The team here at Neurophotometrics is very excited for the upcoming launch of Version 2 (V2/FP3002), our second-generation fiber photometry system. This is more than just a slightly better Version 1 (FP3001) — it’s an entirely new system.
We’ve designed this new system based upon feedback from current users of Version 1 in order to achieve a user-driven optimization of the rig. Hundreds of conversations about dream experiments and existing pain points were taken into consideration while designing Version 2. The result is an all-new fiber photometry system that is unparalleled in its functionality and sensitivity.
Before we jump into the details of what is new in Version 2, we must acknowledge that we could not have done this alone. We devoted thousands of hours to this project in-house, but it was working with developers at Bonsai and engineers at Open Ephys that really made this possible. Thanks to this huge collaborative effort, we are proud to introduce what we believe will become the next generation of fiber photometry.
Towards a Quantitative Photometry System
The guiding force behind the design of V2 was the need for a more quantitative method of doing fiber photometry. Currently, fiber photometry data is collected and measured in arbitrary fluorescence units whose values range considerably between projects and labs. Our vision for the next generation of fiber photometry is to take a step closer towards something more quantitative. We accomplished this step by focusing our innovation efforts in three primary areas:
- Normalization of data to a “true” baseline
- Enhanced sensitivity
- Greater stability and consistency of the light source
Like its predecessor, V2 will have three excitation wavelengths for fiber photometry recordings: a 470nm LED for imaging of green indicators (e.g. GCaMP), a 560nm LED for imaging of red-shifted indicators (e.g. RCaMP), and a 415nm LED for an interleaved isosbestic control to eliminate motion artifacts (i.e. activity-independent signals).
Normalization to a “true” baseline
Defining baseline is a deceptively tricky task in fiber photometry. Some labs use a portion of the recorded trace during which cells appear inactive as their baseline, while others use the relative amount of signal when excitation lights are off. To create a standard baseline, the V2 system references a brief intraoperative recording from a fiber once it is implanted to create a lookup table between excitation levels and emission, resulting in a more reliable F0 value.
20x Increase in sensitivity
Several new additions give V2 outstanding signal-to-noise capabilities. Redesigned custom filters for red emission increase the absolute signal by 63%. A brand new knife’s edge image splitter gives the V2 clearer images and eliminates smearing, increasing the efficiency with which the camera can convert photons to electrons. And speaking of cameras, an upgraded board-level sCMOS camera with a custom heat sink and fan further reduces noise. For reference: the camera on V1 could pick up a single neuron firing a single action potential with high SNR in slice tests. The camera on V2 has 16x less noise. Plus, it can run at 160 Hz with a very high dynamic range; no need to worry about adjusting the gain between experiments.
Ultra-stable scientific LEds
The most pervasive contributor of noise to photometry signals is instability of light source intensity. LEDs (and lasers) are affected by their environment and temperature has a large impact on their efficiency.
Have you ever wondered why the decay of your photometry data is best fit with a biexponential when photobleaching is a monoexponential? The fast component of this fit is heat-mediated LED decay.
A large part of the development process for V2 went into the design of ultra-stable scientific LEDs that will mitigate this decay. Custom LED circuit boards, heat sinks, and fans make these LEDs more stable. A flexible closed-loop photodiode feedback system updates the current sent to the LED, and that information is stored so that you can be absolutely sure your LED is stable over time. Each LED is also fitted with a variable neutral density filter, allowing you to adjust the dynamic range of your LED.
Integrated Optogenetic Stimulation
Successful combination of fiber photometry and optogenetic experiments historically require a mixture of various equipment: a custom photometry system, lasers, collimators, drivers, pulse generators, etc.
Now, you can do combined fiber photometry and optogenetic experiments with just one piece of equipment: the V2. Every V2 system comes standard with a laser for optogenetic stimulation — your choice between a 450nm and 635nm laser, or you can even upgrade to a system that has both lasers built in! You can also remove the variable neutral density filter from the 560nm LED to get sufficient power for halorhodopsin experiments.
All lasers come with an ultra-stable driver, custom control circuitry, and a built-in software-controlled pulse generator. Control of the lasers is very flexible; they can be arbitrarily stimulated, interdigitated between photometry pulses, used for closed-loop experiments, and more. The lasers are also fiber-coupled and collimated on an XY translator, allowing users to stimulate individual fibers within an array.
A very narrow filter allows for both recording red emission and stimulating with the 635nm laser at the same time. Therefore, you can record GCaMP in one cell population, jRCaMP in another population, and stimulate a red-shifted channelrhodopsin simultaneously in the same brain region!
If you’re not doing fiber photometry, the V2 can be used as a standalone opto stimulation rig.
Software control through Bonsai
Through our longtime partnership with Bonsai, the V2 system will be entirely software controlled. This provides a large increase in the flexibility and repeatability of experiments performed with this system. Bonsai will now feature custom NPM photometry nodes. From there, you can adjust every setting and recording parameter you can dream of, work with a dynamic visualization of your data, and save the experiment in an easy-to-read .csv file.
Integrated digital I/Os
There are two digital inputs and two digital outputs — one of which can be controlled for pulse width modulation. These are time-stamped at the board level for high temporal resolution and can be used to flexibly trigger the system.
Timing Synchronization in/out
Recording data across multiple devices sucks! V2 makes it easier. Through collaboration with the team at Open Ephys, V2 has a timing sync in and out port that lets you synchronize the clocks across multiple devices.
Magnetic patch cord mounting system
No more 5-axis translator for alignment! We’ve engineered a new one-axis magnetically coupled patch cord mounting system. Swapping out patch cords takes seconds and alignment is as easy as turning a single knob — you don’t even have to open the system.
We can’t predict the future, so we’ve equipped the system with expansion ports and a multi-pin GPIO for the next advances that come down the pipeline.
Place Your Order for the V2 Today!
Contact us anytime for a quote, and join us in the next generation of fiber photometry!
This post was written by Sage Aronson and Caroline Sferrazza.