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-contrastDark-field שיטות להגברת ניגודיות

Fluorescence techniques Standard techniques:wide-field confocal 2-photon Special applications:FRET FLIM FRAP Photoactivation TIRF

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

Filters How 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 ) 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 filter b) Dichroic mirror (beamsplitter) a) Emission filter

The cube

Excitation / emission excitation 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 EfficiencyFluorescent Probe Excitation Efficiency (Olympus jave tutorial) Choosing Fluorophore Combinations for Confocal MicroscopyChoosing 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 cm 2 /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 IlluminationPoint Illumination Point illumination

Light sources for point illumination Sample Objective lens Excitation light Excitation light must be focused to a diffraction limited spot Could be done with an arc lamp and pinhole – but very inefficient Enter the laser: Perfectly collimated and high power

Fluorescence Illumination of a single point Sample Objective lens Excitation light Tube 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 light Tube 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.

Confocal Use: 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 Widefield100 Spinning-Disk Confocal25 Laser-scanning Confocal1 See Murray JM et al, J. Microscopy 2007 vol. 228 p

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

ways to measure: Acceptor emission Detect 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!

DisadvantagesAdvantages Free fluorophors can mask energy transfer Cheap implementation pH sensitivehigh resolution (1-10nm) Weak effectLiving cells Location of fluorophors is critical Real time 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 concentration Oxygen concentration pH Protein-protein interactions ∆t=lifetime

FLIM - advantages In 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1 2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 intensifier Photon inefficient Time-correlated single 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 back decay 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-stained with 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 transport Idea: bleach in one area, watch recovery by transport from other areas FRAP (Fluorescence Recovery After Photobleaching)

Measuring Cdc42 diffusion constant in yeast Result: d f = (0.036 ± 0.017) μm 2 /s Marco et al Cell 129: FRAP

Intense illumination with 405 laser bleaches the sample within the selected region  observation of the recovery before0.65 s0.78 s Use: to measure the mobility/dynamics of proteins under different conditions

FLIP (Fluorescence Loss in Photo-bleaching) Need: probe connectivity Idea: 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 area Watch fluorescence spread Look for weak light against dark background Instead of slight dimming of 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: photoactivation

Tracking actin flow with Dronpa Kiuchi, T. et al. J. Cell Biol. 2007;177: 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  nm 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/comparison methodexcitationdetectionsectioninguse Wide field Whole sample No sectioning Simple fluorescence samples confocal Whole sampleOne z-plane nm High contrast images, optical sectioning 2-Photon One z-plane nm Deep tissue imaging, optical sectioning FRET Protein interactions, small distances FLIM Environmental changes, protein interactions FRAP/FLIP + photoactivation dynamics/mobility TIRF Only bottom plane nm Membrane dynamics

Light source for fluorescence microscopy Arc lamps Xenon Mercury UVIR Laser types Argon Blue diode Helium-Cadmium Krypton-Argon Green Helium-Neon 543 Yellow Helium-Neon 594 Orange Helium-Neon 612 Red Helium-Neon 633 Red diode Ti:Sapphire