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Bi 1 Lecture 11

Tuesday, April 16, 2006

Better Microscopes and Better Fluorescent Proteins

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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 reefsmolecular biology labs

3. Multiphoton microscopy

Some examples with 2-photon microscopes

Today’s data look noisy.

Pioneering data are always noisy.

3Little Alberts Panel 1-1

exciting light only

emitted light only

beam-splitting(“dichroic”)

mirror

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Confocal Microscope

Big Alberts Figure 9-18 © Garland

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Na+-coupled cell membrane neurotransmitter transporters:

Antidepressants (“SSRIs” = serotonin-selectivereuptake inhibitors):Prozac, Zoloft, Paxil, Celexa, Luvox

Drugs of abuse: MDMA

Attention-deficit disorder medications:

Ritalin, Dexedrine, Adderall,Strattera (?)

Drugs of abuse: cocaine amphetamine

Na+-coupledcell membrane serotonintransporter

Na+-coupledcell membrane dopamine transporter

NH

HO NH3+

HO

HO

H2C

CH2

NH3+

cytosol

outside

major targets for drugs of therapy and abuse

Presynapticterminals

From Lecture 5

Trademarks:

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Antiepileptic

Na+-coupledcell membrane GABAtransporter

cytosol

outside

Presynapticterminal

GABA

Na+-coupled cell membrane neurotransmitter transporters: “focus” on a transporter for GABA, a major inhibitory neurotransmiter

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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

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COOH

NH2

A fusion protein: GABA transporter-GFP

extracellular

intracellular

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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

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<- Anti-GABA transporter fluorescence

GFP fluorescence ->

1150 m

Confocal micrograph of mouse brain with GABA transporter-GFP fusion

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Confocal micrograph of GABA transporter-GFP fusion reveals presynaptic inhibitory terminals

50 m

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-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.60

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40

60

80

100

120

Re

lati

ve

flu

ore

sc

en

t in

ten

sit

y (

%)

D istance (m)

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

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In cultures from hippocampus, 10-15% of cells are inhibitory

fluorescence fluorescence + bright-field bright-field

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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 reefsmolecular biology labs

An experiment with FRET: this week’s problem set

3. Multiphoton microscopy

Some examples with 2-photon microscopes

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Chemiluminescence in jellyfish (Aequorea victoria):

what produces the exciting light?

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Chemiluminescence in jellyfish

blue photonmax = 470 nm

aequorin + coelenterazine + O2

triggered by Ca2 entry

aequorin + coelenteramide + CO2 + hv

protein: aequorin

< 20% of the reactions produce a photon

small molecule: coelenterazine

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Chemiluminescence resonance energy transfer in jellyfish

“virtual”blue photon

green photonmax = 509 nm

Efficiency depends on dipole orientation and on(1/distance)6; increases by 3-5 fold

aequorin + coelenterazine + O2

triggered by Ca2 entry

aequorin + coelenteramide + CO2 + hv

GFP

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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)

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exciter filter:blue light only

barrier filter:no blue light

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exciter filter:blue light only

barrier filter:no blue light

for autofocus:“continuous” dive light

1 battery replaced by a blinker7 seconds on; 1.5 seconds off

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1st photos of fluorescent coralon the Great Barrier Reef,

AustraliaBen Lester, 2000

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no filters

exciter filter only

exciter plus barrier filter

© Charles Mazel

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Normal white-light photograph of Caribbean giant anemone,

Condylactis gigantea, Key West, Florida

©  Charles Mazel

Blue-light fluorescence photograph of the anemone

©  Charles Mazel

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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

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Another way to find new fluorescent proteins: Site-Directed Mutagenesis

RNA

Gene (DNA)

measure

“Express” theprotein with an altered side chain(s)

Hypothesis about an important side chain(s)

Mutate the desired codon(s)

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A pH-sensitive EGFP mutant reveals

synaptic vesicle movements

mutated GFP

synaptic vesicle proteinmutated EGFP

GFP

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At rest Action potentials

Stochastic vesicle release measured optically

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Enhanced fluorescent proteins: site-directed GFP mutants

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Another look at site-directed GFP mutants

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Fluorescence resonance energy transfer (FRET)

32Cyan Fluorescent Protein (CFP)

blue photon

(virtual)cyan photon

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< 10 nm

Fluorescence resonance energy transfer (FRET) detects proximity

Cyan Fluorescent Protein (CFP) Yellow Fluorescent Protein (YFP)

blue photon

virtualcyan photon

yellow photon

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Detecting protein-protein contacts with FRET

CFP YFP

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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 reefsmolecular biology labs

An experiment with FRET: this week’s problem set

3. Multiphoton microscopy

Some examples with 2-photon microscopes

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The multiphoton fluorescence microscope

E = h

ground state

excited state

“simultaneous”, within ~1/4 cycle. At a wavelength of 1 m, 1 cycle is c10-6 m)/(3 x 108 m/s)/= 3 x 10-15 sTherefore 2 photons must hit within ~ 10-15 s = 1 fs.

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computer

A two-photon Microscope

Dichroic mirror

Objective lensPhotodetector

titanium-sapphirelaser

X-Y scanningmirrors

duty cycle is 10-5

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Two-photon excitation eliminates out-of-plane bleaching, because excitation varies with the square of the power intensity

single-photon excitation

two-photon excitation

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Dichroic Mirror

Pinhole

Photodetector

Objective lens

Neuron in a scattering slice

many blue rays scatter few red rays scatter

Pinholenot required

Scattering causes minimal distortion in a 2-photon microscope.Very important for real tissue!

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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 18:351-7http://www.neuron.org/cgi/content/full/18/3/351

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Two-photon image of a neuron filled with a harmless dye

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

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voltage-gated Ca2+ channel

Electricity, then chemistry triggers synaptic vesicle fusion

Ca2+

docked vesicle

neurotransmitternerve impulse

from Lecture 9

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Calcium-sensitive fluorescent dyes

fluo-3

fluo-3

from Lecture 10

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Two-photon images of Ca2+ entering a presynaptic terminal within a slice of brain

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End of Lecture 11