2. 1 Bi 1 Lecture 11 Tuesday, April 16, 2006 Better Microscopes and Better Fluorescent Proteins

3. 2 1.The confocal microscope An experiment with the confocal: GFP-tagged GABA transporters 2.Fluorescence resonance energy transfer (FRET) In search of better fluorescent proteins for FRET: coral reefs molecular biology labs 3.Multiphoton microscopy Some examples with 2-photon microscopes Today’s data look noisy. Pioneering data are always noisy.

4. 3 Little Alberts Panel 1-1 exciting light only emitted light only beam-splitting (“dichroic”) mirror

5. 4 Confocal Microscope Big Alberts Figure 9-18 © Garland

6. 5 Na + -coupled cell membrane neurotransmitter transporters: Antidepressants (“SSRIs” = serotonin-selective reuptake inhibitors): Prozac, Zoloft, Paxil, Celexa, Luvox Drugs of abuse: MDMA Attention-deficit disorder medications: Ritalin, Dexedrine, Adderall, Strattera (?) Drugs of abuse: cocaine amphetamine Na + -coupled cell membrane serotonin transporter Na + -coupled cell membrane dopamine transporter cytosol outside major targets for drugs of therapy and abuse Presynaptic terminals From Lecture 5 Trademarks:

7. 6 Antiepileptic Na + -coupled cell membrane GABA transporter cytosol outside Presynaptic terminal GABA Na + -coupled cell membrane neurotransmitter transporters: “focus” on a transporter for GABA, a major inhibitory neurotransmiter

8. 7 Express DNA The biologist’s method for fluorescent labeling of living cells: attach a fluorescent protein Gene for your favorite proteinGene for GFP protein From Lecture 10 DNA sequences assure expression in the correct cells; Parts of the protein assure transport to the correct subcellular location

9. 8 COOH NH 2 A fusion protein: GABA transporter-GFP extracellular intracellular

10. 9 Hippocampus (memory) cerebellum (movement) Mouse expressing GABA transporter-GFP: all inhibitory neurons fluoresce, because they all express the GABA transporter, because they all use GABA as a neurotransmitter Pleasure system

11. 10 <- Anti-GABA transporter fluorescence GFP fluorescence ->

12. 11 50  m Confocal micrograph of mouse brain with GABA transporter-GFP fusion

13. 12 Confocal micrograph of GABA transporter-GFP fusion reveals presynaptic inhibitory terminals 50  m

14. 13 terminals calibration beads 1  m The limits of optical resolution: all the fluorescence is on the cell membrane... but... some researchers now resolve structures 10-fold smaller with optical microscopes

15. 14 In cultures from hippocampus, 10-15% of cells are inhibitory fluorescence fluorescence + bright-field bright-field

16. 15 1.The confocal microscope An experiment with the confocal: GFP-tagged GABA transporters 2.Fluorescence resonance energy transfer (FRET) In search of better fluorescent proteins for FRET: coral reefs molecular biology labs An experiment with FRET: this week’s problem set 3.Multiphoton microscopy Some examples with 2-photon microscopes

17. 16 Chemiluminescence in jellyfish ( Aequorea victoria) : what produces the exciting light?

18. 17 Chemiluminescence in jellyfish blue photon max = 470 nm aequorin + coelenterazine + O 2 triggered by Ca 2 entry aequorin + coelenteramide + CO 2 + hv protein: aequorin < 20% of the reactions produce a photon small molecule: coelenterazine

19. 18 Chemiluminescence resonance energy transfer in jellyfish “virtual” blue photon green photon max = 509 nm Efficiency depends on dipole orientation and on(1/distance) 6 ; increases by 3-5 fold aequorin + coelenterazine + O 2 triggered by Ca 2 entry aequorin + coelenteramide + CO 2 + hv GFP

20. 19 Hunting for new fluorescent proteins: Dr. Charles Mazel (MIT) Ph D in marine biology; Designs electronics for underwater instruments. also founded Nightsea (http://www.nightsea.com)http://www.nightsea.com

21. 20 exciter filter: blue light only barrier filter: no blue light

22. 21 exciter filter: blue light only barrier filter: no blue light for autofocus: “continuous” dive light 1 battery replaced by a blinker 7 seconds on; 1.5 seconds off

23. 22 1 st photos of fluorescent coral on the Great Barrier Reef, Australia Ben Lester, 2000

24. 23 no filters exciter filter only exciter plus barrier filter © Charles Mazel

25. 24 Normal white-light photograph of Caribbean giant anemone, Condylactis gigantea, Key West, Florida © Charles Mazel Blue-light fluorescence photograph of the anemone © Charles Mazel

26. 25 Burrowing anemone, Anthopleura artemisia Monterey Bay ©Jack Sullins Anemone with clownfish (note that the clownfish is not fluorescent, and appears black) Indonesia ©Stuart and Michele Westmorland Additional fluorescent cnidarians phylum, “stingers”, previously coelenterates

27. 26 Another way to find new fluorescent proteins: Site-Directed Mutagenesis RNA Gene (DNA) measure “Express” the protein with an altered side chain(s) Hypothesis about an important side chain(s) Mutate the desired codon(s)

28. 27 A pH-sensitive EGFP mutant reveals synaptic vesicle movements mutated GFP synaptic vesicle protein mutated EGFP GFP

29. 28 At rest Action potentials Stochastic vesicle release measured optically

30. 29 Enhanced fluorescent proteins: site-directed GFP mutants

31. 30 Another look at site-directed GFP mutants

32. 31 Fluorescence resonance energy transfer (FRET)

33. 32 Cyan Fluorescent Protein (CFP) blue photon (virtual) cyan photon

34. 33 < 10 nm Fluorescence resonance energy transfer (FRET) detects proximity Cyan Fluorescent Protein (CFP)Yellow Fluorescent Protein (YFP) blue photon virtual cyan photon yellow photon

35. 34 Detecting protein-protein contacts with FRET CFP YFP

36. 35 1.The confocal microscope An experiment with the confocal: GFP-tagged GABA transporters 2.Fluorescence resonance energy transfer (FRET) In search of better fluorescent proteins for FRET: coral reefs molecular biology labs An experiment with FRET: this week’s problem set 3.Multiphoton microscopy Some examples with 2-photon microscopes

37. 36 The multiphoton fluorescence microscope E = h ground state excited state “simultaneous”, within ~1/4 cycle. At a wavelength of 1  m, 1 cycle is  c  10 -6 m)/(3 x 10 8 m/s)/= 3 x 10 -15 s Therefore 2 photons must hit within ~ 10 -15 s = 1 fs.

38. 37 computer A two-photon Microscope Dichroic mirror Objective lens Photodetector titanium-sapphire laser X-Y scanning mirrors duty cycle is 10 -5

39. 38 Two-photon excitation eliminates out-of-plane bleaching, because excitation varies with the square of the power intensity single-photon excitation two-photon excitation

40. 39 © Cell Press Dichroic Mirror Pinhole Photodetector Objective lens Neuron in a scattering slice many blue rays scatterfew red rays scatter Pinhole not required Scattering causes minimal distortion in a 2-photon microscope. Very important for real tissue!

41. 40 Figure 1. Imaging in Scattering Media Without multiphoton excitation, one has to choose between resolution and efficient light collection when imaging in scattering samples. Nonlinear excitation imaging lifts that constraint as is illustrated here in a comparison to confocal 1-photon imaging (the scan optics are omitted for clarity). Typical fates of excitation (blue and red lines) and fluorescence (green lines) photons. In the confocal case (left), the excitation photons have a higher chance of being scattered (1 and 3) because of their shorter wavelength. Of the fluorescence photons generated in the sample, only ballistic (i.e., unscattered) photons (4) reach the photomultiplier detector (PMT) through the pinhole, which is necessary to reject photons originating from off-focus locations (5) but also rejects photons generated at the focus but whose direction and hence seeming place of origin have been changed by a scattering event (6). Excitation, photobleaching, and photodamage occur throughout a large part of the cell (green region). In the multiphoton case (right), a larger fraction of the excitation light reaches the focus (2 and 3), and the photons that are scattered (1) are too dilute to cause 2-photon absorption, which remains confined to the focal volume where the intensity is highest. Ballistic (4) and scattered photons (5) can be detected, as no pinhole is needed to reject fluorescence from off-focus locations. from Denk & Svoboda Neuron. 1997 18:351-7 http://www.neuron.org/cgi/content/full/18/3/351

42. 41 Two-photon image of a neuron filled with a harmless dye

43. 42 Two-photon images of synaptic spines moving within a slice of brain (EGFP-labelled neurons)

44. 43 voltage-gated Ca 2+ channel Electricity, then chemistry triggers synaptic vesicle fusion Ca 2+ docked vesicle neurotransmitter nerve impulse from Lecture 9

45. 44 Calcium-sensitive fluorescent dyes fluo-3 from Lecture 10

46. 45 Two-photon images of Ca 2+ entering a presynaptic terminal within a slice of brain

47. 46 End of Lecture 11