Fluorescence microscopy tools and applications
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Fluorescence microscopy Tools and applications. Sylvie Le Guyader Live Cell Imaging Unit, Karolinska Institute, Huddinge [email protected] How can we force specific molecules to fluoresce in live cells? How can we image several fluorophores with a black and white detector?

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Fluorescence microscopy tools and applications

Fluorescence microscopyTools and applications

Sylvie Le Guyader

Live Cell Imaging Unit, Karolinska Institute, Huddinge

[email protected]


Fluorescence microscopy tools and applications

  • How can we force specific molecules to fluoresce in live cells?

  • How can we image several fluorophores with a black and white detector?

  • Imaging volumes: 3D microscopy

  • F-words: Examples of microscopy techniques

  • What is super resolution?

  • Trends in the fluorescence microscopy field


What is and isn t fluorescence microscopy applied to biology

What is and isn’t fluorescence microscopy applied to Biology?


I will only talk about fluorescence microscopy applied to biology

I will only talk about fluorescence microscopy applied to Biology

  • Within microscopy

    • material imaging

    • medical imaging

    • electron microscopy (transmission or scanning)

    • scanning probe microscopy

    • light microscopy

      • bright field microscopy

      • fluorescence microscopy

        • widefieldmicroscopy

        • confocal microscopy


Fluorescence microscopy is not electron microscopy

Fluorescence microscopy is NOTelectron microscopy

  • Uses electron density, not light

  • Very high resolution (=how well are two points separated) 1 nm-1Å

  • No live imaging

  • Localization of up to 2 proteins using gold labeled antibodies

Transmission EM

zebrafish eye

Scanning EM

ant antenna


Fluorescence microscopy tools and applications

Fluorescence microscopy is NOTscanning probe microscopy

  • Atomic Force or Photonic Force microscopes

  • Solid probe tip in the vicinity of the object

  • The probe ‘feels’ the surface by measuring local forces

  • Forces: contact, capillary, chemical bonding, electrostatic, magnetic…

  • nm resolution

  • Surface imaging only

http://en.wikipedia.org/wiki/Atomic_force_microscope


Fluorescence microscopy is not bright field microscopy

Fluorescence microscopy is NOTbright field microscopy

  • Uses light absorption. Transmitted (= not absorbed) light is imaged.

  • Resolution ~200 nm limited by light wavelength

  • Low contrast enhanced using optical elements or colored dyes

  • Fixed or live cells

DIC

Haematoxylin/eosin stains

Phase contrast


I will only talk about fluorescence microscopy applied to biology1

I will only talk about fluorescence microscopy applied to Biology

  • Within microscopy

    • material imaging

    • medical imaging

    • electron microscopy (transmission or scanning)

    • scanning probe microscopy

    • light microscopy

      • bright field microscopy

      • fluorescence microscopy

        • widefield microscopy

        • confocal microscopy


Light is an electromagnetic wave created by photons

Light is an electromagnetic wave created by photons

  • A point charge at rest produces an electric field

  • A point charge moving with constant speed produces an electric field and a magnetic field

  • A point charge moving with a varying speed (acceleration) produces an electromagnetic wave which can travel at 3.108m/s though empty space

  • Light is an electromagnetic wave created by accelerating photons

  • How the speed of the charge changes determines the length of the wave, how energetic it is and how focussed the beam can be


Fluorescence microscopy tools and applications

Light is an electromagnetic wave created by photons

Wavelength l

microwaves


Fluorescence is a cycle of photon absorption and emission

Fluorescence is a cycle of photon absorption and emission

  • Only some molecules can fluoresce: fluorophore/fluorochrome

  • Absorption, loss and emission of energy: Stokes shift

  • Phosphorescence, bioluminescence and fluorescence

http://www.olympusmicro.com/primer/lightandcolor/fluorointroduction.html


Imaging live cells in many dimensions

Imaging live cells in many dimensions

  • Volume: xyz

  • Specific molecules: l

  • Live cells: t

  • Experiments: cell types, drugs…

  • Super resolution (20 nm)

  • Contrast: single molecule

  • Incubator

  • Photobleaching!

  • Bleed through!

  • Light toxicity!

  • Live cells move!

  • Automation

  • Data analysis and management

http://www.microscopyu.com/articles/livecellimaging/fpimaging.html


We can learn a lot from fluorescence microscopy

We can learn a lot from fluorescence microscopy!

  • Where is this protein synthesized?

  • Does it get degraded if I add a drug?

  • Are these 2 proteins in the same cell compartment at the same time?

  • Do they physically interact with each other?

  • Do they phosphorylate each other?

  • Is this protein part of a larger molecular complex?

  • How can one side of a cell send a signal to another? (e.g. Ca2+ waves)

  • How fast is each molecule removed and replaced by a new one?

  • Can we use imaging to find new drugs?


How can we make specific molecules fluoresce in live cells

How can we make specific molecules fluoresce in live cells?


Some fluorophores are small chemicals

Some fluorophores are small chemicals

Quantum dots

  • Small and highly conducting crystals that fluoresce

  • Highly photostable, very bright

  • Excited by blue light

  • Toxic

    Organic dyes

  • FITC, TRITC, Rhodamine, Cy3/5, DiI, Alexa...

  • Markers for pH, calcium, membrane, cytoplasm...

Few antibody applications for live cells

FITC

http://qt.tn.tudelft.nl/grkouwen/qdotsite.html


Some fluorophores are proteins produced by cells after genetic modification

Some fluorophores are proteins produced by cells after genetic modification

GFP

Fluorescent fusion proteins

  • Plasmid with a cDNA coding for the fluorophore fused to the protein of interest

  • Transfected cells express the fusion protein

    FPs require overexpression of a highly modified protein which can lead to artifacts

http://www-bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html


Fps come in many colours

FPs come in many colours

  • Photoswitchable(green to red)

  • Photoactivatable(non fluorescent to fluorescent)

  • Conformation sensors

Miyawaki et al. Nat Rev Mol Cell Biol 2003

Shaner et al Nat Biotech 2004

mRFP1 derivatives


Fluorescence microscopy is beautiful

Fluorescence microscopy is beautiful! 

Hoechst

ReAsh

Antibody-QD565

GFP

Antibody-Cy5

actin

nuclei

mitochondria

microtubules

Golgi

Giepmans et al

Science 2006


How can we image several fluorophores with a black and white detector

How can we image several fluorophores with a black and white detector?


Fluorescence microscopy tools and applications

Signals coming from different fluorophores must be separated

  • GFP is excited by 350-530 nm light

  • 488 nm light (blue) is most efficient to excite GFP but it also excites mCherry

  • GFP emits light at 480-600 nm but most light is emitted at 512 nm (green)

  • Lasers (narrow bandpass) help reduce bleed through

Omega Optical Filters webpage/Curvomatic


The signals are separated using a set of 3 filters

The signals are separated using a set of 3 filters

  • 3 filters per filter set:

    • Excitation filter

    • Dichroic mirror

    • Emission filter

  • Why do we need filters?

    • The dichroic mirror prevents the excitation light reflected by the object from reaching the detector.

    • The excitation and emission filters allow separation of several signals in a sample labeled with several fluorophores.

Objective

Emitter

Exciter

Dichroic

mirror


Only the green fluorophore is imaged by the green filter set

Only the green fluorophore is imaged by the green filter set

Ex

Eliminates reflected Ex

D

Em

Eliminates red EM (bleed through)


Only the red fluorophore is imaged by the red filter set

Only the red fluorophore is imaged by the red filter set

Ex

D

Em


Filters allow imaging of several fluorophores

Filters allow imaging of several fluorophores

Bright field (DIC)

GFP-Myo10

DsRed-actin

overlay

filopodium


How to choose a fluorophore

How to choose a fluorophore?

  • I think that human cancer cells produce more of my favourite protein P than normal cells in the surrounding tissue. How can I show that?

  • P is known to bind DNA. I hypothesize that when cells touch each other, P goes out of the nucleus, briefly binds the plasma membrane then goes back to the nucleus. How can I investigate this?

  • When P is at the plasma membrane, the whole cell suddenly moves in the opposite direction. I suspect that P communicate with the whole cell via fast calcium pulses. How can I check that?


Can we image fluorescent volumes

Can we image fluorescent volumes?


Improving resolution in x y and z

Improving resolution in x, y and z

  • Widefield microscopy

    • Deconvolution

  • Confocal microscopy

    • Laser point scanning microscopy

    • Spinning disk microscopy

    • Two-photon microscopy

  • Super resolution (= nanoscopy)

    • Total Internal Reflection Fluorescence (TIRF)

    • Structured Illumination (SIM)

    • Stimulated Emission Depletion (STED)

    • Stochastic Optical Reconstruction (PALM-STORM)


Images of fluo volumes are blurry

z

y

x

XZ

XY

Images of fluo volumes are blurry

Blue laser

  • The image of a fluorescent dot passing through a lens is mis-shaped (hour glass) according to a known mathematical transformation called Point Spread Function (PSF)

  • The image of a fluorescent volume is blurry!

Plane of interest

  • Fluorescence from objects above and below the plane creates a haze

http://en.wikipedia.org/wiki/Point_spread_function


Widefield imaging is fast but volume images are blurry

Widefield imaging is fastbut volume images are blurry

  • The whole field is illuminated at once

  • The whole emission is captured at once by a digital camera

  • Similar to flash photography

  • (+) Very fast

  • (+) Sensitive

  • (-) Volume images are blurry


Widefield volumes can be deblurred by deconvolution

Widefield volumes can be deblurred by deconvolution

Before

After

Schizosaccharomyces pombe (3-4 um)

  • Images acquired at different z levels with a widefield microscope

  • Computational calculation done at each z level

  • Reassign light from each blurry spot back to a dot using 1/PSF

  • (+) Allows volume imaging in widefield

  • (-) Can create artifacts

  • (-) Requires heavy computation after acquisition

http://micro.salk.edu/dv/dv.html


Fluorescence microscopy tools and applications

Confocal microscopy allows immediate volume imaging

  • Laser scanning confocals: the laser excites the sample point by point, line by line

  • A pinhole in front of the detector eliminates the light coming from out of focus planes

  • ‘Optical sectioning’ of the sample: throwing away most light!

  • Laser scanning confocal (‘confocal’) or spinning disk confocal

pinhole


Fluorescence microscopy tools and applications

Laser scanning microscopes are slower but more versatile

  • (+) Immediate high resolution in x, y and z

  • (+) Flexibility in resolution, zoom, scan speed…

  • (+) Allows bleaching/activating of small regions

  • (+) The emission light can be split into its spectral component

  • (-) Slower

  • (-) Detectors are slightly less sensitive than cameras

  • (-) Stronger excitation required (more bleaching, light toxicity...)


Confocals give z information without image processing

Confocals give z information without image processing


In two photon microscopy only the plane of interest is excited

In two-photon microscopy, only the plane of interest is excited

  • Laser scanning microscope

  • Tunable Infra-Red pulsed laser

  • The density of light at the focal plane allows fluorophores to absorb 2 photons of low energy (high l) almost simultaneously

  • Fluorophore excitation occurs only at the focal plane

http://www.udel.edu/bio/research/facilities/microscopy/equipment/multimgs.html


Penetration is enhanced but resolution is lower

Penetration is enhanced but resolution is lower

Confocal

Two-photon

(+) Low energy light (IR) is less toxic

(+) No out of focus light is produced

(+) Low energy light is more penetrating

(-) Less specific excitation

(-) The longer excitation l limits the resolution

http://www.udel.edu/bio/research/facilities/microscopy/equipment/multimgs.html


The many f words of microscopy

The many F-words of microscopy!


Fluorescence microscopy tools and applications

FRAP shows how fast molecules are replaced within a structure

  • Fluorescence Recovery After Photobleaching

  • What for?

    • Shows how fast molecules are replaced within a structure (turn-over)

    • The turn-over slows down when molecules bind to each other

  • Method?

    • Done with a laser scanning confocal

    • Photobleaching of the fluorophore in a small region

    • Measurement of the recovery of fluorescence at the bleaching point


Fluorescence microscopy tools and applications

FRAP

  • GFP:

    • Diffuses freely in the cytoplasm

    • Recovers very fast

  • mRFP1-Myosin-X:

    • Attached to an unknown structure

    • Recovers only when the structure is replaced

Bleached region


Fluorescence microscopy tools and applications

FRET shows if 2 proteins are in direct contact

  • Fluorescence Resonance Energy Transfer

  • What for?

    • Shows the distance between two proteins or two domains of the same protein (10 nm)

    • Indicates direct binding, conformation changes…

  • Method?

    • Done with widefield or confocal microscopes

    • The two proteins of interest are fused to fluorescent proteins that are able to transmit energy from donor to acceptor


Fluorescence microscopy tools and applications

Intra/intermolecular FRET


Intramolecular fret with biosensors

Intramolecular FRET with biosensors

Redhigh FRET

Blue low FRET

Green intermediate

http://web.mit.edu/chemistry/Ting_Lab/movies.html


What is super resolution

What is super resolution?


The importance of contrast

The importance of contrast

  • When the best possible resolution is optically achieved, improving contrast can still add information

  • Fluorescence is an excellent contrasting method (luminous object in a dark background)


Super resolution microscopy

Super-resolution microscopy

  • TIRF

  • Light shaping techniques

    • SIM

    • STED

  • Single molecule localization techniques

    • PALM

    • STORM


Stimulated emission depletion sted

Stimulated emission depletion STED

  • Confocal microscope

  • 2 lasers

  • Emission depletion

  • Improved xyz resolution

  • ‘A guide to super-resolution fluorescence microscopy’ Schermelleh et al, Journal of Cell Biology, 190(2):165-175, 2010


Photoactivated localization stochastic optical reconstruction palm storm

Photoactivated localization/ Stochastic optical reconstruction PALM/STORM

  • Widefield microscope

  • Photoswitching fluorophores

  • 20,000 images/ final image

  • Best xyz resolution

  • ‘A guide to super-resolution fluorescence microscopy’ Schermelleh et al, Journal of Cell Biology, 190(2):165-175, 2010


Super resolution microscopy1

Super resolution microscopy

Confocal

  • ‘A guide to super-resolution fluorescence microscopy’ Schermelleh et al, Journal of Cell Biology, 190(2):165-175, 2010


Fluorescence microscopy tools and applications

Trends in the field of fluorescence microscopy


Important issues for cell biology and biomedicine

Important issues for cell biology and biomedicine

  • Live imaging: 3D cultures, in vivo, ex vivo...

  • Sensitivity: single molecule imaging

  • Space: Intracellular localization

    colocalization of one or several molecules, compartments...

  • Time: Dynamics (time lapse in live cells)

    • how molecules move relative to each other, are synthesized, degraded, how fast do they turn over in a structure?

  • Quantitative analysis: Statistics

    • Imaging of binding, activation, phosphorylation, change of conformation...

    • Large screens


Excellent websites and reviews

Excellent websites and reviews

  • Interactive tutorials:

    http://micro.magnet.fsu.edu/primer/index.html

  • Microscopy tutorials on the Nikon (MicroscopyU), Olympus (Microscopy resource centre), Zeiss (Campus) and Leica websites

  • ‘Fluorescence microscopy’, Lichtman and Conchello, Nature Methods 2 (12):910, 2005

  • ‘Optical sectioning microscopy’, Conchello and Lichtman, Nature Methods 2 (12):920, 2005


Live cells in a collagen matrix

Live cells in a collagen matrix

Hamdah Abbasi, KI

Andrew Paterson, KI


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