Fluorescence microscopy Tools and applications

1. Fluorescence microscopyTools and applications Sylvie Le Guyader Live Cell Imaging Unit, Karolinska Institute, Huddinge sylvie.le.guyader@ki.se

2. 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

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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. Light is an electromagnetic wave created by photons Wavelength l microwaves

11. 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

12. 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

13. 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?

14. How can we make specific molecules fluoresce in live cells?

15. 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

16. 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

17. 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

18. Fluorescence microscopy is beautiful!  Hoechst ReAsh Antibody-QD565 GFP Antibody-Cy5 actin nuclei mitochondria microtubules Golgi Giepmans et al Science 2006

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

20. 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

21. 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

22. Only the green fluorophore is imaged by the green filter set Ex Eliminates reflected Ex D Em Eliminates red EM (bleed through)

23. Only the red fluorophore is imaged by the red filter set Ex D Em

24. Filters allow imaging of several fluorophores Bright field (DIC) GFP-Myo10 DsRed-actin overlay filopodium

25. 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?

26. Can we image fluorescent volumes?

27. 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)

28. 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

29. 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

30. 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

31. 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

32. 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...)

33. Confocals give z information without image processing

34. 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

35. 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

36. The many F-words of microscopy!

37. 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

38. 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

39. 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

40. Intra/intermolecular FRET

41. Intramolecular FRET with biosensors Red high FRET Blue low FRET Green intermediate http://web.mit.edu/chemistry/Ting_Lab/movies.html

42. What is super resolution?

43. 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)

44. Super-resolution microscopy • TIRF • Light shaping techniques • SIM • STED • Single molecule localization techniques • PALM • STORM

45. 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

46. 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

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

48. Trends in the field of fluorescence microscopy

49. 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

50. 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