Konjugierte Ebenen im Lichtmikroskop - ZMB UZH · 1) Lamp focused on the front aperture of the condenser. 2) Focus the specimen. 3) Focus the condenser to see the field stop diaphragm

Retina

Zwischenbildebene

Objektebene

Leuchtfeldblende

Eintrittspupille des Kondesors

Austrittspupille des Objektivs

Iris

Lampen Filament

Auge

Okular

ObjektivObjekttisch

Kondensorlinse

Kollektorlinse

Image planes

Konjugierte Ebenen im LichtmikroskopBildebenen Diffraktionsebenen

1) Lamp focused on the front aperture of the condenser.

2) Focus the specimen.

3) Focus the condenser to see the field stop diaphragm.

4) Adjust the condenser diaphragm (also phase rings) using the eyepiece telescope.

Köhler Illumination

Markings on Objectives

Amplitude and phase objects: only amplitude objects can be seen by eye!

Frits Zernike (1888 – 1966)Nobel prize 1953 for work on phase contrast1930

Phase contrast microscopy

Bright field Phase contrast

LensGrating

Object plane Image planeBack focal plane

Planar wave

0th

ordern

thorder




Phase contrast microscopyInterpretation of images

Light Path and Optical Elements in Different Microscopic Techniques

Bright Field Microscopy Phase Contrast Microscopy

Phase Ring

Condenser

ObjectivewithPhase Ring

Condenser

Objective

Alignment:Köhler illuminationCondenser aperture: close max 20%Field aperture: illuminaton of field of view

Alignment:Köhler illuminationCondenser aperture fully openField aperture: illuminaton of field of viewAdjust correct phase rings

y

z

x

Polarization of Light

E

B

Splitting of an incident linerar polarized ray into O- and E-ray components by a birefringent crystal

Differential interference microscopy(DIC)

Wollaston prism: wedge-shapedslabs of quartz: spliting and recombining of polarized light

Differential interference microscopy

Differential interference microscopy(DIC)


Phase contrast DIC



Bright Field Microscopy Phase Contrast MicroscopyDifferential InterferenceMicroscopy

Wollaston Prism

Wollaston Prism

Condenser

Objective

Phase Ring

Condenser


Condenser

Objective

Alignment:Köhler illuminationCondenser aperture: close max 20%Field aperture: illuminaton of field of view

Alignment:Köhler illuminationCondenser aperture: close max 20%Field aperture: illuminaton of field of viewAdjust polarizers and wollaston prisms

Polarizer

Polarizer

Alignment:Köhler illuminationCondenser aperture fully openField aperture: illuminaton of field of viewAdjust correct phase rings

Fluorescence microscopy


Annexin V staining(apoptotic cells –

phosphatidyl serinexposure)

PhiphiluxV staining(apoptotic cells –

caspase 3 activation)


DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C


Chlamydia pneumonia, live

stain

Generation of fluorescence

Common Fluorochromes - FITC

Immunfluoreszenz: Markierung mit Fluoreszenzfarbstoff gekoppelten Antikörpern

FITC

Direkte Immunfluoreszenz

FITC

indirekte Immunfluoreszenz

Antigen

AntigenGrösse des Stoke’s Shift

Molarer Extinktionskoeffizient

Quantumeffizienz

Resistenz gegenüber Photobleaching

Anzahl von Farbstoffmolekülen / Antikörper

Quenching

Common Fluorochromes - FITC

Stoke’s Shift: 20nm for Fluorescein (> 200nm for porphyrins

Molar extinctions coefficient: potential to absorb photon quanta

Quantum efficiency: fraction of absorbed photon quanta that is re-emitted by fluorescent photons (fluorescein: 0.9 in alkaline pH and in solution, 0.3 – 0-6 coupled to antibodies)

Quenching: interaction with other neighboring dye or aromatic molecules

Photobleaching: permanent loss of fluorescence by photon induced damage

Photobleaching

Photobleaching: permanent loss of fluorescence by photon induced damage

Mechanism: after excitation to a singlet state transit to a triplet state followed by complex reactions with other molecules

Examples: reactions with molecular oxygen permanently destroys the fluorochrome (free oxygen radicals destroy other molecules in sample)

Solution: reducing oxygen concentration by addition of n-propyl gallate, phenylendiamine, ….. Or commercially available embedding media

Fluorescent Proteins – GFP and Variants

Fluorescent Proteins – GFP

GFP

• Composed of 238 amino acids• Each monomer composed of a central -helix surrounded by an

eleven stranded cylinder of anti-parallel -sheets• Cylinder has a diameter of about 30Å and is about 40Å long• Fluorophore located on central helix inside cylinder• Fluorophore protected in very stable -can barrel structure • Autocatalytic formation of fluorophore

Andere fluoreszente Proteine

• DsRed: (obligates Tetramer), grüne Phase • HcRed1: co-aggregation mit DsRed, weniger hell

Fluorescent Proteins

• Living and fixed samplesGene expressionReporter assaysLocalisation studies……

• Fixation: Formaldehyd, Methanol, Ethanol, Aceton

Never: Glutaraldehyde

• Disadvantage: some fluorescent proteins tend to form oligomers (DsRed!), size (GFP: 28 kDa)

Fluorescent dyes: examples

DAPI

A popular nuclear and chromosomecounterstain, DAPI emits bluefluorescence upon binding to AT regionsof DNA. Although the dye is cellimpermeant, higher concentrations will enter a live cell.

DAPI binds to theminor groove of DNA


Hoechst 33342

A popular cell-permeant nuclearcounterstain that emits blue fluorescencewhen bound to dsDNA. This dye is oftenused to distinguish condensed pycnoticnuclei in apoptotic cells and for cell cyclestudies in combination with BrdU. Hoechst binds to the minor groove of DNA

Hoechst 33342DAPI


Lectins

• Lectins bind to oligosaccharides. Although most abundant on the cell surface, oligosaccharide residues are sometimes also found covalently attached to constituents within the cell.

• Fluorescent derivatives have been used to detect cell-surface and intracellularglycoconjugates by microscopy.

The 2 most important are:

Concanavalin A: Con A selectively binds to -mannopyranosyl and -glucopyranosyl residues

Wheat Germ Agglutinin: WGA binds to sialic acid and N-acetylglucosaminylresidues.

• Disadvantage: Distribution of staining is variable between cell types


Ion sensitive dyes

• Fura-2: popular Ca2+ sensitive dye • Measurement:ratio imaging excitation 340 / 380 nm


Dyes with preferential uptake into selective cellular compartments

• Mitochondria: selective dyes that stainsmitochondria in live cells and itsaccumulation is dependent uponmembrane potential. Some dyes arewell-retained after aldehyde fixation(e. g.: Mitotracker (several colors))

• Lysosomes: Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes.(e. g.:

FluorescenceFilter Cube

Ocular

Sample PlaneObjectives

Condenser

Z Focus

Light Source

FluorescenceLight Source

Phase RingWollaston Prism

Wollaston Prism

Bright Field Microscopy(including DIC / Phase Contrast)

Fluorescence Microscopy

Fundamental Setup of Light Microscopes

Polarizer

Polarizer

Fluorescence filter cube

Fluorescence filters


Bright Field Microscopy Phase Contrast Microscopy Fluorescence MicroscopyDifferential InterferenceMicroscopy

Wollaston Prism

Wollaston Prism

Condenser

Objective

Phase Ring

Condenser


FluorescenceCube

Objective

Condenser

Objective

Alignment:Köhler illuminationCondenser aperture: close max 20%Field aperture: illumination of field of view

Alignment:Köhler illuminationCondenser aperture: close max 20%Field aperture: illumination of field of viewAdjust polarizers and wollaston prisms

Polarizer

Polarizer

Alignment:Köhler illuminationCondenser aperture fully openField aperture: illumination of field of viewAdjust correct phase rings

Alignment:Correct alignment of fluorescence lamp

Background in fluorescence

• Usually 15 - 30 % of maximum signal intensity

• Less than ideal performance of filter sets

• Specimen preparation: autofluorescence / non specific binding / unbound fluorochrome

• Reflection and scattering in the optical pathway

Bleed through

• Multiple stained specimens!

• Excitation spectra of fluorochromes are broad and overlap

• Emission spectra are broad and overlap

• Emission of one dye stimulate a second longer wavelength dye

Microscopy in 3 Dimensions

Why confocal microscopy?Image acquired with a widefield microscope

Image acquired with a confocal microscope

Low signal to noise ratio Very high signal to noise ratio

Why confocal microscopy?Image acquired with a widefield microscope

Image acquired with a confocal microscope

Low definition of depth of field

High definition of depth of field

Why confocal microscopy?Image acquired with a confocal microscope

Data is 3 dimensional

Why confocal microscopy?Image acquired with a confocal microscope

Data is 3 dimensional

Principal Light Pathways in Confocal Microscpoy


Light source pinhole aperture

Laser

Detector pinhole aperture

Photomultiplier detector

Dichroic mirror

Objective

Focal planes in specimen



Laser



Dichroic mirror

Objective


Retina

Zwischenbildebene

Objektebene

Leuchtfeldblende

Eintrittspupille des Kondesors

Austrittspupille des Objektivs

Iris

Lampen Filament

Auge

Okular

ObjektivObjekttisch

Kondensorlinse

Kollektorlinse

Diffraction planesImage planesKonjugierte Ebenen im Lichtmikroskop

DiffraktionsebenenBildebenen



Laser



Dichroic mirror

Objective


Spatial resolution in confocal fluorescence microscopy

• Definition: NA = n sin • In fluorescence microscopy for

self luminous points: d = 0.61 NA

• In confocal fluorescencemicroscopy: resolution dependson excitation and emissionwavelength (smallest resolvabledistance is proportional to 1/1 + 1/2)

dx,y ≈ 0.4

NA

dz ≈ 1.4NA2

Spatial resolution in x,y and z

1 µm

Crossectionfocal plane 0.1 µm bead

Rea

lity

Theo

ry

Airy Disk – The Image of a Point

The image of point in the microscopy

a) Airy disk surrounded by diffraction rings

b) The first minima is dependent on the NA of the objective

Spatial resolution (point) – measurement

0

50

100

150

200

250

0 0.5 1

Intensity profile of bead

Full width at half maximum height is measured (d)

1/2

d

Spatial Resolution – Rayleigh Criterion(regular light microscopy)

d = 0.61λ/NA – Point objects which are self luminous

Two adjacent points are resolved when the central diffraction spot (Airy Disk) of one point coincides with the first diffraction minimum ot the other point (b)).

Influence of NA on resolution in z (CLSM with a pinhole size of 1 airy

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Numerical Aperture

dz (

m)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

§

Influence of pinhole diameter on resolution in z (CLSM with an objective of 1.25 NA

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.0 0.2 0.4 0.6 0.8 1.0

Pinhole in Object Plane (m)

dz (

m)

1 Airy

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.0 0.2 0.4 0.6 0.8 1.0


dz (

m)

Influence of pinhole diameter on resolution in z (CLSM with an objective of 1.4 NA

1 Airy



Laser



Dichroic mirror

Objective


Influence of pinhole diameter on resolution in z (CLSM with an objective of 1.25 NA or 1.4 NA

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.0 0.2 0.4 0.6 0.8 1.0


dz (

m)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

0.0 0.2 0.4 0.6 0.8 1.0


dz (

m)

NA 1.25 NA 1.4

Influence of wavelength on resolution in z (CLSM with an objective of 1.4 NA

525 nm 700 nm

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Numerical Aperturedz

( m

)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

§

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Numerical Aperture

dz (

m)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

§

Resolution is only dependent on the NA of objectives

dxy (NA)

0.000.200.400.600.801.001.201.401.601.802.00

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Numerical Aperture

dx,y

( m

)

§

Spatial Resolution – Rayleigh Criterion(electronic light microscopy)

d = 0.61λ/NA – Point objects which are self luminous

Two adjacent points are resolved when the central diffraction spot (Airy Disk) of one point coincides with the first diffraction minimum ot the other point (a)).

Resolution improvement by electronic imaging: adjustment of gain and offeset(b)

Important: the dip (dashed curve) in the summation of the individual intensitieshas to be detected.

Quality of confocal (electronic) images

• Spatial resolution

• Resolution of light intensity

• Signal to noise ratio

• Temporal resolution

Signal to noise ratio

Longer integration times with CCD cameras: more electrons / well are generated

Longer dwell times per pixel (lower scan rate) with CLSM: more electrons / pixel aregenerated

Several images (frames) are accumulated and averaged

1 Frame

20 Frames

Signal to noise ratio

• 50 – 100 photons /s/pixel are detected from a moderate to brightspecimen by CLSM

• S/N (Signal to noise) → 25

• Improve S/N•

→ averaging→ slower scan rates (longer dwell times/pixel resulting in morephotons but increases bleaching)→ increase size of pinhole

Temporal resolution – Nipkow disk (spinning disk –tandem) scanning microscopy

Temporal resolution – Nipkow disk (spinning disk –tandem) scanning microscopy

• Thousands of small pinholes• Thousands of small scanning points

• Generation of a real confocal image recorded on camera

• Time resolution is improved (up to 360 frames /s)(a conventional CLSM: 12-36 frames/s)

Confocal parameters: image intensity and spatialresolution

↑ Intensity

↑ Spatial resolution

↑ Objective NA

↓ Intensity

↑ Temporal resolution

↑ Scan rate

↑ Intensity

↑ Spatial resolution

↑ Zoom

↑ Intensity

↓ Spatial resolution

↑ Pinhole diameter

EffectParameter

Photobleaching

• Electrons are excited above singlet state with a probability to entertriplet states

Molecules in triplet states are chemically reactive, generation of radicals

Photobleaching

• Reduce photobleaching:

Employ anti bleach solutions and mounting media (anti free radicalreagents)

Reduce laser power

Scan at a faster rate

Lower zoom factor (larger area)

open pinhole to allow shorter dwell times

capture more of the emission spectra (long pass filters)

Reduce frame averaging

FRAP (Fluorescence Recovery After Photobleaching)

• FRAP measures turnover time of molecules in membranes, cytoplasm, or chromatin, the dynamics of proteins and macromolecules in living cells. Briefly,

1. A photobleach mark is placed on a cell. The shape of the bleach mark is a spot or a vertical slit.

2. Fluorescence recovery is monitored with a CCD camera or with a confocal laser scanning microscope.

3. Kinetics are modeled to determine the “half-time of recovery”.

FRAP – Fluorescence recovery after photobleaching

Resolution in Time


Monitoring the recovery of fluorescence afterphotobleaching.

BleachingMonitoring the fluorescence before photobleaching.

Graphical presentation of datacollected during a FRAP experiment.


Binding, Dissociation and FRAP

Proteins ina structure

Proteins freein the cytosol

Before Photobleaching

Binding (k+)

Dissociation (k-)


After Photobleaching


Partial fluorescence Recovery

Recovery Curve

• Kinetics modeling: perturbation- relaxation • Ft = Fi + (Ff - Fi)(1 - e-kt)

Finitial

Ffinal

Ftime = t

FRET (Fluorescence Resonance Energy Transfer)

• FRET imaging can give information about the precise location andnature of interaction between specific molecular species.

• Background: • Energy from one exited fluorochrome is transferred without emission

of photons to a second fluorochrome of longer wavelength

• Distance between the two fluorochromes must be in the range of several nanometer (Förster Radius)

• Emission spectra of the donor and excitation spectra of the acceptor must overlap

TIRF Microscopy

• TIRF = Total internal reflection microscopy

• Used for single molecule and vesicle trafficking studies at membrane surfaces

• Principle: different refractive indexes:glass (n = 1.518) vs sample (n = 1.33 – 1.37)

TIRF Microscopy

1. Laser excitation light is directed at a tissue sample through a glass slide at a specific, oblique angle (critical angle)

2. Most of the light is reflected at the interface between glass and the tissue sample (total internal reflection)

3. Induction of a evanescent wave parallel to the slide

4. Decay of the evanescent wave over 200 nm

1 2

3

4

TIRF Microscopy

Documents

Konjugierte Ebenen im Lichtmikroskop - ZMB UZH · 1) Lamp focused on the front aperture of the condenser. 2) Focus the specimen. 3) Focus the condenser to see the field stop diaphragm