nb & b – functional imaging section 1: microscopic imaging applications – from molecules to...
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NB & B – Functional Imaging
Section 1: Microscopic Imaging
Applications – from molecules to rats (and frogs)
Motivations to develop functional single-channel Ca2+ 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 Ca2+
Imaging single Ca2+ channel gating:Fluorescent probe (Fluo-4) of ion (Ca2+) flux
High (a few mM) concentration of Ca2+ in the extracellular fluid or ER lumen
Very low (ca. 50 nM) resting free cytosolic Ca2+ concentration
High gain – many Ca2+ ions pass through a channel, so fluorescence canbe excited from many probe molecules
Large, localized increase in [Ca2+] around channel mouth
Ca2+ signals are large and fast near the channelmouth, 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.
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.
Total Internal Reflection (TIRF) MicroscopyA 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
TIRFM imaging of single-channel Ca2+ signals : Ca2+ entry through plasma membrane channels
expressed in Xenopus oocytes
Imaging can give information about the AMPLITUDES of signals
e.g. Neuronal 42 nAChRs show multiple Ca2+ permeability levels whereas muscle
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
In the presence of IP3, positive and negative feedback of Ca2+ on the IP3R generate repetitive, regenerative waves
cytoplasm
Ca2+
IP3
+
IP3 receptor
+-
[Ca
2+]c
yt
Global cellular Ca2+
Waves© Jim Lechleiter,
U. Texas
ER
time
IP3 receptors are clustered on ER, so Ca2+ interactions can
take place on 2 different distance and time scales;
Local (tens of nm) scale between IP3R to generate Ca2+ puffs
Longer range (a few m) interactions between clusters to propagate Ca2+ waves
TIRF imaging + EGTA loading gives fluorescence signals that track Ca2+ flux (current) rather than a
‘leaky integral’ of Ca2+ accumulation in the cytosol
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
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
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
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
Tracking the motility of single molecules in cells
IP3R tagged with a photoactivatable fluorescent protein
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
Imaging by spatially defined STIMULATION
e.g. caged compounds (neurotransmitters, second messengers)
“optogenetics”: e.g channel rhodopsin
Mapping the dendritic field of neurons in a brain slice byrecording epsps evoked by local photorelease of glutamate
at different sites
Callaway & Katz, PNAS 90;7661
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
Mapping neuronal projections by local subcellular activation of ChR2
Leopoldo Petreanu, Daniel Huber, Aleksander Sobczyk & Karel SvobodaNature Neuroscience 10, 663 - 668