1. Алексей Васильевич Семьянов
Кальциевый имиджинг
Алексей Васильевич Семьянов

2. Introduction. Calcium in brain cellsAP AP
EPSP
Ca2+
Extrasynaptic membrane
backpropagating action potentials
morphological changes
“extrasynaptic” plasticity
Synapse
release of neurotransmitter
postsynaptic Ca2+ entry
synaptic 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

3. I. Calcium indicators

4. 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)

5. BAPTA based chelators as Ca2+ indicators
+ 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

6. Single wavelength measurements
Absorption-emission spectra of fluo-4
Changes in fluorescence intensity
because of Ca2+ binding

7. Single wavelength measurements
(1)
where Kd – dissociation constant of the indicator
Fmin fluorescence at zero Ca2+ concentration
Fmax fluorescence at saturating Ca2+ levels
depend on dye
concentration
DF/F=(F-F0)/F0
- relative fluorescence change
(independent of dye concentration)
(2)
where F0 – prestimulus fluorescence level
where DF/F<<(DF/F)max
(3)

8. Dual-wavelength excitation measurements
changes with Ca2+ signal
dye concentration independent 1 2
F1 or F2 depending
on excitation wavelength
where
Rmin – ratio in Ca2+ free solution
Rmax – ratio at Ca2+ saturating levels
Keff – effective binding constant
Ratiometric method can be used with mixtures of non-ratiometric dyes
calibration constants
Excitation (detected at 510 nm) and
emission (excited at 340 nm) spectra of fura-2
A – Ca2+ saturated
B – Ca2+ free

9. Calibration of fura-2 Simple calibration Calibration in the cell
Spectral response of fura-2 in solutions
containing 0–39.8 µM Ca2+
Ca2+ independent point
zero Ca2+ - Rmin
high Ca2+ - Rmax
intermediate Ca2+ - Keff= [Ca2+]intx(Rmax-Rint)/(Rint-Rmin)
Problem – intracellular behaviour
of the dye is different
viscous cytosolic environment
intracellular binding and uptake
Calibration in the cell
pipettes containing different [Ca2+]
strong stimulation
Problem – difficult to obtain stable clamp of Ca2+
cell loading might take long time
extrusion mechanisms
Ca2+ independent point – you get equal fluorescent signal with any Ca2+ concentration
So you can use it as a reference point to measure Ca2+ concentration.

10. Dual-wavelength emission measurementsF1 F2
Spectral response of indo-1 in solutions
containing 0–39.8 µM free Ca2+
Absorption-emission (excited at 338 nm) spectra
of Ca2+-saturated (A) and Ca2+-free (B) indo-1

11. Fluorescent resonance energy transfer (FRET)
FRET principle

12. 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

13. Measurement of calcium in a dendrite using Yellow Chameleon 3.6

14. 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.

15. 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

16. 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
Typical life time is 5-10 nanoseconds

17. How to do FLIM Take different time measures after laser pulse
Subtract one measurement from another
Obtain D1/D2
If fluorescence life time >>
Interval between laser pulses
– laser will transfer more energy
than necessary to the preparation

18. 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)

19. 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
Structure of a Qdot nanocrystal
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.

20. Relative size of Qdot nanocrystals

21. 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.

22. 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

23. II. Ca2+ imaging: points for consideration
preparation (in vivo imaging, slice, cell culture)
appropriate equipment
specific preparation of the sample for imaging

24. 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

25. 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

26. Astrocytes stained with sulforodamine 101
Two photon image

27. Astrocytes loaded through patch pipette

28. 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

29. Organelle specific markers
Fluorophore attached to a target-specific 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
Lysosome tracer
Mitochondria tracer
Endoplasmic reticulum tracer

30. 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

31. 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)

32. Time scale of Ca2+ signal requires different imaging technique
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)
Spontaneous activity in astrocytes
of CA1 astrocytes loaded with Oregon Green-AM
Lebedinskiy et al., unpublished
Time lapse (hundreds of seconds)

33. III. Loading cells with Ca2+ indicators

34. Loading cells with acetoxymethyl (AM) esters of Ca2+ indicators
Problems
Generation of potentially toxic by-products (formaldehyde and acetic acid)
Compartmentalization:
AM esters accumulate in structures within the cell
indicators in polyanionic form are sequestered within organelles via active transport
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
Leakage: extrusion of anionic indicators from cells by organic ion transporters
fura-2 AM ester

35. Sequestration of AM dyes in organelles
Ca2+ indicator
cell with mitochondria
both cytoplasm and
mitochondria filled
with the indicator
cytoplasm filled
with the indicator

36. Use of selective fluorescent marker for organelles
(with different emission/excitation spectra)
indicates mitochondria
cell filled with Ca2+ indicator
Simultaneous recording cytoplasmic and mitochondrial Ca2+ signals

37. 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
brain slice
solution
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

38. Spontaneous activity in astrocytes of hippocampal slice
Oregon Green AM – “preferentially” stains astrocytes
60x times accelerated movie

39. Oregon Green AM and sulforodamine 101
OG AM +
SR101
Frame width 450 mm

40. Software for automatic actrocyte detection using reference image
Alexey Pimashkin
Nizhny Novgorod University

41. Biolistic dye loading Biolistic – biological ballistic
(Left) Spherical 1.6 µm gold particles.
(Right) M25 tungsten particles (~1.7 µm)
particles coated with calcium dye
stains all cells (astrocytes and neurons, young and old animals)
multiple cell staining with polar dye
Helios Gene Gun

42. Tissue stained with biolistic technique
Lebedinskiy et al, unpublished
P.Kettunen et al 2002

43. Loading dyes with patch pipets
pipete
with Ca2+ indicator
cell
Removing positive and applying negative pressure to break through the membrane when gigaom contact is formed
Whole-cell configuration Ca2+ indicator diffuses into the cell
Approaching the cell with positive pressure

44. Using morphological tracer to identify small compartments
Fluo 4 (200 mM)
Alexa594 (20 mM)
50mm
50mm
filter: nm nm
Same excitation wavelength, different emission

45. 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