Prizmatix Innovative Application
High-intensity LEDs offer bright excitation light and fast
switching for simultaneous sodium and calcium imaging in neurons
William N. Ross, Ph.D., of New York Medical College and the Marine Biological Laboratory, uses imaging
techniques to study synaptic integration and plasticity in dendrites — branched extensions of neurons
that receive in coming signals from different parts of the brain. To learn more about the calcium and
sodium interactions that take place in neurons, Ross and his colleagues developed a technique for
imaging both sodium and calcium ion signals in quick succession. Incorporating high intensity LEDs from
Prizmatix into their imaging setup made it possible to achieve the fast excitation light switching and high
signal-to-noise ratios needed to capture small, fleeting changes in fluorescence intensity.
To image calcium and sodium simultaneously, the researchers loaded calcium and sodium
fluorescence indicators into a single neuron and then quickly switched between the
excitation wavelengths for each indicator at 500 Hz. The use of LEDs to provide the
excitation light was advantageous because they could be turned on and off
instantaneously without a shutter, which cannot respond this fast and can
introduce vibrations. To take full advantage of the fast LED switching,
the researchers synchronized it to the frame rate of their high-speed camera.
“This rapid switching let us alternately illuminate different indicator dyes very rapidly,” said Ross.
The outputs of the two Prizmatix LEDs are combined with a dichroic mirror.
Because the LEDs are modular, the researchers easily, and cost-effectively,
incorporated various LED combinations into the excitation port of their fluorescence microscope.
“We found it useful that the Prizmatix LEDs could
be controlled by gating, a variable intensity dial,
or analogue input. There was no extraneous
computer control, which makes some other
systems more expensive without providing a real
Prizmatix offers LEDs that have among the
highest intensity of any on the market — five to
ten times brighter than arc lamps in the narrow
wavelength bands used for exciting fluorescent
dyes. This high intensity allowed the researchers
to acquire better measurements since, in the small
compartments of single neurons, the signal-to-noise
ratio is proportional to the square root of the light intensity.
Through test experiments, the researchers
showed that their method produced an
improved signal-to-noise ratio compared to
approaches other researchers had used for
simultaneous imaging of sodium and calcium
ions. While conducting these experiments,
the researchers realized that the LEDs came
with another advantage: the cooling fans
turned on only after a few second delay. Since
this delay was usually longer than the
duration of the measurements, there was no
disturbance from fan vibrations.
The top image shows part of a pyramidal neuron dendrite in a
brain slice from the rat hippocampus with a region of interest
(red) marked near a branch point. The patch electrode on the
soma contained 200 µM ANG-2 (sodium indicator) and 200 µM
bis-fura-2 (calcium indicator). The recording on the bottom left
shows the envelope of the combined signal from the region of
interest, with the two arrows indicating changes resulting from
an extracellular stimulus. The recording on the right shows the
separated signals, which go in opposite directions as the
fluorescence changes from the two indicators either increase
(ANG-2) or decrease (bis-fura-2) with increasing ion
concentrations. The black trace on the bottom shows the
electrical recording from the soma taken at the same time as
the optical traces. Figure courtesy of William N. Ross.
“The Prizmatix LEDs met all our expectations
and have been very reliable,” said Ross. “In
fact, the intensity of the LEDs was much
higher than we originally expected, allowing
us to make more precise measurements.”
After experiencing success with using one pair
of LEDs, the researchers designed new
experiments that take advantage of Prizmatix
LEDs that optimally excite other indicator dyes.
They now have four different LEDs, covering
different spectral ranges, that can be used for
“I found the people at Prizmatix very helpful.
They clearly understood what I was trying to
accomplish with the LEDs,” said Ross. “They let
me try different components before purchasing
them and supplied all the pieces needed to
connect the LEDs to our microscope.”
Miyazaki K. Ross WN. Simultaneous Sodium and Calcium Imaging from Dendrites and Axons.
eNeuro 14 October 2015, 2 (5) ENEURO.0092-15.2015
UHP-LED for Sodium and Calcium Imaging
Prizmatix Products for Research
In this research Dr. Ross used Prizmatix Ultra High Power Collimated LEDs (UHP-T-LEDs) at wavelengths 385 nm, 460 nm and 520 nm.
Since then Prizmatix improved and expanded UHP-T-LED product lines and now we offer more advanced models.
Currently main product lines are:
UHP-T-MP – Most powerful collimated LED with rectangular shape LED emitter (about 3x4mm).
Providing rectangular beam for applications such as microplate illumination.
UHP-T-DI - Powerful collimated LED with square shape LED emitter (3x3mm).
Providing square beam for application such as epifluorescence microscopy,
petri dish illumination, optional 5mm core light guide coupling.
UHP-T-EP – Powerful and high brightness collimated LED with square shape LED emitter (2x2mm).
Providing square beam for application such as epifluorescence microscopy, DMD, Optogenetics,
optional 3mm core light guide coupling.
UHP-T-SR – Ultra Bright collimated LED with small LED emitter (several sizes available).
Providing extra brightness for application such as epifluorescence microscopy, Optogenetics,
optional 3mm core light guide coupling and fiber coupling.
Advantages of UHP-T series:
Location of high current LED driver
In UHP-T-LED the high current LED driver is located inside LED head rather than in controller.
In similar products from other vendors the high current driver located in the controller box.
When high current flows from controller to LED head high RFI/EMI interference
may disturb delicate measurements. In UHP-T-LED models the LED head is grounded and
serve as Faraday cage reducing the RFI/EMI interference.
Optically Isolated TTL and Analog Inputs
The controller of UHP-T-LED features, as standard, TTL input for very fast triggering
(or ON/OFF strobing) of LED light without need of a shutter. The Analog input provides
simple way to control the LED light power from computer. Both inputs are independent
and optically isolated to eliminate ground loops.
Low Optical Noise Option
Most modules can be purchased with Low Noise (-LN) option. The low optical noise
light source is very important in experiments involving such measurements as
cell Sodium and Calcium imaging. In many preparations the change of fluorescence signal may be
just few percent. If the excitation light source exhibits high intensity fluctuations
the small fluorescence changes may be obscured. UHP-T-LED-LN has intensity fluctuations of
less than 0.01% RMS between DC to 1MHz, enabling detection of small changes in fluorescence intensity.
The contemporary LED light source for Sodium and Calcium imaging is UHP-T-EP-LN.
This model provides besides powerful illumination low RFI/EMI interference,
low optical noise and convenient TTL and Analog inputs.
All these features are important in LED light-source for Ephys rig environment.