Transcript From Lecture 10

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 reefs
molecular biology labs
3.
Multiphoton microscopy
Some examples with 2-photon microscopes
Today’s data look noisy.
Pioneering data are always noisy.
2
emitted light only
beam-splitting
(“dichroic”)
mirror
exciting light only
Little Alberts Panel 1-1
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Confocal Microscope
Big Alberts Figure 9-18
© Garland
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From Lecture 5
Na+-coupled cell membrane neurotransmitter transporters:
major targets for drugs of therapy and abuse
Antidepressants
(“SSRIs” =
serotonin-selective
reuptake inhibitors):
Prozac, Zoloft, Paxil,
Celexa, Luvox
Drugs of abuse:
MDMA
Na+-coupled
cell membrane
serotonin
transporter
Attention-deficit
disorder medications:
Trademarks:
Ritalin, Dexedrine,
Adderall,
Strattera (?)
Presynaptic
terminals
Drugs of abuse:
cocaine
amphetamine
Na+-coupled
cell membrane
dopamine
transporter
cytosol
NH 3+
HO
outside
HO
N
H
HO
H2
C
C
H2
NH 3+
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Na+-coupled cell membrane neurotransmitter transporters:
“focus” on a transporter for GABA,
a major inhibitory neurotransmiter
Antiepileptic
Presynaptic
terminal
Na+-coupled
cell membrane
GABA
transporter
cytosol
outside
GABA
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From Lecture 10
The biologist’s method for fluorescent labeling of living cells:
attach a fluorescent protein
DNA
Gene for GFP
Gene for your favorite protein
Express
protein
DNA sequences assure expression in the correct cells;
Parts of the protein assure transport to the correct subcellular location
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A fusion protein: GABA transporter-GFP
extracellular
intracellular
NH2
COOH
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Mouse expressing GABA transporter-GFP:
all inhibitory neurons fluoresce, because they all express the GABA transporter,
because they all use GABA as a neurotransmitter
cerebellum
(movement)
Hippocampus
(memory)
Pleasure system
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<- Anti-GABA transporter
fluorescence
GFP fluorescence ->
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Confocal micrograph of mouse brain with GABA transporter-GFP fusion
50 m
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Confocal micrograph of GABA transporter-GFP fusion reveals presynaptic inhibitory terminals
50 m
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The limits of optical resolution: all the fluorescence is on the cell membrane
terminals
Relative fluorescent intensity (% )
120
calibration beads
100
1 m
80
. . . but . . .
60
some researchers now resolve
structures 10-fold smaller with
optical microscopes
40
20
0
-1.2 -0.8 -0.4 0.0
0.4
0.8
1.2
1.6
Distance (  m )
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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 reefs
molecular 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
protein:
aequorin
small molecule:
coelenterazine
aequorin + coelenterazine + O2
triggered by Ca2 entry
aequorin + coelenteramide + CO2 + hv
blue photon
lmax = 470 nm
< 20% of the reactions produce a photon
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Chemiluminescence resonance energy transfer in jellyfish
aequorin + coelenterazine + O2
triggered by Ca2 entry
green photon
lmax = 509 nm
aequorin + coelenteramide + CO2 + hv
“virtual”
blue photon
GFP
Efficiency depends on dipole orientation and on(1/distance)6; increases by 3-5 fold
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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 blinker
7 seconds on; 1.5 seconds off
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1st photos of fluorescent coral
on the Great Barrier Reef,
Australia
Ben Lester, 2000
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no filters
exciter filter only
© Charles Mazel
exciter plus
barrier filter
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Normal white-light photograph of
Caribbean giant anemone,
Condylactis gigantea, Key West,
Florida
Blue-light fluorescence
photograph of the anemone
© Charles Mazel
© Charles Mazel
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Additional fluorescent cnidarians
phylum, “stingers”,
previously coelenterates
Anemone with clownfish
(note that the clownfish is not
fluorescent, and appears black)
Indonesia
©Stuart and Michele Westmorland
Burrowing anemone,
Anthopleura artemisia
Monterey Bay
©Jack Sullins
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Another way to find new fluorescent proteins: Site-Directed Mutagenesis
Gene (DNA)
Hypothesis about an
important side chain(s)
Mutate the desired codon(s)
RNA
“Express” the
protein with an
altered side
chain(s)
measure
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mutated
GFP
A pH-sensitive EGFP mutant
reveals
synaptic vesicle movements
GFP
synaptic vesicle protein
mutated EGFP
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Stochastic vesicle release measured optically
At rest
Action potentials
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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)
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blue photon
(virtual)
cyan photon
Cyan Fluorescent Protein (CFP)
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Fluorescence resonance energy transfer (FRET) detects proximity
blue photon
yellow photon
< 10 nm
virtual
cyan photon
Cyan Fluorescent Protein (CFP)
Yellow Fluorescent Protein (YFP)
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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 reefs
molecular biology labs
An experiment with FRET: this week’s problem set
3.
Multiphoton microscopy
Some examples with 2-photon microscopes
35
The multiphoton fluorescence microscope
excited state
E = hn
ground state
“simultaneous”, within ~1/4 cycle.
At a wavelength of 1 m, 1 cycle is
l/c = (10-6 m)/(3 x 108 m/s)/= 3 x 10-15 s
Therefore 2 photons must hit within ~ 10-15 s = 1 fs.
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X-Y scanning
mirrors
A two-photon Microscope
titanium-sapphire
laser
duty cycle is 10-5
Dichroic mirror
computer
Objective lens
Photodetector
37
Two-photon excitation eliminates out-of-plane bleaching,
because excitation varies with the square of the power intensity
two-photon
excitation
single-photon
excitation
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Scattering causes minimal distortion in a 2-photon microscope.
Very important for real tissue!
Photodetector
Pinhole
Pinhole
not required
Dichroic Mirror
Objective lens
Neuron in a
scattering slice
many blue rays scatter
few red rays scatter
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© Cell Press
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
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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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from Lecture 9
Electricity, then chemistry triggers synaptic vesicle fusion
docked vesicle
Ca2+
nerve impulse
neurotransmitter
voltage-gated
Ca2+ channel
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from Lecture 10
fluo-3
Calcium-sensitive fluorescent dyes
fluo-3
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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
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