It takes two to tango: using Rhod-2 for
multiplexed calcium imaging
James Drew, Lucie Bard, Dmitri A Rusakov
Clinical Neuroscience PhD
UCL Institute of Neurology
Email: james.drew.14@ucl.ac.uk
Calcium is an important intracellular signaling molecule in the
brain. Both neurons and glial cells maintain low intracellular
calcium levels and influx of calcium is used to initiate a diverse
range of signaling pathways. Fluorescent imaging of calcium
using green calcium-sensitive dyes, such as Fluo-4, has been the
primary diagnostic tool for measuring intracellular calcium levels
and exploring these signaling cascades. However, use of a single
calcium dye has limitations when trying to record multiple
calcium transients in distinct compartments, such as pre- and
postsynaptic cells or neurons and astrocytes, due to the inability
to distinguish between the two signals. To do this, it is
preferential to use two dyes with contrasting emission spectrums.
The red dye Rhod-2, traditionally used as a mitochondrial calcium
indicator, is a candidate for pairing (or multiplexing) with Fluo-4.
However, it is unclear how sensitive Rhod-2 is to cytoplasmic
calcium. This project aims to test whether Rhod-2 is a suitable
tool for measure cytoplasmic calcium and therefore multiplexed
calcium recordings.
A closer look at Rhod-2
Rhod-2 is a rhodamine-based calcium
indicator first produced by Roger Tsien’s
lab [1]. It is non-ratiometric (Fig. 3) and so
the recorded fluorescence is dependent on
both the calcium concentration and the
concentration of Rhod-2.
Importantly, the excitation/emission
spectrum of Rhod-2 is shifted to longer
wavelengths than green indicators [2], with
maximum emission at 570nm. This has a
number of potential advantages:
Reduced autofluorescence
Some cellular components fluoresce independently. This phenomenon adds
noise to fluorescence imaging and limits imaging in deeper tissue. Lowerenergy dyes like Rhod-2 reduces the impact of autofluorescence
Ability to multiplex with green dyes
Methods and results
Simultaneous use of green and red dyes can be used to accurately measure
calcium in two overlapping compartments in a single slice
Acute transverse slices of hippocampus were taken from rats aged P15-21 (Fig. 1).
Brains were submerged in gassed (95% O2 and 5% CO2) ice-cold slicing solution,
and transverse slices (350µm thick) were made. Slices were incubated for an hour in
Ringer solution at room temperature prior to experimentation and used within 6 hours
of sacrifice of the animal.
A
B
Str. oriens
Str. pyramidale
C
Str. radiatum
Fig 1. A: schematic setup of tissue slice and patch recording. B: Digital Image Correlation
(DIC) image showing a band of CA1 cells visible under 20x magnification. C: DIC at 60x
magnification showing patched CA1 cell
Cells were identified before patching by morphology and location in DIC. Patched cells
were loaded with Alexa dye to visualise cell processes. Cell types were then confirmed
by cell morphology and electrophysiology. A range of cells were chosen (Fig. 2) in
different regions of the hippocampus.
A
Fig. 3: Emission spectrum of Rhod-2
at 540nm excitation at different
concentrations of calcium [2]
B
Reduced damage to tissue
Excitation/emission wavelengths are lower energy for Rhod-2 than the green
dyes. It is therefore less harmful to the tissue and better suited for longer
studies
Future directions
Test Rhod-2 response to a range of cytoplasmic calcium dynamics
The next step is to load patched brain cells with Rhod-2 and test the fluorescent
response to a range of physiologically-relevant calcium dynamics. In neurons, calcium
influx can be evoked by injecting a depolarising step to elicit action potentials. In
astrocytes, calcium influx can be induced by puffing glutamate next to the cell,
producing prolonged calcium waves. In both cases, intracellular calcium levels have
been reported to rise to µM levels. As the Kd of Rhod-2 around 550nm, it would be
expected that these dynamics will be suitable for interrogation. It will also be useful to
co-load cells with both Rhod-2 and Fluo-4 to directly compare the fluorescence
responses of the two dyes.
Multiplex Rhod-2 and Fluo-4
It will then be useful to test if Rhod-2 and Fluo-4 can be multiplexed to simultaneously
measure calcium dynamics in multiple compartments (Fig. 4).
•  Rhod-2 (in the AM form) has a long history as a mitochondrial calcium indicator [3]
and there is a large body of research that has used Rhod-2 with fluo dyes to record
mitochondrial and cytoplasmic calcium in single cells (Fig. 4A).
•  Simultaneous measurement of pre- and post-synaptic calcium dynamics (Fig. 4B)
could be useful for interrogating processes such as synaptic plasticity, for which
calcium plays an essential role.
•  Additionally, the last decade has seen an explosion of interest in the the ‘tripartite
synapse’ and the active roles that astrocytes play in synaptic transmission.
Measurement of calcium dynamics in neurons and associated astrocytes (Fig. 4C)
is a potentially powerful tool for investigating how astrocytes interact with neurons
to modify neuronal activity.
Ca
Cb
A
B
C
Postsynaptic
neuron
Presynaptic
neuron
Astrocyte
Mitochondria
Fig 2. Fluorescence imaging of Alexa loaded cells A: Granule cell in the dentate gyrus with a
close up of dendritic spines (arrows). B: Pyramidal cell in CA1. C: Astrocyte in the stratum
radiatum (Ca) and at lower magnification (Cb) showing gap junction-coupled cells (arrows)
Fig 4. Uses for multiplexing red and green calcium dyes. A: Cytoplasmic and mitochondrial
calcium in a single cell. B: Presynaptic and postsynaptic cytoplasmic calcium. C: Neuronal
and astrocytic calcium.
References
1.  Minta A., Kao J. P., Tsien R. Y. (1989) J. Biol. Chem. 264:8171–8178.
2.  http://www.b2b.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Indicators-for-Ca2-Mg2-Zn2-and-Other-Metal-Ions/Fluorescent-Ca2-Indicators-Excited-with-Visible-Light.html#head2
3.  Muriel M.P., Lambeng N., Darios F., Michel P.P., Hirsch E.C., Agid Y., Ruberg M. (2000) J Comp Neurol. 426(2):297-315