Frequently Asked Questions

Is this System a Kit?

No, our system is completely plug and plug. It will be shipped with all parts assembled. All you will need to buy in addition is a patch cord and fiber optic cannulae.

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.

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 version of Bonsai should I be using?

Make sure you have installed Bonsai 2.3. 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 installing them all!

Why isn’t Bonsai recognizing FlyCap?

We are not sure why this happens sometimes, but we suspect a recent software update may have caused the problem. 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.

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.

How do I align my system?

We align your system before we send it, and then adjust the top-left knob to remove the objective and package it safely. After opening your system and installing your objective, hook up a patch cord and turn the system on. Open the FlyCap software, slect your camera, and click OK. At first, only adjust the top-left knob, allowing the patch cord to tilt towards the objective. You should see a number of clear, in-focus circles corresponding to the number of fiber cores in your patch cord. These circles should visibly flicker if you touch the end of your patch cord. If necessary, make small tweaks to the other four knobs to achieve the best possible alignment.

Example of a 2x patch cord w/SMA connector. This may look slightly different with other patch cords or connectors.

Do you sell fiber optic cannulae?

Yes — we offer fiber optic cannulae with black ceramic ferrules that have been optimized for fiber photometry. 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.

Do you sell patch cords?

We only sell patch cords for our multi-fiber arrays. For all other patch cord needs, we strongly endorse those made by Doric Lenses.

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.

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 1.25mm 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.

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.

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.

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.

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 10 hours.

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,:);

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.

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.