Frequently Asked Questions

For version 6 to recognize older Doric devices, you also need to update the firmware of your devices. Download instructions Here. In case a manual update is required, you can find the firmware specific to our different devices on this webpage. 

Doric Neuroscience Studio (DNS) is an intuitive software that controls Doric hardware beside recording and analyzing optogenetics, fiber photometry, electrophysiology, and behavior camera data.

Any other behavior data (for instance pressing a key in the arena) that is obtained in voltages using a BNC cable (maximum 10 V) can be recorded by the Doric console either via DIO ports (if the data is on/off mode) or AIN port (for sinusoidal data) simultaneous with the photometry recording and be aligned in time. Above this voltage an extra adaptor is required to adjust the voltage level.

All information coming to console and then DNS will be saved as a single file with .Doric format, containing subfolders for each channel, and they can be loaded and analyzed all together later.

DNS software can livestream the data collection for all channels simultaneously.

The software also contains the following data analysis modules:

• Signal Analysis module (fiber photometry ΔF/F, filters, arithmetics, spike finding, ...)
• Behavior Analysis module (open-field tracking, motion score, and speed)

The negative signals in the image below are what we call ‘packet-loss artifacts’. These types of artifacts can occur when data points are lost during data acquisition.  

Troubleshooting packet-loss artifacts:

Set the software to high-priority, as in DNS-priority.png. (This makes Windows prioritize Doric Neuroscience Studio over any operations running in the background that can tax the computer.)

    Double-check that the console is directly connected to the computer via a USB 3.0 or USB SS (superspeed) port. USB 2.0 ports on the computer are also too slow and often lead to data loss.

     Do not use USB Hub (even if it is USB 3.0). Most off-the-shelf USB hubs do not have the bandwidth and/or speed to properly transfer data from the console to the computer without loss. If required, we sell USB hubs that are thoroughly tested with our equipment (or you can re-arrange USB connections so that the console is directly connected to USB 3.0 of the computer.)

     Avoid: recording data using Remote Access or Zoom or other taxing software when recording data; Remote Access in particular leads to poor data quality and many pack-loss artifacts.

In the new versions of DNS (starting from v 6.4.2.0), we added an image lost counter at the bottom of the microscope view and replace the dropped frame by a copy of the previous one. The number of dropped frame and their timestamp are also saved in an attribute of the dataset:

Here are instructions showing how to use 405 nm (isosbestic signal) to remove motion artifacts from a red signal in danse:

1. Decimate signal (not necessary if using default Lock-In settings in DNS, which automatically decimate by 200x).
2. Baseline Correction (remove exponential decay due to photobleaching to flatten the signal).
3. Align signals (this standardizes isosbestic and biosensor signal so that they can be properly subtracted)
4.  Subtract isosbestic from biosensor signal
5. Filter the signal to remove high-frequency noise (optional)

See example of this is correction, which successful calculated dFF for red signal and remove of motion artifacts.

See the example of pipeline and parameters used in danse™ software to correct the above example:

Instruction for using Statistic Analysis:

1. Calculate photometry signal dF/F: Check this video for more information. 
2. Find Spikes: Check this video for more information. 
   - Note video does not include updated features like interation (which allows the identification of smaller spike when there are very big spikes present in the recording)
3. Create TimePoints (as in image1). Note that the timepoints must be in seconds (not minutes).
4. Select Analysis button (like for PeriEvent), but instead of selecting the Analysis = PeriEvent, select Statistics
5. Then choose spikes as the signal (like image2)
6. Then select Timeperiods and what analysis you would like between spike amplitude, spike frequency or spike count (as in image3)

Note that the Statistic operation can also be used for Events instead of timeperiods.

Image1:

Image2:

Image3:

Here are the instructions:

1. Calibrate the video (set pixel-to-dimension conversion factor for proper speed measurements).
2. Draw Area
3. Animal Tracking (Body)
4. Update Animal Tracking Coordinates (if needed, for example, to label nose to more easily identify interactions)
5. Draw Zones and or distance from points
6. Computer Prescence in Zone / Distance from points
7. Create Events from Prescence in Zone / Distance from points (see image)
   

A useful guide presenting and comparing our different photometry systems may be found here. This guide, in addition to going through their specificities, may also guide you towards a system that may better fit your own experiment. If you need further assistance with this, contact one of our specialists at sales@doriclenses.com.

The Technical Support Tab of our website now contains several tutorial videos providing some help with photometry systems installation and set-up. If you need further assistance with setting-up your system, contact one of our specialists at sales@doriclenses.com. 

If you are using a standard basic fiber photometry setup read information below: 

In general, the standard fiber photometry consists of multiple pieces including: computer interface, console, LED driver, light source, mini-cube, detector, fiber and cannula. A diagram below shows the interconnection of these components.

All commands initiated in the software first reach to the console. Console is typically the control hub of the system, managing all the connections. The console is equipped with three types of ports: the analog output (AOUT), analog input (AIN) and digital input-output (DIO). Upon receiving software commands, the console converts them into voltages and transmits them to the LED drivers through the AOUT ports. The driver then converts voltages into preset current intensities to trigger LED light excitation. Depending on these current, the light sources (LEDs or lasers) will generate the excitation light spectrum. The excitation light then passes through filters and dichroic mirrors inside a cube and reaches to the tissue by optical fibers. The fiber and cube also return the collected neuronal fluorescence emission to the detector which converts light intensities into currents and amplifies them. Those currents are finally sent back to the console via AIN ports.

Remarkably, as mentioned before the console also contains DIO port that are useful for connecting external devices such as an optogenetics system, camera or even a second photometry system, …. DIO ports in this case are useful for synchronizing the additional devices with the photometry system.

Depending on the system, the expression level of the recorded fluorophores, and a bunch of external factors, these values will always be different and must be optimized for your specific conditions.  A good starting point is to look up papers in the brain region of interest and/or that used your fluorophore. The LED power is usually reported and can give you an idea of a more specific range of power for your case. We do recommend though that the isosbestic power be half the power of the experimental signal. This will reduce photobleaching. You should measure the LED power of each LED one at a time (turn off LEDs in the LED Driver module). 

While the isosbestic point of GCaMP fluorophores have been well characterized and 405 and 415nm being widely used in green photometry, red photometry is still in its infancy. There are some who do not use an isosbestic point at all for either color of photometry (even though it is a nice way to control for background fluorescence/artifacts, especially in freely moving animals). 

• FRJ_1x1_PT: 2% peak-to-peak
• FRJ_2x2_PT: 3% peak-to-peak
• AFRJ_2x2_PT: 3% peak-to-peak

Following references list the isosbestic point for frequently used calcium indicators: 

To properly be warned of signal saturation, make sure to specify the right detector type in the Doric Neuroscience Studio (DNS), as different detectors will saturate at different maximum voltages (e.g. Doric = 5 V, while Newport = 7.5 V). Specifically, in the Analog Input setter, set the Saturation parameter to the appropriate detector name. The saturation level is a feature defined by detector hardware and it cannot be modified.

• If the gain is at 100x: reduce it to 10x.  
• If the gain is already at 10x: 
  1. Reduce LED excitation power (see solution 1b)  
  2. If reducing the power did not solve the saturation: reduce gain to 1x.

Photobleaching is among the commonly observed issues during fiber photometry, as a steep drop in photometry signal intensity during the first few seconds of recordings. This decrease is caused by various factors including the light-induced patch cord bleaching, fluorescent reporters, and autofluorescence from the tissue itself. 

Signal disappearing due to photobleaching

When combining blue optogenetic stimulation with red photometry usually researchers report huge artifacts appearing in their recordings. In fact, blue light naturally causes autofluorescence from the silica inside the patch cord at red spectrum (reference, Figure 7b). This artifact is proportional to the blue excitation power used. So, a first potential solution would be to decrease the power of your optogenetic excitation.

If you are using a frequency-division (Lock-In mode) strategy for photometry and TTL pulses (square pulses) for optogenetics stimulation this does not work well with the algorithm and every time the laser opto-stim is turned on or off, it will increase the baseline noise. Instead, you can try using analog signals (sine waves instead of square pulses, at the same frequency), as analog signals work better with the demodulation algorithm.

In addition, check whether you are saturating the red detector. Blue optogenetic light is usually much stronger than blue photometry excitations (mW vs uW). Saturation of the detector will prevent detection of any peaks, as the detector is already at maximum.

Possible Solutions:

1.     Reducing blue excitation power.

2.     Instead of using square pulses for your optogenetic excitation, try converting it to an analog signal with sine waves.

3.     Check that you are not saturating any detectors. (If you are saturating the detector, then you should again reduce opto excitation power.)

4.     Use Interleave mode instead of the Lock-In mode (time-division instead of frequency-division), such that your optogenetic excitation and photometry recording are never at the same time.

5.     Change your experiment design: use red opsin with green photometry instead.

In fact, green fluorophores, commonly have excitation (E) peak at 490 nm. However, as their fluorescent emission (F) is very close to E range, it is an industry-standard to excite slightly off-peak and then maximize the detector filter to obtain a better signal. This range has the added benefit of avoiding the tail-end excitation of the red biosensors.

The excitation and emission filters are predesigned inside the cubes and cannot be modified. As so, using an LED with a peak excitation (490 nm) that is not matching the cube filter peak range (475 nm), might lead to weak excitation power. Plus, the 490 nm LED has relatively less power intensity compared to the 475 nm LED, therefore overall, the power will be lower. This can probably be adjusted using lower attenuating patch cord to compensate and keep the power of excitation high enough.

Besides centering the excitation filter around 490 nm excitation can lead to crosstalk with emission spectrum inside the cube.

The image below is an example for GCaMP6f, comparing two different conditions for E and F ranges. To prevent the excitation light from being picked up by the detector (cross-talks), we need a minimum distance between E and F for 10 nm. So, the more the excitation is shifted to the right (second image), the more the detector filter range will have to also shift to the right, which can lead to significant signal loss (see red arrow). As shown in the second image area under the curve (AUC) is smaller for F detection, meaning that the photometry signal will be weaker.

* Image from https://www.fpbase.org/

This paper reported the isosbestic range of a large number of sensors. It is shown that not every sensor has an isosbestic point. One alternative is to use a non-dynamic fluorophore as a control (like tdTomato), instead of the isosbestic point. You can read more about this option in the same paper above.

The new Doric photometry system comes with additional choices of isosbestic points: 350, 405, 415, 425, 430, 440 nm, etc. We also offer a custom ‘flexible isosbestic’ option, for new photometry systems compatible for diverse set of biosensors. Both of these options require special filters inside the cube that support multiple isosbestic points (so it cannot be retrofitted for older photometry systems).

Regardless, some people have published using 405nm isosbestic as a control signal even, if it is not the true isosbestic point of their sensor (this is quite common for dopamine sensors in the literature). However, it requires further investigation to make sure it is still valid to say that peaks common to red and isosbestic are not real signals, especially when correcting for motion artifacts.

That being said, if 405 is not the true isosbestic signal, care should be taken so that subtraction does not cause artifacts or artificially amplify the signal. This is especially crucial if the signal is quite strong, as very strong signal will lead to measurable ‘downpeaks’ in the isosbestic basically mirroring the biosensor signal (see image below). If this occurs, we recommend reducing the excitation power to minimize ‘mirroring effect’ of isosbestic as much as possible.

The images obtained with Doric Neuroscience Studio are saved in a .doric format, which is a HDF5 type format. They can be visualized using softwares supporting HDF5 format such as Doric Neuroscience Studio (using the Image Analyzer plugin), ImageJ (Import function and various plugins) or a HDF viewer. Code examples are also provided here on our website and allow to read the images in Python, Matlab and Octave. 

We recommend that the optical fiber Patch Cord is of equal or shorter length than the microscope Electrical Cable when connected to the Assisted Opto-Electric Rotary Joint. Even if the cable is looped, the distance from rotary joint to patch-cord connector should be shorter than the length of the electrical cable. 

These two components are meant to be glued together after installation. If they have not been glued during installation, add a drop of quick-drying glue on the border between the Cannula and Protrusion Adjustment Ring. 

Fill the interior of the Cannula with KWIK-CAST (WPI) to act as a cap. After removal of the dried sealant, clean the Rod Lens outer surface using a cotton swab lightly dipped in isopropyl alcohol. 

You can test this compatiblity by downloading the Doric Neuroscience Studio (it's free) and connecting the camera to the same computer. If the camera is recognized in the software, it will appear in the Device Selection window (image 1). If the camera is recognized, it is recommend to test whether it can properly record data.

If the camera is not detected by the software, you can still synchronize your camera using the DIO port of the Doric console by sending a TTL pulse to drive your the camera acquisition. However, you will need to record the video footage with other software (like the one that comes with the camera), and will not be able to use Doric Neuroscience Studio.

Please note to have a single interface to simultaneously record behavior and neural data, we recommend our Beh Camera.

Image 1

1. Make sure your camera’s Iris settings matches lighting conditions. Note that you can use the little screws (yellow box in image) to fix the iris in place.
   
2. For normal ambient light: the white dot of the Iris should be near “1.4”, as in image above
3. For recording in the dark or near-dark: the white dot on the Iris should be near “C”.
4. Make sure your camera’s Focus settings are not drastically out of alignment. The white dot can be anywhere within the blue box, but we recommend centering it between the infinity sign and the first white line. (NOTE: We will optimize the focus after calibration).
5. In the software create a Camera Channel like in image (or Connect the Camera in Device Selection window if you want to use the camera in standalone mode)
   
   
6. NOTE: You can specify FPS in the Camera Option (#3 in image above)
7. Then, as in image, select the “Beh Camera Tab” and click on “Calibrate”.
   
   
8. Under the Beh Camera Tab, you can also adjust the FPS
9. Wait 5-15 seconds until the calibration is complete. (The calibration is complete when the camera image becomes fixed and the “Calibrate” button is no longer selected.)
10. Click ‘Live’ under the Acquisition Tab to adjust the focus to make the image crystal clear. Fix the Focus in place with the screws (yellow box).