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NB & B – Functional Imaging Section 1: Microscopic Imaging Applications – from molecules to rats (and frogs) Imaging the function of singlechannels Single-channel recording techniques the very first records… and 30 years on Single-channel recording techniques the very first records… and 30 years on Motivations to develop functional single-channel Ca2+ imaging 1. To study the functioning of calciumpermeable 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 Ca2+ Imaging single Ca2+ channel gating: Fluorescent probe (Fluo-4) of ion (Ca2+) flux Very low (ca. 50 nM) resting free cytosolic Ca2+ concentration High (a few mM) concentration of Ca2+ in the extracellular fluid or ER lumen Large, localized increase in [Ca2+] around channel mouth High gain – many Ca2+ ions pass through a channel, so fluorescence can be excited from many probe molecules Ca2+ signals are large and fast near the channel mouth, but small and slow only 1 mm away. So, to get a faithful record of channel gating, we need to record local, near-membrane signal. Optimal compromise between kinetic resolution and noise level achieved with sampling volumes of tens of atto liter Kinetic resolution improves with ever decreasing sampling volume. 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. How might we actually achieve this? 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 Through-the-lens TIRF microscopy TIRFM imaging of single-channel Ca2+ signals : Ca2+ entry through plasma membrane channels expressed in Xenopus oocytes Optical single-channel recording: Single Channel Ca2+ Fluorescence Transients (SCCaFTs) Visual presentation How to condense 1 GB of information into a single image - the ‘channel chip’ Imaging can give information about the AMPLITUDES of signals e.g. Neuronal a4b2 nAChRs show multiple Ca2+ permeability levels whereas muscle abgd nAChRs have (mostly) uniform Ca2+ permeability …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 …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 Advantages of optical single-channel Ca2+ 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 Ca2+ permeability Applicable to channels in both the cell membrane and in intracellular organelles Allows spatial mapping of the functional ion channels and measurement of their motility Advantages of optical single-channel Ca2+ 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 Ca2+ permeability Applicable to channels in both the cell membrane and in intracellular organelles Allows spatial mapping of the functional ion channels and measurement of their motility So, should you throw away your patch-clamp ??? 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 Responses of neurons in visual cortex during stimulation by moving bars at different orientations Reid. Nature Sharply-defined boundaries between areas with cells showing different orientation selectivity Reid. Nature Breaking the diffraction limit Ways to ‘sidestep’ the resolution limit set by the wavelength of light The ‘classical’ resolution limit of optical microscopy © Molecular Expressions Microscopy Primer 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 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 Excitation laser 532 nm Activating laser 405 nm inactive state Fluorescence emission active state Bleached state Repeat thousands of times 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 Example of PALM • Super-resolution imaging of actin tagged with a photo-activatable protein Eos-actin TIRF Eos-actin PALM Imaging by spatially defined STIMULATION e.g. caged compounds (neurotransmitters, second messengers) Precise control of intracellular [IP3] by photorelease from caged IP3. 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 ChannelRhodopsin Light-activated channels originally isolated from an algae. Nonselective cation channel, so opening induced by blue light can be used to depolarize neurons transfected to express ChR Mapping neuronal projections by local subcellular activation of ChR2 Leopoldo Petreanu, Daniel Huber, Aleksander Sobczyk & Karel Svoboda Nature Neuroscience 10, 663 - 668