1. NB & B – Functional Imaging Section 1: Microscopic Imaging Applications – from molecules to rats (and frogs)

2. Imaging the function of single- channels

3. Single-channel recording techniques the very first records… and 30 years on

5. Motivations to develop functional single-channel Ca 2+ imaging 1. To study the functioning of calcium- permeable channels themselves – previously possible only by the electrophysiological patch-clamp technique. Patch-clamping has limitations including - lack of spatial information regarding channel location; inability to obtain simultaneous, independent recordings from multiple channels; need for physical access of pipette; inaccessibility of intracellular channels in the intact cell 2. To image the spatial locations of functional channels, and the resulting distribution of cytosolic Ca 2+

6. Imaging single Ca 2+ channel gating: Fluorescent probe (Fluo-4) of ion (Ca 2+ ) flux High (a few mM) concentration of Ca 2+ in the extracellular fluid or ER lumen Very low (ca. 50 nM) resting free cytosolic Ca 2+ concentration

7. High gain – many Ca 2+ ions pass through a channel, so fluorescence can be excited from many probe molecules Large, localized increase in [Ca 2+ ] around channel mouth

8. Ca 2+ signals are large and fast near the channel mouth, but small and slow only 1  m away. So, to get a faithful record of channel gating, we need to record local, near-membrane signal.

9. Optimal compromise between kinetic resolution and noise level achieved with sampling volumes of tens of atto liter How might we actually achieve this? But “molecular shot noise” increases as the number of Ca-bound dye molecules decreases. Molecular shot noise predominates over other noise sources: e.g. photon shot noise, camera dark noise, camera read-out noise. Kinetic resolution improves with ever decreasing sampling volume.

10. Total Internal Reflection (TIRF) Microscopy A way to excite fluorescence in a very thin (~100 nm) layer next to a coverglass. Imaging can then be done with a camera (i.e. unlike confocal and 2-photon, not a scanning technique) © Molecular Expressions Microscopy Primer

11. Through-the-lens TIRF microscopy

12. TIRFM imaging of single-channel Ca 2+ signals : Ca 2+ entry through plasma membrane channels expressed in Xenopus oocytes

13. Optical single-channel recording: Single Channel Ca 2+ Fluorescence Transients (SCCaFTs)

14. Imaging can give information about the AMPLITUDES of signals e.g. Neuronal  4  2 nAChRs show multiple Ca 2+ permeability levels whereas muscle  nAChRs have (mostly) uniform Ca 2+ permeability

15. …and about the KINETICS of signals Factors influencing kinetic resolution: Engineering constraints – how fast is your camera? Biological and probe constraints – how fast is your signal? Signal-to-noise constraints – the faster you record, the smaller the signal

16. …and, imaging provides (near) simultaneous information from multiple, spatially separated entities (molecules/cells/brain regions); whereas classical techniques (patch-clamp/microelectrode recording) monitor only one at a time. e.g. nominally identical nAChR channels (expressed from the same cloned gene) display widely varying properties

17. Imaging intracellular IP 3 receptor/channels at the single- channel level

18. In the presence of IP 3, positive and negative feedback of Ca 2+ on the IP 3 R generate repetitive, regenerative waves cytoplasm Ca 2+ IP 3 + IP 3 receptor + - [Ca 2+ ]cyt Global cellular Ca 2+ Waves © Jim Lechleiter, U. Texas ER time

19. Local and global IP3-mediated calcium signals Calcium puffs and waves

20. IP 3 receptors are clustered on ER, so Ca 2+ interactions can take place on 2 different distance and time scales; Local (tens of nm) scale between IP 3 R to generate Ca 2+ puffs Longer range (a few  m) interactions between clusters to propagate Ca 2+ waves

21. TIRF imaging + EGTA loading gives fluorescence signals that track Ca 2+ flux (current) rather than a ‘leaky integral’ of Ca 2+ accumulation in the cytosol

22. Puffs evoked by photoreleased IP 3 in SH-SY5Y cells sec:ms

23. Puffs imaged by TIRF show ‘quantal’, stepwise variations in amplitude

25. Quantal analysis of step amplitudes during puffs and blips

26. Advantages of optical single-channel Ca 2+ imaging Massively parallel - simultaneous and independent recording from many hundreds ion channels with time resolution approaching that of patch-clamp recording Applicable to both voltage- and ligand- gated ion channels with partial Ca 2+ permeability Allows spatial mapping of the functional ion channels and measurement of their motility Applicable to channels in both the cell membrane and in intracellular organelles

27. Advantages of optical single-channel Ca 2+ imaging Massively parallel - simultaneous and independent recording from many hundreds ion channels with time resolution approaching that of patch-clamp recording Applicable to both voltage- and ligand- gated ion channels with partial Ca 2+ permeability Allows spatial mapping of the functional ion channels and measurement of their motility So, should you throw away your patch-clamp ??? Applicable to channels in both the cell membrane and in intracellular organelles

28. Tracking the motility of single molecules in cells IP3R tagged with a photoactivatable fluorescent protein

29. IP3R diffuse within the ER membrane

30. Two-photon calcium imaging in cerebral cortex Monitoring activity in multiple individual neurons in the brain of anesthetized animals via calcium imaging Load Ca indicator into neurons by injecting a bolus of AM ester dye via a micropipette Konnerth. PNAS

31. Responses of neurons in visual cortex during stimulation by moving bars at different orientations Reid. Nature

32. Sharply-defined boundaries between areas with cells showing different orientation selectivity Reid. Nature

33. Imaging by spatially defined STIMULATION e.g. caged compounds (neurotransmitters, second messengers) “optogenetics”: e.g channel rhodopsin

34. Precise control of intracellular [IP3] by photorelease from caged IP3.

35. Mapping the dendritic field of neurons in a brain slice by recording epsps evoked by local photorelease of glutamate at different sites Callaway & Katz, PNAS 90;7661

36. Channel Rhodopsin Light-activated channels originally isolated from an algae. Non- selective cation channel, so opening induced by blue light can be used to depolarize neurons transfected to express ChR

37. Mapping neuronal projections by local subcellular activation of ChR2 Leopoldo Petreanu, Daniel Huber, Aleksander Sobczyk & Karel Svoboda Nature Neuroscience 10, 663 - 668