jpg)
Rädler SS08 Biophysik der Zelle 43
Purification of cells and organells by flowcytometry
Requires fluorescent tag for desired cell
![Page 44: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/44.jpg)
Rädler SS08 Biophysik der Zelle 44
Purification of cell parts
• Understanding the roles of each each cell component depends on methodsto break open (lyse) cells and separate cell components for analysis
• Cell lysis is accomplished by various techniques:blender, sonication, tissue homogenizer, hypotonic solution
• Separation of cell components generally involves centrifugation
![Page 45: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/45.jpg)
Rädler SS08 Biophysik der Zelle 45
Cell fractionation by differential centrifugation
![Page 46: Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion Microscope The helium ion beam has a DeBroglie wavelength that is approximately 300](https://reader033.fdocuments.in/reader033/viewer/2022041702/5e427d59efade5533e661a48/html5/thumbnails/46.jpg)
Rädler SS08 Biophysik der Zelle 46
Organelle separation by equilibriumdensity-gradient centrifugation