Кальциевый имиджинг

1. Кальциевый имиджинг Алексей Васильевич Семьянов

2. Introduction. Calcium in brain cells Ca2+ Ca2+ AP AP Ca2+ EPSP 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 asCa2+ 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

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

9. Ca2+ independent point Spectral response of fura-2 in solutions containing 0–39.8 µM Ca2+ Calibration of fura-2 Simple calibration 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

10. 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

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

17. How to do FLIM • Take different time measures after laser pulse • Subtract one measurementfrom 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 Qdotnanocrystals

21. Tuneability of Qdotnanocrystals Five different nanocrystal solutions are shown excited with the same long-wavelength UV lamp; the size of the nanocrystal determines the color.

22. QdotBioconjugates 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 structureswithin the cell • indicators in polyanionic form are sequesteredwithin 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 both cytoplasm and mitochondria filled with the indicator cell with mitochondria cytoplasm filled with the indicator

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

37. solution brain slice 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 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 sulforodamine 101 OG AM + SR101 Oregon Green AM 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 • particles coated with calcium dye • stains all cells (astrocytes and neurons, young and old animals) • multiple cell staining with polar dye (Left) Spherical 1.6 µm gold particles. (Right) M25 tungsten particles (~1.7 µm) 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 configurationCa2+ 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: 500-560 nm 580-620 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