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Troubleshooting

My LEDs are not turning on

Several factors affect whether the LEDs will turn on. First, check the that you have Detected your RZ10(x) Lux banks. Next, check that the appropriate Drivers and Sensors are active in your Fiber Photometry gizmo, and that you have the appropriate Lux bank active. Then, check the DC Offset setting in Synapse and make sure this is large enough to drive an LED. If this is still an issue, please check your BNC connections from the RZ10(x) to your photodetector and the connections from your LEDs through to your cannula.

My Fi1r signal is always very high (6V or more) or a flat line at a high voltage

If your signal is clipping on the high-end, try turning off the lights in your room. On the benchtop, ambient lighting gets picked up by the cannula and can add a lot of power to the photosensor signal. Ambient lighting will not be a problem in vivo because the brain is dark. If ambient lighting is not the cause of this issue, then adjust the power level of your LED driver down. If there is still a problem, then refer to the next FAQ point.

There is a very narrow range of LED Driver currents or Level settings that gives me a stable Fi1r signal. Outside of that, the LED is either off or I have high-end clipping.

The most likely issue is that too much power is going through your patchcords from the light source. Try using the PM1 to lower the current output on your LEDs to an appropriate target level, using 50 mA Max mode in the Drivers, or putting an attenuation coupler on the output of your LEDs.

My demodulated data stream has a steady downward slope in my subject

You are likely experiencing bleaching or patchcord autofluorescence. One of the benefits of having an isosbestic control is that you can detrend signal bleaching in post processing using a 1st-order polyfit of the control to the GCaMP data (code in the Fiber Photometry Workbook Example). However, it is best to reduce bleaching as much as possible online. Try reducing the power of your lights first and give it a few minutes to stabilize. If that doesn't help, there may be autofluorescence in your patchcords. To reduce this, photobleach your cables.

I pick up 465 nm fluorescence on my 560 nm photosensor (crosstalk)

This is normal due to the filter bandwidths in the fluorescent ports or Minicubes. If you are using two photosensors (one for 405 + 465 or just one for 465, one for 560) and you are modulating the LEDs at different non-multiplicative frequencies (e.g frequency parameter set to 330 Hz, 450 Hz), then this is ok, because lock-in amplification will only extract the contributions of the relevant LED driver signal on each sensor. Just make sure that the 560 photosensor is not being saturated.

My demodulated signals have low-frequency or high-frequency sinusoidal artifacts in them

If you are experiencing very fast (> 2 Hz) or very low-frequency sinusoidal artifacts (\< 1 Hz) in one or both of your demodulated data streams, then it could be because your DC Offset is too low or your Quality-Score is too low in general. Tech Note 0991 has more information about this issue. Please read the Adjusting the LED Parameters section for more details about properly setting the DC Offset and Level.

I have tried all sorts of stimuli and levels, but I cannot get a response

This is not an uncommon result, especially when you are getting up and running. Many factors can attribute to this, not all of which include:

Fluorophore expression - this is dependent on injection accuracy and virus uptake. Histology should be done on all subjects after the completion of experiments to verify expression.

Targeting accuracy - If the cannulas are not within approximately 1 mm of the injection site, then the ability to detect a signal will be compromised. Cannula targeting can be verified during histology.

Time since infection - Levels of GCaMP expression will decrease over time. The longer the time post infections, the lower the overall expression will be.

Photobleaching - Long-term low-level or high intensity light exposure can cause photobleaching of the GCaMP proteins. With photobleaching, users will see a decrease in response from the GFP and the response will be at a constant lower level.

Low Light Power - Under driving the LEDs can make it difficult to pick up a noticeable response during Run-Time. Try slowly increasing the light levels and retest. Do not increase the level too much, or else you may photobleach any GCaMP that is in the area.

Try recording from different animals in the same cohort if you prepped multiple animals. If problems persist, consider trying an easier or more common target to demonstrate that the system and your methodology can work, then try targeting different areas.

What do I need to add a second animal or second site?

The general rule is one photosensor and one set of dichromatic mirrors or Minicube per site/subject. The RZ10(x) can control up to 6 independent light sources. A two animal, fully-independent 405 nm + 465 nm setup would have: four LED Driver channels, two 405 nm and two 465 nm LEDs, two sets of proper dichromatic mirrors or Minicubes, and two photosensors. Each subject would use its own Fiber Photometry gizmo and LUX bank. Multi-site setups on the same animal could share LED sources using a bifurcating cable going from the LED to each minicube or set of mirrors, but this is not recommended because you will lose independent LED and power control.

I want to receive digital TTL communication from an external device, such as MedAssociates. How do I do this?

This is a common feature that customers add to their Synapse experiment when doing behavioral work. The RZ10(x) has 24 bits of digital I/O communication. Four BNC ports are accessible on the front panel of the unit that correspond to Bits C0 - C3. Adding epoc markers to timestamp digital communication in real time is easy in Synapse by enabling Bit Input , Word input, or using the User Input Gizmo (v90 or greater).

I want to add optogenetic or some other external TTL-triggered stimulation to my experiment

The Pulse Gen or User Input gizmos may be used to accomplish this. Be sure to route the gizmo outputs to the desired Digital I/O port on your RZ. Pulse Gen can be set up to trigger pulse trains based on gizmo inputs or external TTL inputs.