Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion...

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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

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

Grundlagen: optisches Mikroskop

Δ: Tubuslänge

Vergrößerung des Sehwinkels

Optisches Mikroskop

OkularObjektiv

2-100x10-20x

Immunhistochemie

Anfärbung durch „Elektroadsorption“Schichtdicken ca. 10µm

bright field phase contrast

Normaski DIC dark field

Phase Contrast Microscopy

Abbesche Abbildungstheorie

Entstehung einesFourierbildes in derBrennebene einer Linse

Darstellung der Hin-und Rücktransformationdurch Anordnung zweierLinsen

OptischeDatenverarbeitungim Fourier-Raum

Originalbild

Bild mit Rasterpunkten

Entfernung der periodischenRasterpunkten durch Blendeim Fourierbild

Das Phasenkontrastmikroskop

Kondensor

Ringblende

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

http://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html

Fluoreszenz

495 nm 520 nm

Stokes Shift is 25 nmFluorescein

Wavelength

Chromophore (Farbstoffe) enthalten große delokalisierte π-Elektronensysteme. Oft sind dies aromatische Ringe.

Fluoreszenz

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

[Vibrational sublevels]

Jablonski Termschema

Vibrational energy levelsRotational energy levelsElectronic energy levels

Singlet States Triplet States

Dichroitischer Spiegel

Objektiv

Bogenlampe

Emissionsfilter

Anregungsblende

Okular

Anregungsfilter

EPI-Beleuchtung

Fluoreszenzmikroskop

!" #= 22,1min

Lage des ersten Beugungsminimums

Beugung an einer kreisrunden Öffnung

Auflösungsbegrenzung durch Beugung:

Für zwei selbstleuchtende Objekte istder kleinste auflösbare Abstand Δx :

61.0min

Wobei λ die Wellenlänge undn sinα die numerische Apertur ist.(α: Öffnungswinkel der Linse)

Objective

n=1.52

n = 1.52

Specimen

Coverslip

n=1.33

n = 1.52

n = 1.0

n = 1.5

Ölimmersion erhöht die numerische Apertur

Wellencharakter massiver Teilchen:

Elektronen Mikroskop

(de Broglie Beziehung)

Ue !=2

Uem !=2

1vElektronenbeschleunigung

" nm[ ] =h

2mee #U

U V[ ]

z.B. 100keV λ~3pm

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

Transmission Electron Microscope (TEM)

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

A) REMB) opt. Mic. C) TEM

Drei Bildgebende Techniken im Vergleich

Negative staining and freeze fracture

actin filaments

Myosin molecules

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

TEM Bilder sind Projektionen !

A deconvolution algorithm subtractsout-of plane image information usingstacks of image data

DasKonfokaleMikroskop

Objektiv

Emissionsblende

Anregungsblende

Emissions-filter

Anregunsfilter

KonfokalKonventionellesFluoreszenzmikroskop

Two-Photon Excitation

<P> ≈ mW P > MW => ps Pulse

Virtuallevel

Point-spreadfunction!

1.5µm

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).

Femtosecond pulsetrain @ 80 MHz

Two photon excitation is spatially localized

0 5 10 15 20 25

STED-Mikroskopie schlägt das BeugungslimitSTED:Stimulated Emission Depletion

Der Anregungsfokus wird „maßgeschneidert“.

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)

Purification of cells and organells by flowcytometry

Requires fluorescent tag for desired cell

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

Cell fractionation by differential centrifugation

Organelle separation by equilibriumdensity-gradient centrifugation

Microscopy in Cell Biophysics - uni-muenchen.de...Rädler SS08 Biophysik der Zelle 32 Helium-Ion...

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