Fluorescence microscopy

Transcription

1 Fluorescence microscopy 1

2 Fluorescence microscopies basic fluorescence, fluorophores Deconvolution Confocal Two-photon/multi-photon 4Pi Light sheet Total internal reflection STED FRAP/FLIP/FCS FRET PALM/STORM/iPALM

3 Jablonski diagram bleaching quantum yield = light emitted/light absorbed Q ~ 0.8 fluorescein ~ 0.3 rhodamine

4 ex: UV em: blue ex: blue em: green ex: green em: red Cox Fig. 3.5

5 Stokes shift

6 ReAsH and FlAsH: fluorescent arsenical helix-binders Membrane permeant Highly specific to tetracysteines Only fluoresce when bound to 4-Cys target Toxic Catalyzes photoconversion of DAB into an insoluble osmiophilic precipitate (light on FlAsH in the presence of oxygen produces radicals that cause localized polymerization of diamino benzidine (DAB), which in turns binds osmium, which is visible in the EM)

7 FlAsH/ReAsH can be used for correlated light and electron microscopy Connexins in a gap junction Gaietta et al. (2002). Science 296:503

8 eapex monomeric 28 kda peroxidase engineered to oxidize diaminobenzidine (DAB) genetically-encoded active in all cellular compartments does not require light (just add DAB and hydrogen peroxide - allows use deep inside tissues). withstands strong EM fixation to give excellent ultrastructural preservation. minisog is a similar tag Nat Biotechnol Nov; 30(11): PLoS Biol 9(4): e doi: /journal.pbio

9 GFP and its derivatives Lippincott-Schwartz and Patterson (2003). Science 300:87 * from the jellyfish Aequoria victoria, whose tentacles glow * highly stable: 8 M urea, 1% SDS, aldehyde fixation, ph , 65 * 27 kda, single gene, no post-transcriptional mods or co-factors needed * does require maturation time (side chains react to form fluorophore) * now other jellyfish and corals found with variants * disadvantage is that it can disrupt the localization and function of targets it is fused to

10 Fluorescent proteins now available in many colors Excitation Emission Lippincott-Schwartz and Patterson (2003). Science 300:87

11 Quantum dots as markers for correlated light and electron microscopy

12 Basic fluorescence microscope

13 Illuminators and their emission spectra Murphy Fig. 3-1

14 Acousto-optical tunable filter (AOTF) Frequency of sound input determines which incoming wavelength is deflected into the microscope. The strength of the signal (loudness) determines the percent of the selected wavelength that is deflected. Cox Fig. 5.11,13

15 lasers emit single wavelength, eliminating need for first filter Cox Fig. 5.11,13

16 Example fluorescent light microscope images Reider and Khodjakov Science 300 cover ~2 microns Moller-Jensen et al., (2003). Mol Cell 12:1477

17 Point spread function Object Image Cox Fig. 10.1, 3

18 Point spread function is 3-D If a fluorophore is just above or below the focal plane, a different cross-section through the 3-D PSF is seen

19 First way to reduce 3-D blurring: Deconvolution Compute model of what might have generated the image Image blurred by PSF Compare and iterate Compute how model would be blurred by PSF

20 Polytene chromosome from Drosophila stained with fluorescent DNA-binding dye before After deconvolution

21 Second way to reduce 3-D blurring: confocal microscopy

22 Excitation and detection both fall off by the distance from the focus squared. Result is optical sectioning with sensitivity falling off by (distance from the focus) 4.

23 confocal deconvolution Stephens and Allan (2003). Science 300:82

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25 Cox Fig. 5.15

26 Photon multiplier tube Cox Fig. 5.18

27 Spinning disk

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29 Excitation and detection both fall off by the distance from the focus squared. Result is optical sectioning with sensitivity falling off by (distance from the focus) 4. Bad news is that regions not generating the image are still illuminated

30 Two-Photon Excited Fluorescence Very low probability: requires intense pulsed laser light Requires two photons, so excitation is a function of [exciting light] 2 Intensity of exciting light falls off by (distance from focus) 2 Thus, emission falls off by (distance from focus) 4 Courtesy Scott Fraser

31 2-photon excitation Confocal excitation

32 4Pi microscopy Idea is to make excitation focus smaller and collect twice as many fluorescence photons by having two objective lenses

33 Immunostained Hela cell (Alexa488 on an anti-alpha-tubulin) Regular confocal 4Pi 4Pi + deconvolution Cox Fig

34 Light sheet microscopy Image by Zeiss Microscopy via Wiki Commons Chen et al. Lattice light-sheet microscropy: Imaging molecules to embryos at high spatiotemporal resolution. Science. 2014

35 Total internal reflection microscopy (TIRF) Following Snell s law, there is a critical angle at which the refracted beam never leaves the sample, but there is an evanescent wave that penetrates Sin(alpha)/sin(exit) = n water /n glass Where does exit = 90? sin(exit) = 1, and alpha = sin -1 (n water /n glass ) Stephens and Allan (2003). Science 300:82

36 GFP-tagging can lead to mislocalizanon Caulobacter proteins (~2,800: 74% of predicted ORFs that were tagged successfully and imaged) Same localizanon 58 N-terminal GFP fusions that localized 165 C-terminal fusions that localized 187 Different 5 data from Werner et al., PNAS 106:7858 (2009)

37 Shih et al., PNAS 2003

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39 Stimulated emission depletion (STED) Cox Fig

40 Super-resolution STED Stephan Hell Nobel Prize

41 Membrane of Bacillus megaterium, imaged by 4Pi/STED microscopy Dybe & Hell, Phys. Rev. Lett. 2002

42 Lippincott-Schwartz and Patterson (2003). Science 300:87

43 Fluorescence correlation spectroscopy Cox Fig

44 FRET: fluorescent resonance energy transfer R0 is the so-called Forster radius, which is the distance at which transfer is 50% efficient. FRET strength theoretically falls off as 1/distance 6, so has potential to indicate closeness, but in practice transfer depends heavily on orientation of dipole moments and perhaps other factors, so in practice is more of a close or far measurement if it works at all.

45 Example use of FRET: Cameleon Ca2+ sensor Adapted from Brasselet et al., 2000, J. Phys. Chem. B 104,

46 Photo- acnvatable and photo- switchable fluorophores Mechanism #1: decarboxylation Examples: PA-GFP from Aequorea victoria 100 fold increase PS-CFP from Aequorea cooerulescens 1500 fold increase PA-mRFP from Discosoma sp (DsRed) 70 fold increase Lukyanov et al., Nature Reviews Molecular Cell Biology, 6:885 (2005)

47 Mechanism #2: backbone cleavage meosfp and Dendra2 Lukyanov et al., Nature Reviews Molecular Cell Biology, 6:885 (2005)

48 Mechanism #3: Reversible cis-trans chromophore isomerizanon Dronpa, rsfaslime and mtfp0.7 Lukyanov et al., Nature Reviews Molecular Cell Biology, 6:885 (2005)

49 Photoactivation localization microscopy (PALM) and Stochastic optical reconstruction microscopy (STORM)

50 Photo-activation localization microscopy (PALM) Eric Betzig,

51 Stochastic optical reconstruction microscopy (STORM)

52 Interferometric (i)palm Schtengel and Hess et al., PNAS 2009

53 Brightfield fluorescence PALM ipalm Harald Hess,

54 Who is doing it at Caltech and why? 54

55 Concept check questions What is a Jablonski diagram? What kinds of molecular structures fluoresce? How are small molecule fluors different from fluorescent proteins? What are FlAsH? ReAsH? Quantum dots? eapex? How is a fluorescence microscope different from the others we ve covered so far, for instance a bright field compound microscope? What is a point-spread-function? What is a contrast-transfer-function? What is convolution? How do deconvolution, 4-pi, confocal, and two-photon microscopy work? What are the advantages of light sheet microscopy? What is TIRF, FRET, and FCS? What is meant by a super-resolution technique? How are PALM/STORM, STED, and ipalm all different? What kinds of chemical mechanisms drive photo-activation? 55