מיקרוסקופיה פלואורסנטית

Contrasting techniques - a reminder…

• Brightfield - absorption

• Darkfield - scattering

• Phase Contrast - phase interference

• Polarization Contrast - polarization

• Differential Interference Contrast (DIC) - polarization + phase interference

• Fluorescence Contrast - fluorescence

חוב מהשיעור הקודם

Bright-field DIC

Phase-contrast Dark-field

שיטות להגברת ניגודיות

Fluorescence techniques

• Standard techniques: wide-fieldconfocal2-photon

• Special applications: FRETFLIMFRAPPhotoactivationTIRF

Fluorescence

Fluorescence

Excited state

Ground state

excitation

shorter wavelength, higher energy

emission

longer wavelength, less energy

Stoke’s shift

Fluorophores (Fluorochromes, chromophores)

• Special molecular structure

• Aromatic systems (Pi-systems) and metal complexes (with transition metals)

• characteristic excitation and emission spectra

Fluorophores

FiltersHow can we separate light with specific wavelength from the rest of the light?

Filters

Filter nomenclature• Excitation filters: x• Emission filters: m• Beamsplitter (dichroic mirror): bs, dc, FT

• 480/30 = the center wavelength is at 480nm; full bandwidth is 30 [ = +/- 15]

• BP = bandpass, light within the given range of wavelengths passes through (BP 450-490)

• LP = indicates a longpass filter which transmits wavelengths longer than the shown number and blocks shorter wavelengths (LP 500)

• SP = indicates a shortpass filter which transmits wavelengths shorter than the shown number,

and blocks longer wavelengths

Filter nomenclature

Filters

Multiple Band-Pass Filters

Basic idea

Basic design of epi fluorescence

Objective acts as condenser; excitation light reflected away from eyes

The cube

Excitation/emission spectra always a bit overlapping

filterblock has to separate them

a) Exitation filterb) Dichroic mirror (beamsplitter)a) Emission filter

The cube

Excitation / emissionexcitation and emission spectra of EGFP (green) and Cy5 (blue)

excitation and emission spectra of EGFP (green) and Cy2 (blue)

No filter can separate these wavelengths!

Where to check spectra?You can plot and compare spectra and check spectra compatibility for many fluorophores using the following Spectra Viewers.

Invitrogen Data Base BD Fluorescence Spectrum Viewer University of Arizona Data Base Fluorescent Probe Excitation Efficiency (Olympus jave tutorial)

Choosing Fluorophore Combinations for Confocal Microscopy (Olympus java tutorial)

Photobleaching• Photobleaching - When a fluorophore permanently loses the ability

to fluoresce due to photon-induced chemical damage and covalent modification.

Photobleaching• At low excitation intensities, pb occurs but at lower rate.

• Bleaching is often photodynamic - involves light and oxygen.• Singlet oxygen has a lifetime of ~1 µs and a diffusion coefficient

~10-5 cm2/s. Therefore, potential photodamage radius is ~50 nm.

Standard techniques

• wide-field• Confocal• Spinning disk confocal• 2-photon

Wide-field fluorescence

• reflected light method

• Multiple wavelength source (polychromatic, i.e. mercury lamp)

• Illumination of whole sample

Wide-field vs confocal

Wide-field image confocal image

Molecular probes test slide Nr 4, mouse intestine

Widefield Illumination Point Illumination

Point illumination

Light sources for point illumination

Sample

Objective lens

Excitation light

Excitation light must be focused to adiffraction limited spot

Could be done with an arc lampand pinhole – but very inefficient

Enter the laser: Perfectly collimated and high power

Fluorescence Illumination of a single point

Sample

Objective lens

Excitation lightTube lens

Emission light

Problem – fluorescence is emitted along entire illuminated cone, not just at focus

Camera

Point illumination

The confocal microscope

Sample

Objective lens

Excitation lightTube lens

Emission light

Pinhole

Detector

• method to get rid of the out of focus light less blur

• whole sample illuminated (by scanning single wavelength laser)

• only light from the focal plane is passing through the pinhole to the detector

The confocal microscope

Scanning

Sample

Objective lens

Changing entrance angle of illumination moves illumination spot on sample

The emission spot moves, so we have to make sure pinhole is coincident with it

Improved PSF and pinhole size

PSF for the focal plane and planes parallel to it: (a) conventional diffraction pattern (b) Confocal case.

Why can’t it be as small as possible?

• Reduced number of photons that arrive at the detector from the specimen may lead to a reduced signal-to-noise ratio.

• Raising the intensity of the excitation light can damage the specimen.

• Optical sectioning does not improve considerably with the pinhole size below a limit that approximates the radius of the first zero of the Airy disk.

How big should your pinhole be?• Width of point spread function at pinhole =

Airy disk diameter × magnification of lens = 1 Airy unit= resolution of lens × magnification of lens × 2

– 100x / 1.4 NA: resolution = 220nm, so 1 Airy unit = 44 m

– 40x / 1.3 NA: resolution = 235nm, so 1 Airy unit = 19 m

– 20x / 0.75 NA: resolution = 407nm, so 1 Airy unit = 16 m

– 10x / 0.45 NA: resolution = 678nm, so 1 Airy unit = 14 m

How big should your pinhole be?• A pinhole of 1 airy unit (AU) gives the best signal/noise.• A pinhole of 0.5 airy units (AU) will often improve resolution

IF THE SIGNAL IS STRONG.

ConfocalUse:• to reduce blur in the picture high contrast

fluorescence pictures (low background)• optical sectioning (without cutting);

3D reassembly possible

Careful: increasing image size (more pixels) does not mean that the objective can resolve the same!!! (resolution determined by NA, a property of the objective)

Spinning Disk Confocal

Spinning Disk

• Fast – multiple points are illuminated at once • Photon efficient – high QE of CCD• Gentler on live samples – usually lower laser

power

• Fixed pinhole• Small field of view (usually)• Crosstalk through adjacent pinholes limits

sample thickness

Relative Sensitivity

• Widefield 100• Spinning-Disk Confocal 25• Laser-scanning Confocal 1

• See Murray JM et al, J. Microscopy 2007 vol. 228 p390-405

Excited state

Ground state

2-photon microscopy

Excitation: long wavelength (low energy)

Each photon gives ½ the required energy

Emission: shorter wavelength (higher energy) than excitation

2-photon microscopy

Advantages: IR light penetrates deeper into the tissue than shorter wavelength

2-photon excitation only occurs at the focal plane less bleaching above and below the section

Use for deep tissue imaging

Use of lower energy light to excite the sample (higher wavelength)

1-photon: 488nm

2-photon: 843nm

Special applications:

• FRET and FLIM

• FRAP/FLIP and photoactivation

• TIRF

FRET (Fluorescence Resonance Energy Transfer)

• method to investigate molecular interactions• Principle: a close acceptor molecule can take the excitation energy

from the donor (distance 1-10 nm)

Donor (GFP)

FRET situation: Excitation of the donor (GFP) but emission comes from the acceptor (RFP)

Exited state

Ground state

Acceptor (RFP)

Exited state

Ground state

Energy transfer, no emission!

Exited state

Ground state

No FRET

• Both Acceptor and Donor are fluorescent• The Donor is excited and its emission excites the Acceptor

Ex(D)

Ex(A)Em(D)

Em(A)

FRET

• FRET is a competing process for the disposition of the energy of a photo excited electron.

• Donor emission decreases

• Donor lifetime decreases

• Acceptor emission increases

FRET

Energy transfer efficiency

• Depends on: Donor emission and acceptor absorption spectra, relative orientation of D and A

FRET

FRET

FRETways to measure:

• Acceptor emissionDetect the emission of the acceptor after excitation of the donor, e.g. excite GFP with 488 but detect RFP at 610 (GFP emission at 520)

• Donor emission after acceptor bleaching take image of donor, then bleach acceptor (with acceptor excitation wavelength - RFP:580nm), take another image of donor should be brighter!

Advantages Disadvantages

Cheap implementation Free fluorophors can mask energy transfer

high resolution (1-10nm) pH sensitive

Living cells Weak effect

Real time Location of fluorophors is critical

FRET

FLIM (Fluorescence Lifetime Imaging Microscopy)

• measures the lifetime of the excited state (delay between excitation and emission)

• every fluorophore has a unique natural lifetime

• lifetime can be changed by the environment, such as: Ion concentrationOxygen concentrationpHProtein-protein interactions

∆t=lifetime

FLIM - advantagesIn this method we measure the lifetime of the excited

state and not the fluorescence intensity, therefore: • We can separate fluorophores with similar spectra.• We minimize the effect of photon scattering in thick

layers of sample.

1 2

1/e

lifetime = ½ of all electrons are fallen back

FLIM - Measurement approaches

•Frequency domain• Modulated excitation• Lock-in detect emission phase

•Time domain (pulsed exc.)

•Gated intensifierPhoton inefficient

•Time-correlatedsingle photon counting

Very efficient one photon per pulse slow Time gates

FLIM

Excitation of many electrons at the same time count the different times when they are falling back down (i.e. photons are emitted)

lifetime = ½ of all electrons are fallen backdecay curve

Lifetime histogram

Example of FLIM-FRET measurement

GFP expressed in COS 1 cell: average lifetime of 2523 ps

fused GFP-RFP expressed in COS 1 cell: average lifetime of 2108 ps

Joan Grindlay, R7

FLIM

Hepatocyte membrane-stainedwith NBD, which has a

hydrophobicity-dependent lifetime(TCSPC, 3 minutes for 300x300 pixels )

For FLIM-FRET you still need: a suitable FRET-pair with the right orientation of the π-orbitals

Interaction of proteins is not enough, because fluorophores have to be close enough and in the right orientation!

Use of FLIM: measurements of concentration changes (Ca+2), pH change etc, Protein interactions

FLIM

Special applications:

• FRET and FLIM

• FRAP/FLIP and photoactivation• TIRF

Need: to probe transportIdea: bleach in one area,watch recovery by transport from other areas

FRAP (Fluorescence Recovery After Photobleaching)

Measuring Cdc42 diffusion constant in yeast

Result: df = (0.036 ± 0.017) μm2/s Marco et al. 2007 Cell 129:411-422

FRAP

FRAP

• Intense illumination with 405 laser bleaches the sample within the selected region observation of the recovery

before 0.65 s 0.78 s

Use: to measure the mobility/dynamics of proteins under different conditions

FLIP(Fluorescence Loss in Photo-bleaching)

Need: probe connectivityIdea: bleach in one compartment,watch loss in connected compartments by exchange

Bleach one area repeatedly. Entire ER dims. ER is contiguous

Photoactivation(Better?) FRAP/FLIP alternative

Some fluorophores can be activated by light

• Photo-uncagable dyes• GFP-family proteins

Activate a small areaWatch fluorescence spread

Look for weak lightagainst dark backgroundInstead of slight dimmingof bright background

photoactivation• Fluorophore only becomes active (= fluorescent) if

excited (e.g. with 405 laser) due to structural change

Pictures taken from a activation movie: activation of a line trough the lamellipodia of the cell, activated GFP_F diffuses quickly

Photoactivation - Proteins

Off-On•PA-GFP, PS-CFP

Color change•Kaede, KikGR, Eos,•Dendra (activatable by blue)

Reversibly Switchable•asCP, KFP (tetrameric)•Dronpa

Activate

green red

before

after

Dendra2 demo

Dronpa – photoswitchable on and off

Ando et al. 2004, Science 306: 1370-1373

photoactivation

Tracking actin flow with Dronpa

Kiuchi, T. et al. J. Cell Biol. 2007;177:465-476

photoactivation

Special applications:

• FRET and FLIM• FRAP/FLIP and photoactivation

• TIRF

TIRF (Total Internal Reflection Fluorescence)

You need:

• TIRF objectives with high NA

• TIRF condensor, where you are able to change the angle of illumination

• Glass coverslips

TIRF

micro.magnet.fsu.edu

Result: very thin section at the bottom of the sample 150-200nm

Use: to study membrane dynamics (endocytosis, focal adhesions, receptor binding)

Nikon TE 2000

TIRF vs epi

FAK-lasp in epi mode (wide field)

FAK-lasp in tirf mode (wide field)Heather Spence, R10

TIRF vs epi

Lasp in TIRF mode

Lasp in confocal sectioning

Heather Spence, R10

Summary/comparisonmethod excitation detection sectioning use

Wide field Whole sample Whole sample No sectioningSimple fluorescence samples

confocal Whole sample One z-plane 350-500nmHigh contrast images, optical sectioning

2-Photon One z-plane One z-plane 500-700nmDeep tissue imaging, optical sectioning

FRETProtein interactions, small distances

FLIMEnvironmental changes, protein interactions

FRAP/FLIP + photoactivation

dynamics/mobility

TIRFOnly bottom plane

Only bottom plane

150-200nm Membrane dynamics

Light source for fluorescence microscopyArc lamps

XenonMercury

UV IR

Laser types

Argon 351 364 457 477 488 514

Blue diode 405 440

Helium-Cadmium 354 442

Krypton-Argon 488 569 647

Green Helium-Neon 543

Yellow Helium-Neon 594

Orange Helium-Neon 612

Red Helium-Neon 633

Red diode 635 650

Ti:Sapphire 720-980