1 bi 1 lecture 11 tuesday, april 16, 2006 better microscopes and better fluorescent proteins
TRANSCRIPT
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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
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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
39© Cell Press
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