NB & B – Functional Imaging

Section 1: Microscopic Imaging

Applications – from molecules to rats (and frogs)

Imaging the function of single-channels

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 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

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)

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

Imaging intracellular IP3 receptor/channels at the single-

channel level

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

Local and global IP3-mediated calcium signals

Calcium puffs and waves

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

Puffs evoked by photoreleased IP3 in SH-SY5Y cells

sec:ms

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

amplitude

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

amplitude

Quantal analysis of step amplitudes during puffs and blips

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

IP3R diffuse within the ER membrane

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

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

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