Алексей Васильевич Семьянов Кальциевый имиджинг Алексей Васильевич Семьянов

Introduction. Calcium in brain cells AP 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

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 + 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

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

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)

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

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.

Dual-wavelength emission measurements F1 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

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 Typical life time is 5-10 nanoseconds

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

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 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.

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 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

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 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)

III. Loading cells with Ca2+ indicators

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

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

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

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

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

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

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) particles coated with calcium dye stains all cells (astrocytes and neurons, young and old animals) multiple cell staining with polar dye Helios Gene Gun

Tissue stained with biolistic technique Lebedinskiy et al, unpublished P.Kettunen et al 2002

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

Using morphological tracer to identify small compartments Fluo 4 (200 mM) Alexa594 (20 mM) 50mm 50mm filter: 500-560 nm 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