Frequently Asked Questions

FP3001 General Information

Where can I find the FP3001 user manual?

You can find a user manual for the FP3001 to download here.

Is this System a Kit?

No, our system is completely plug and play. It will be shipped with all parts assembled. All you will need to buy in addition is a patch cord, fiber optic cannulae, and an adapter to connect the patch cord to your fibers (i.e. ceramic split sleeves).

How many channels can I record from?

You can simultaneously record from red, green, and isosbestic channels.

How many branches can I record from?

You can record from a maximum of 14 different branches from a 200um fiber optic implant or 8 different branches from a 400um fiber optic implant.

How long can I record in a single session?

This will vary based on your signal-to-noise ratio (SNR). In some cases, the SNR is so high that you can reduce the light power, which would allow you to record continuously with very little photobleaching of your fluorophore. A good time range to start your experiments is between 30 minutes and 4 hours. The longest recording with one of our systems to date was 15 hours.

Additional Required Components

What are the system requirements for the computer I will use with this system?

Hard Drive200 MB
RAM8 GB minimum; 16 GB recommended
PortsUSB-3 required
Operating SystemWindows or Linux (32- or 64-bit)
CPU3.1 GHz or equivalent

Do you sell fiber optic cannulae?

Yes — we offer fiber optic cannulae (FOC) that have been optimized for fiber photometry. Our FOC have black ceramic ferrules, which have higher efficiency and lower autofluorescence than other FOC. They are available with 200um or 400um cores and 1.25mm or 2.5mm diameter ferrules. Please visit our product page for pricing and more information.

How do I connect my patch cord to the fiber optic cannula?

You should use a mating sleeve to connect your patch cord to the ferrule. We offer mating sleeves made of white ceramic, which allows you to more easily detect light loss at the connection point when plugged into your test subject. Please visit our product page for pricing and more information.

My fibers are too long. How should I cut them?

Fibers from Neurophotometrics are 10mm long so that you can cleave them in lab to whatever length you need for your experiments. We do offer in-house fiber cleaving services for $3/fiber if needed. We recommend that customers start out by cleaving their own fibers until they have experimentally validated the length of fiber that they’ll be using throughout a project. We have published instructions on how to cleave fibers effectively on the FP Academy.

Do you sell patch cords?

We only sell patch cords for our multi-fiber arrays. For all other patch cord needs, we have published purchasing guidelines here.

What kind of connector should my patch cord have?

All Neurophotometrics systems produced after December 1, 2018 have FC connectors. If your patch cords have SMA connectors, we can provide an adapter for you.

Hardware

What is the difference between the trigger modes on your system?

Trigger mode 1 (BSC1) records with 470 and 560 LEDs in phase while 415 is out of phase.
Trigger mode 2 (BSC2) records with 470 and 560 LEDs out of phase. This trigger mode does not record from the 415 (isosbestic) channel).
Trigger mode 3 (BSC3) records with all excitation channels (560, 470, and 415) out of phase.

What are the TTL modes?

The TTL modes behave similarly to their BSC counterparts, but will only be active as long as a 5V TTL is sent to BNC1. Please do not use a TTL stronger than 5V — you may fry the electronics inside!

What should my camera settings be?

The camera should arrive with the optimum settings pre-programmed and should not be changed. If you suspect your camera settings were changed, refer to the settings below.

What should the gain be set to?

The gain should be high during alignment and lowered during recording. Gain is a digital amplification that uniformly increases signal and noise. Thus, increasing gain during alignment will help to visualize fibers. Decreasing the gain to zero during recording will maximize dynamic range (as increases do not affect SNR).

How do I align my FP3001 system?

We align your system before we send it, but it may need to be aligned to your specific patch cord. Read instructions for aligning the FP3001 here.

Example of a 2x patch cord w/SMA connector. This may look slightly different with other patch cords or connectors. See the manual for more details on alignment.

My 470nm LED is too strong. Can I fix this?

Some of the earliest Neurophotometrics systems have a minimum power output from the 470nm LED that may be too high for your experimental needs. If this has been a problem for you, contact us and we will send you a free neutral density filter (NDF). Swapping in an NDF is a quick process — about 5 minutes — and it will lower the output power to a more suitable range. We work with customers on an individual basis to determine how much power reduction is appropriate.

Software

What version of Bonsai should I be using?

Make sure you have installed Bonsai 2.4. After installation, ensure that the application you use is Bonsai (x64).

What additional software packages should I install?

The most important package is the PointGrey package, but some other video and visual packages are required. We recommend checking out our Getting Started with Bonsai guide for a more complete list of packages to install.

Why isn’t Bonsai recognizing FlyCap?

A recent software update caused this problem in some cases. First, uninstall the FlyCap/PointGrey additional package in Bonsai. Then, uninstall the FlyCapture software. Reinstall using this version of FlyCapture and make sure to install the associated USB drivers. Finally, reinstall the PointGrey package in Bonsai. PLEASE NOTE: FlyCap has been replaced by Spinnaker, so if you are experiencing issues with FlyCapture, we recommend making the shift to Spinnaker.

Experimental Design

How long should I wait for my virus to express?

This depends on your viral serotype, titer, and promoter. We generally recommend waiting 2-3 weeks for good expression. The fastest that we have ever seen good GCaMP expression under a human synapsin (hSyn) promoter in AAV8 was 9 days post-injection.

Where should I buy my virus?

We recommend buying viruses from Addgene. For dLight, we recommend either buying from Addgene or contacting Dr. Lin Tian’s lab directly.

Which GCaMP/RCaMP should I use?

Both GCaMP6s and GCaMP7s work very well. For RCaMP, we have had good results with jRCaMP1b. If you plan to use both GCaMP and RCaMP in the same experiment, we recommend using GCaMP6s instead of GCaMP7s to minimize the difference in kinetics between the two fluorophores.

Analysis

How do I de-interleave my 415nm and 470nm data from trigger mode?

You can separate your data by evens and odds. Example code for this might look like:

dataEven=photometryData(2:2:end,:);
dataOdd=photometryData(1:2:end,:);

Please note that the camera buffer should be turned on to avoid dropped frames that could throw off your data. If the camera buffer is off, you could have a skipped frame.

I see huge increases and decreases in my data after I de-interleave the signals! What is going on?

This can happen if your camera skips a frame. You can fix the problem during analysis by finding the interval between frames and deleting the frames that follow especially high inter-frame intervals. Example code for this might look like:

IFI = diff(data(:,1)); %finds time between frames
plot(IFI) %use this graph to set your threshold
[PKS LOCS] = findpeaks(IFI,’MinPeakProminence’,0.05); %Edit the 0.05 threshold based on plot of IFI. Finds skipped frames
droppedFrames = LOCS
data(droppedFrames,:)=[]; %deletes skipped frames

If you have been having repeated issues with dropped frames, contact us, as we we may be able to help change your camera settings to avoid this problem altogether.