Ion Signalling Microscopy
Challenge of Imaging Ions
Techniques for the study of intracellular ions are used widely in biology, for example,
to identify spatial variations in calcium levels within living cells, to measure
the concentrations of intracellular ions including cell pH, and to monitor how these
concentrations change with time. Monitoring intracellular ion changes is vital for
our understanding of signalling and functional pathways in cellular systems, central
to many fundamental processes such as muscle contraction as well as synaptic nerve
signal transmission.
Such measurements have been central to a range of research programs, including drug
discovery research. Calcium channels are representative of an important class of
cell membrane molecules known as ion channels. As these open or close, e.g. in response
to extracellular messenger molecules, intracellular ion concentrations can also
change, altering how the cell behaves. Therapeutic agents that act directly on these
ion channels may provide effective treatments for many diseases. For example, drugs
that target calcium channels are useful in treating a variety of cardiovascular
disorders.
Fluorescent dyes are designed to have affinity to specific ions; for example, FURA-2
is specific to calcium ions. When they bind to an ion, their absorption or fluorescent
properties are altered. The principle of ion concentration determination is such
that, when the free ion concentration changes, the equilibrium between free indicator
dye and ion-bound dye also changes, resulting in a change in the photophysics of
the indicator dye.
Ratiometric microscopy is one approach for such measurements - by quantitatively
interpreting the changes in fluorescent properties by looking at both bound and
unbound dye ratio, the concentration of the ion being investigated can be measured.
However, there are some common non-ratiometric dyes also, such as Fluo-4.
Selected fluorescence images, taken with the Revolution 488, from a 1500 frame kinetic
series at 30 frames/sec, showing the progression of a Ca2+ wave as it progresses
though a rabbit urethral muscle cell. Kinetic plots derived from offset ROIs within
the cell show temporal progression of the Ca2+ flux. Using the same field of view
and no binning, frame rates were then pushed to 120fps for better temporal resolution
of events. Courtesy of Dr Mark Hollywood, Smooth Muscle Research Centre, Dundalk
Institute of Technology.
In many tissue types, ion flux changes occur rapidly, very often in the low millisecond
time domain. As such, ion-binding fluorescence dyes tend to have rapid responses,
enabling changes in concentrations of free ions to be imaged with high temporal
resolution, requiring a means of fast quantitative imaging of these fluorescence
intensity changes.
Ultimately however, we are practically limited by the binding constants of these
dyes – the rate of imaging does not need to not exceed the time taken for dyes to
diffuse, bind and respond to free ions, irrespective of how rapidly the ion concentration
changes. One is better determining this binding constant of the dye, and using an
exposure time suited to this. Using too short an exposure time adversely affects
signal to shot noise ratio, the ultimate limiting detection phenomenon (which can
make your image look grainy and unresolved), as would be the case even in the presence
of a "perfect" photon detector (100% Quantum Efficiency (QE) + zero noise floor).
Another factor that must be taken into consideration is the concentration of ion
dye used. Ideally, lowest possible concentrations of dye are used as indicators
of free ion concentrations, such that the dye binds to negligible amounts of ions
as compared to the total quantity of the ion species in question, and so it does
not significantly alter the statistical equilibrium of bound and free ions. The
remedy is to lower the concentration as far as possible whilst still maintaining
an adequate signal to noise ratio for the exposure time (and therefore frame rate)
required.
EMCCD for Ion Signalling
EMCCD technology, whether as a key component in confocal live cell imaging systems,
or as a "stand-alone" EMCCD + imaging software solution, is the proven solution
for ultra-sensitive rapid imaging of ion concentrations. The Signal to Noise (S/N)
achievable at rapid frame rates is ideally suited to intracellular visualization
and measurement of common ion-binding dyes such as the calcium-specific Fluo-4.
EMCCD technology enables readout noise to be completely negated, even at very fast
readout rates, ideally suited to temporal requirements of calcium transient processes,
such as calcium sparks or waves.
Through minimization of excitation powers, whether laser or lamp based, photobleaching
rates of the dyes can be dramatically reduced. In living cell environments, these
"milder" excitation conditions also reduce the phototoxic effects that would otherwise
kill the living cells, enabling them to be studied over longer periods of time.
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