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. Visual presentation How to condense 1 GB of information into a single image - the ‘channel chip’

15. 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

16. …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

17. …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

18. 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

19. 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

20. 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

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

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

23. Breaking the diffraction limit Ways to ‘sidestep’ the resolution limit set by the wavelength of light

24. The ‘classical’ resolution limit of optical microscopy © Molecular Expressions Microscopy Primer

25. BUT – the position of a single point source (e.g. a fluorescent molecule) can be localized with much higher precision, limited only by the number of photons that can be collected. What we then need is to have only sparse sources at any given time, so as to avoid unresolved overlap

26. Photoactivation Localization Microscopy (PALM) (Betzig et al., Science 2006) Express protein of interest tagged with a photoactivatable fluorescent protein (eg.g. EOS) in cell Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function inactive state active state Fluorescence emission Bleached state Activating laser 405 nm Excitation laser 532 nm Repeat thousands of times

27. Photoactivation Localization Microscopy (PALM) (Betzig et al., Science 2006) Express protein of interest tagged with a photoactivatable fluorescent protein (eg.g. EOS) in cell Stochastically photoactivate a low density of molecules per frame and localize using Gaussian function

28. Example of PALM Super-resolution imaging of actin tagged with a photo-activatable protein Eos-actin TIRF Eos-actin PALM

29. Imaging by spatially defined STIMULATION e.g. caged compounds (neurotransmitters, second messengers)

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

31. 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

32. ChannelRhodopsin 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

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