Transcript Document

Кальциевый имиджинг
Алексей Васильевич
Семьянов
Introduction. Calcium in brain cells
Ca2+
Ca2+
AP
AP
Ca2+
EPSP
Extrasynaptic membrane
Synapse
release of neurotransmitter
postsynaptic Ca2+ entry
synaptic plasticity
backpropagating action potentials
morphological changes
“extrasynaptic” plasticity
Ca2+
Ca2+
Intra-organelle Ca2+ changes
mitochondria sequester
Ca2+
shaping cytosolic Ca2+ transients
Calcium in astrocytes
Ca2+ waves in astrocytic network
glutamate release by astrocytes
I. Calcium indicators
Fluorophores
•
chromophore is part of a molecule
responsible for its color
(absorption/reflection of light)
•
fluorophore is a component of a
molecule which causes a molecule to
be fluorescent (absorption-emission of
light)
absorption and emission properties (spectral profile,
maximums, intensity of emitted light)
early 1940s - Albert Coons
developed a technique for
labeling antibodies
with fluorescent dyes
(immunofluorescence)
BAPTA based chelators as Ca2+ indicators
BAPTA
+ fluorophore
+ groups modifying affinity for Ca2+
Fura-2
Fluo-4
Rhod-2
Electron-donating –CH3 or electron-withdrawing –NO2 groups change affinity for Ca2+
Small fluorophores (benzofurans and indoles) are excitable in UV region – shifted excitation/emission peaks
Flurescein and rhodamine fluors operate in visible region – changes in emission intensity
emision
Single wavelength measurements
450
500
550
600
650
700
emission wavelength (nm)
Absorption-emission spectra of fluo-4
Changes in fluorescence intensity
because of Ca2+ binding
Single wavelength measurements
[Ca 2 ]i  K d
F  Fmin
Fmax  F
(1)
where Kd – dissociation constant of the indicator
Fmin fluorescence at zero Ca2+ concentration
Fmax fluorescence at saturating Ca2+ levels
DF/F=(F-F0)/F0 - relative fluorescence change
depend on dye
concentration
(independent of dye concentration)
(2)
where F0 – prestimulus fluorescence level
Kd
D[Ca ]i 
(DF / F )
(DF / F ) max
2
where DF/F<<(DF/F)max
(3)
Dual-wavelength excitation measurements
1
2
R
F1 or F2 depending
on excitation wavelength
F1
F2
-changes with Ca2+ signal
-dye concentration independent
2
[Ca ]i  K eff
R  Rmin
Rmax  R
where
Rmin – ratio in Ca2+ free solution
Rmax – ratio at Ca2+ saturating levels
Keff – effective binding constant
Excitation (detected at 510 nm) and
emission (excited at 340 nm) spectra of fura-2
A – Ca2+ saturated
B – Ca2+ free
calibration constants
Calibration of fura-2
Simple calibration
zero Ca2+ - Rmin
high Ca2+ - Rmax
intermediate Ca2+ - Keff= [Ca2+]intx(Rmax-Rint)/(Rint-Rmin)
Ca2+ independent point
Problem – intracellular behaviour
of the dye is different
1.
viscous cytosolic environment
2.
intracellular binding and uptake
Calibration in the cell
• pipettes containing different [Ca2+]
• strong stimulation
Spectral response of fura-2 in solutions
containing 0–39.8 µM Ca2+
Problem – difficult to obtain stable
clamp of Ca2+
1.
cell loading might take long time
2.
extrusion mechanisms
Dual-wavelength emission measurements
F1
F2
Absorption-emission (excited at 338 nm) spectra
of Ca2+-saturated (A) and Ca2+-free (B) indo-1
Spectral response of indo-1 in solutions
containing 0–39.8 µM free Ca2+
Fluorescent resonance energy transfer (FRET)
FRET principle
Fluorescent resonance energy transfer (FRET) based
Ca2+ indicators
A. Bimolecular fluorescent indicators
Ca2+ binding causes interaction separate GFP and YFP
B. Unimolecular fluorescent indicators
Ca2+ binding conformational change and interaction
GFP and YFP domains – “Chameleons”
Miyawaki A.,Dev Cell. 2003
Measurement of calcium in a dendrite using Yellow
Chameleon 3.6
FRET based Ca2+ indicators
• Indicators are genetically encoded and allow Ca2+ measuring in
specific cell types and organelles.
• May perturb cellular activity
• Overlapping spectra: laser can excite both donor and accepter
molecules.
Fluorescence lifetime
•
The fluorescece lifetime - the time the
molecule stays in its excited state before
emitting a photon. Fluorescence follows
first-order kinetics:
•
•
[S1] is the remaining concentration of
excited state molecules at time t,
[S1]0 is the initial concentration after
excitation.
FLIM – fluorescence lifetime imaging
Binding to calcium changes life time of fluorescence
R=D1/D2 – sensitive to calcium
Single wavelength indicators can
be used for ratiometric concentration
independent measurements
How to do FLIM
1. Take different time
measures after laser pulse
2. Subtract one measurement
from another
3. Obtain D1/D2
If fluorescence life time >>
Interval between laser pulses
– laser will transfer more energy
than necessary to the
preparation
Summary: calcium indicators
•
The binding of Ca2+ results in a shift in excitation and sometimes
emission peaks – ratiometric indicators (fura-2, quin-2, indo-1)
•
The binding of Ca2+ leads to a change in fluorescence intensity but not
change in spectrum (fluo-4, rhod-2, calcium green)
•
The binding of Ca2+ results in changes in fluorescent resonance energy
transfer (FRET e.g. chameleons)
•
The binding of Ca2+ leads to a change in fluorescence life time (FLIM)
Nanocrystal technology
early 1980s - labs of Louis Brus at Bell Laboratories
and of Alexander Efros and A.I. Ekimov of the Yoffe
Institute in Leningrad
Qdot nanocrystals are nanometer-scale atom clusters,
containing from a few hundred to a few thousand atoms
of a semiconductor material (cadmium mixed with
selenium or tellurium) coated with a semiconductor shell
(zinc sulfide) to improve the optical properties
of the material.
These particles fluoresce without the involvement of ->*
electronic transitions.
Structure of a Qdot nanocrystal
Relative size of Qdot nanocrystals
Tuneability of Qdot nanocrystals
Five different nanocrystal solutions are shown excited
with the same long-wavelength UV lamp; the size of the
nanocrystal determines the color.
Qdot Bioconjugates
Qdot nanocrystals coupled to proteins, oligonucleotides,
small molecules, etc.,
The emission from Qdot nanocrystals is narrow and
symmetric; therefore, overlap with other colors is minimal,
yielding less bleed through into adjacent detection channels
and attenuated crosstalk and allowing many more colors
to be used simultaneously
II. Ca2+ imaging: points for consideration
•
preparation (in vivo imaging, slice, cell culture)
– appropriate equipment
– specific preparation of the sample for imaging
II. Ca2+ imaging: points for consideration
•
preparation (in vivo imaging, slice, cell culture)
– appropriate equipment
– specific preparation of the sample for imaging
Helmchen et al., Neuron 2001
brain slice
cell culture
II. Ca2+ imaging: points for consideration
•
preparation (in vivo imaging, slice, cell culture)
– appropriate equipment
– specific preparation of the sample for imaging
•
cell types (inhibitory, excitatory, astrocytes)
– morphological identification of the cells
– use of cell type specific markers
– cell type specific indicator loading techniques
Astrocytes stained with sulforodamine 101
Two photon image
Astrocytes loaded through patch pipette
II. Ca2+ imaging: points for consideration
•
preparation (in vivo imaging, slice, cell culture)
– appropriate equipment
– specific preparation of the sample for imaging
•
cell types (inhibitory, excitatory, astrocytes)
– visual identification of the cells
– use of cell type specific markers
– cell type specific indicator loading techniques
•
cellular compartments (soma, axon, dendrite, glial processes, organelles)
– use of specific markers
– imaging with different resolution
Organelle specific markers
(a)
(b)
(c)
Lysosome tracer
Mitochondria tracer
Endoplasmic reticulum tracer
•
Fluorophore attached to a targetspecific part of molecule that assists in
localizing the fluorophore through
covalent, electrostatic, hydrophobic or
similar types of bonds.
•
May permeate or sequester within the
cell membrane (useful for living cells)
•
Can be used together with calcium
indicators
•
Can be retained after fixation of the
tissue
Mitochondrial Ca2+ imaging with rhod-2
Confocal micrographs of cells after incubation with rhod-2/AM ond MitoTrackerTM Green FM
Hoth et al., J. Cell Biol.1997
II. Ca2+ imaging: points for consideration
•
preparation (in vivo imaging, slice, cell culture)
– appropriate equipment
– specific preparation of the sample for imaging
•
cell types (inhibitory, excitatory, astrocytes)
– visual identification of the cells
– use of cell type specific markers
– cell type specific indicator loading techniques
•
cellular compartments (soma, axon, dendrite, glial processes, organelles)
– use of specific markers
– imaging with different resolution
•
parameters of Ca2+ signal
– time scale of the signal (fast or slow imaging)
– concentration range of Ca2+ (high or low affinity indicators)
Time scale of Ca2+ signal requires different
imaging technique
Spontaneous activity in astrocytes
of CA1 astrocytes loaded with Oregon Green-AM
Lebedinskiy et al., unpublished
Time lapse (hundreds of seconds)
Action potential-evoked Ca2+ influx
in axonal varicosities of CA1 interneurons.
Fluo-4 fluorescence responses
Rusakov et al., Cerebral Cortex 2004
Line scan (hundreds of milliseconds)
III. Loading cells with Ca2+ indicators
Loading cells with acetoxymethyl (AM) esters of
Ca2+ indicators
Problems
fura-2 AM ester
1.
Generation of potentially toxic by-products
(formaldehyde and acetic acid)
2.
Compartmentalization:
•
AM esters accumulate in structures
within the cell
•
indicators in polyanionic form are sequestered
within organelles via active transport
3.
Incomplete AM ester hydrolysis:
partially hydrolyzed AM esters are Ca2+-insensitive,
detection of their fluorescence as part of the total
signal leads to an underestimation of the Ca2+
concentration
4.
Leakage:
extrusion of anionic indicators from cells
by organic ion transporters
Sequestration of AM dyes in organelles
Ca2+ indicator
cell with mitochondria
cytoplasm filled
with the indicator
both cytoplasm and
mitochondria filled
with the indicator
Use of selective fluorescent marker for
organelles
cell filled with Ca2+ indicator
fluorescent marker
(with different emission/excitation spectra)
indicates mitochondria
Simultaneous recording cytoplasmic and mitochondrial Ca2+ signals
Optical probing of neuronal circuits with calcium
indicators
1.
an initial incubation with 2-5 µl of a 1 mM fura-2 AM
in 100% DMSO solution for 2 min
2.
second incubation in 3 ml of 10 µM fura-2 AM
in ACSF for 60 min
solution
brain slice
DIC (A) and fluorescence (B) images of the lower layers of a visual cortex slice
electrical stimulation of one cell will produce Ca2+ signals in synaptically
coupled followers
Spontaneous activity in astrocytes of hippocampal slice
Oregon Green AM – “preferentially” stains astrocytes
60x times accelerated movie
Oregon Green AM and sulforodamine 101
Oregon Green AM
Frame width 450 mm
sulforodamine 101
OG AM + SR101
Software for automatic actrocyte detection using
reference image
Alexey Pimashkin
Nizhny Novgorod University
Biolistic dye loading
Biolistic – biological ballistic
(Left) Spherical 1.6 µm gold particles.
(Right) M25 tungsten particles (~1.7 µm)
Helios Gene Gun
•
particles coated with calcium dye
•
stains all cells (astrocytes and
neurons, young and old animals)
•
multiple cell staining with polar dye
Tissue stained with biolistic technique
Lebedinskiy et al, unpublished
P.Kettunen et al 2002
Loading dyes with patch pipets
pipete
with Ca2+ indicator
cell
1.
Approaching the cell
with positive pressure
2.
Removing positive and
3.
applying negative pressure
to break through the membrane
when gigaom contact is formed
Whole-cell configuration
Ca2+ indicator diffuses
into the cell
Using morphological tracer to identify small
compartments
Fluo 4 (200 mM)
Alexa594 (20 mM)
50mm
filter:
500-560 nm
50mm
580-620 nm
Same excitation wavelength, different emission
Dendrites and spines of the same cell with Fluo 4
and Alexa 594
Fluo 4 (200 mM)
Alexa 594 (20 mM)
Use of morphological tracer:
- identification of small compartments when baseline Ca2+ is low
- the use of DF/G instead DF/F gives better signal-to-noise ratio