Transcript Ian Parker Department of Neurobiology & Behavior, UC Irvine

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