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Fiber Photometry Gizmo

The fiber photometry gizmo is the main interface for setting up and controlling your fiber photometry equipment. There are five tabs to configure your light sensors, light drivers, demodulated data streams, and additional Lux configuration options. Any single fiber photometry gizmo can support up to two sensors and three light sources on a single Lux I/O bank. Additional gizmos can be added to access the second Lux I/O bank for increasing subject or target site count.

Note

Connected Lux power meters (PM1) can accessed at Run-Time through any connected FibPho gizmo no matter which Lux I/O bank the meter is plugged in to.

Driver(s) Tab

This tab is used to configure settings for modulating light sources.

If you have not done so, please perform a Detect Hardware in the Lux tab of the RZ10(x). This will automatically enable detected Light Driver outputs and fill in the Name if a specific LED wavelength is recognized.

Name

This is the name assigned to each light source. For detected Lux LEDs, the Name will autofill with the recognized LED wavelength. If you are using an external LED with an M8 connector, the Name will default to Dv{N}, where N is the light driver output number. Any name can be changed by unchecking Auto ID. The typical convention is to name them after the wavelength of light each source is generating. For example, if Output 1 is your GCaMP signal, then you might provide a name of 465. This will also inform the colors on the runtime interface for the enable buttons and for the demodulated data streams associated with this driver signal. The first three characters of this name will appear on the demodulated data stream store, with the last letter being the first letter of the sensor name.

Max

This is the light driver output range. Options include 50 mA, 200 mA, 500 mA and 1000 mA. The 200 mA, 500 mA, and 1000 mA settings adjust the actual hardware precision to maximize the dynamic range for your desired output signal. Lower max LED currents provide a higher resolution LED output with lower distortion. You should match this setting for your application. You can typically leave this setting as the default 200mA for fiber photometry unless you need to drive higher current outputs to achieve appropriate light power at the fiber tip. If you are using a 400um non-attenuating fiber between the LED and the cube and need finer precision, you should set this value to 50mA. The 50 mA Max uses the 200mA hardware precision settings but gives you 0.1mA precision for the Level and Offset at runtime instead of the default 1mA precision. This increases precision allows for finer control to achieve desired signal output while minimizing distortion.

Defaults

These are adjustable parameters for modulating the light sources. The default values set here in Design-Time will appear the first time the user goes into Preview or Record mode with a new Experiment or Subject, or if the user chooses a 'Fresh' persistence or Run-Time Persistence for the Fiber Photometry gizmo is OFF. At Run-Time, if any of these values are changed, and the user has 'Best' persistence selected, then these values will not be used upon the next Preview or Record. Instead, the last value set in Run-Time will be used. The defaults will, however, not be updated in the Design-Time gizmo settings unless changed by the user.

Frequency - This is the frequency at which the light source will be modulated. Each light source on a subject should be modulated at a different frequency for lock-in amplification to work effectively. Frequency has no effect on the power output. For more on choosing the frequency values, see the Run-Time section.

Level - This is the peak-to-peak amplitude of the light source modulation. This will be the main parameter to adjust when changing power levels. This setting will be adjusted based on the desired light power output or level of response signal observed.

Offset - This is the DC current offset to bias the light source. We will set this to the minimum current that turns the light on through a full modulation cycle and minimizes signal distortion.

Lock Freqs at Run-Time

This option prevents users from accidentally changing the light driver frequencies during Run-Time. Run-Time frequency adjustments are typically only needed for troubleshooting.

Auto-Calc Offsets

Auto-adjust the light driver DC Offset at Run-Time based on the light driver Level. This sets the DC Offset to 10% of the Level (rounding up), with a minimum of 5mA and maximum of 20 mA. This can help reduce signal distortion at higher Level settings.

Launch Power Est

This option will display at Run-Time an estimation of the light power output (in µW) for a connected Lux LED color through a fiber with the fiber core diameter chosen by this setting. This setting can be used in conjunction with a Lux Power Meter PM1 to measure overall light transmission through the entire optical chain.

Sensor(s) Tab

This tab is used to configure settings for connected photosensor signals. If you have not done so, please perform a Detect Hardware in the Lux tab of the RZ10(x). This will automatically enable detected Sensor outputs and fill in the Name.

Note

Connected Lux Power Meters (PM1) will not appear in the Sensor tab. However, if a PM1 is detected, a 'Power Meter' option will appear in the Fiber Photometry Run-Time controls during Preview mode. Please see the Using the PM1 Power Meter section for more details.

Name

This is the name assigned to the photosensor, which is based on the sensor's location in the RZ10(x) (A, B, C, D). The first letter of the sensor name will be appended to the store name of the demodulated data. Any name can be changed by unchecking Auto ID.

Clip Threshold

This value will be set once you know the maximum voltage the photosensor can receive. The clip threshold sets a voltage level above which a red clipping indicator light will turn on in the fiber photometry Run-Time window. The clipping threshold is a dummy light, so it cannot tell when the photosensor is clipping. It must be set correctly, by the user, to be calibrated. For TDT PS1 photosensors, 9.5 V should be accurate. Other external photosensors may have a different clipping threshold. Please refer to the Fiber Photometry Guide for RZ5P Users for more information about adjusting the clipping threshold for non PS1 photosensors.

Demodulator

These settings affect the smoothness of the demodulated data stream. They are applied in real-time, so set these according to how you want the data to be saved.

Filter Order - This setting determines how sharp the low pass filter is that smooths the data. The default 6th order is most used.

Default Low Pass Frequency - This setting will determine the extent of the frequency content in the demodulated data stream. The minimum frequency is 1 Hz and the maximum frequency is 20 Hz. Increasing the low pass corner frequency will add higher frequency content into your demodulated waveform. I prefer the default value of 6 Hz because this provides a nice visualization of Ca++ transients (fast rise and slow decay) during Run-Time. Going below that may be too low, as as Ca++ signals can have a rise time of 100 ms - 300 ms, so some of the response characteristics may be attenuated. Saving the full bandwidth at 20 Hz could be advantageous if later scientific reports show meaningful response dynamics above 6 Hz.

Demodulator(s) Tab

Setup options for signal demodulation and dF/F calculations.

Demodulator Save Options

This cross table (picture, right) is used to configure demodulated data streams. Choose which sensor signals to demodulate at specific light driver frequencies. The appropriate configuration will depend on how many LEDs and sensors are being used and on which subjects.

The above example picture is setup for a subject with 405 nm and 465 nm light sources, and fluorescent responses going to the same photosensor. This configuration will result in two demodulated data streams 405A and 465A that save during Run-Time.

If a second sensor were active in the Sensor(s) tab, then the 'B' column would be active. A typical 3-color configuration is shown to the right. In most cases, one light Driver is only ever crossed with one Sensor, so having both A and B active for any one light driver would not be desired.

Calculated Outputs

These options allow you to perform up to four real-time calculations on the demodulated data streams.

'Source' is a demodulated signal, such as the 465A stream. You can optionally subtract another demodulated signal using the 'Difference with...' column.

A dF/F calculation can be performed on the result of the 'Source' and 'Difference with...' columns. The dF/F calculation, which is a relative change metric, uses a sliding average window as the baseline signal Fo. The 'Window Duration' can be changed from 3 seconds to 120 seconds. The dF/F calculation, which is (F - Fo)/ Fo is performed on each demodulated stream before differencing occurs.

Note

The 'Window Duration' uses an exponential smooth to estimate the mean. Longer windows will have a longer settling time but will provide a cleaner baseline Fo. A 5 - 10 second window should be appropriate for most Run-Time application.

'Saving (1K Rate)' option allows the user to output the calculated signal ('Output Only') or additionally plot ('+Plot') or plot and save ('++Save').

Note

These options do not need to be active to save your regular demodulated stream. This is only for saving the 'Calculated Outputs' stream.

Above is an example output of the 465A demodulated signal plotted above the dF/F of (465A) over a 10 second Window Duration. As you can see, the signals look similar, but the F1c1, which is the dF/F trace, is mean shifted to 0 and normalized to provide a percent change metric of the signal.

Overall, the Calculated Outputs options are useful online visualization tools to give you a general sense of dF/F. They can also be used for sending signals out to other gizmos, such as the Unary Processor or Oscope, for real-time threshold detection and closed-loop stimulation. However, these metrics should not be used as your final dF/F calculations for data analysis. Offline dF/F calculations use more sophisticated signal processing methods and are not as subject to large artifacts and other issues you may encounter at Run-Time.

I recommend that you do both a dF/F of (GCaMP - ISOS) output and a dF/F of (GCaMP) output for comparison. In some cases, such as a very flat Isosbestic signal, the subtraction of the ISOS dF/F from the GCaMP dF/F may add noise to the calculated signal. This is because dF/F is a relative change metric, so for a very flat ISOS signal the baseline fluctuates a significant amount from its Fo, even if it is clean. In this case, just a dF/F of (GCaMP) may be a more accurate representation. If you have a lot of motion artifact, performing a difference will help.

Lux Options Tab

Setup options for Lux bank components and timing controls.

Timing Control

Timing control options are used to cycle the LEDs On and Off for set durations and repeats during Run-Time. This feature is very useful for researchers running long (greater than 1 hour) experiments where photobleaching becomes a concern. The 'Idle When Done' option will return Synapse to Idle mode upon completion of the timing sequence.

Power Meter

This option sets a visual green target range (see blue arrow) set at 75% to 133% of the Target Range for each driver when Display Control Power Meter is active during Run-Time. The target range is total power being read by the Power Meter PM1.

Misc

Assigned Lux I/O Bank - This option informs the Fiber Photometry gizmo which bank of LED Driver outputs and Sensor inputs to target on the RZ10(x), either 'Upper Bank' or 'Lower Bank'.

Important

Your gizmo settings may change depending on which assigned bank is selected. Please check to make sure the assigned I/O bank is the one you want to use with the respective Fiber Photometry gizmo. The Assigned Lux I/O Bank will default to 'Upper Bank' for the first Fiber Photometry gizmo added to the experiment tree. If a second gizmo is added, the Assigned Lux I/O bank will default to 'Lower Bank'.

Legacy Run-time Interface - This option can be enabled if the user wants to use the Fiber Photomery gizmo interface from Synapse v92 and below. Please refer to the Fiber Photometry User Guide for RZ5P users for more details.

Misc Tab

Setup options for default data stores, sampling rate, and driver toggling at runtime.

Required Sample Rate

This option informs the RZ what minimum sample rate this gizmo requires. Typically, 6K is enough. Only increase this if the light driver frequency needs to go beyond 1-2kHz for your experiment, which is rarely done.

Drivers On at Runtime

This option will automatically turn the light driver outputs on when going to Preview or Record mode.

Misc Saves

Store Driver Signals - These data are saved under the store name '{Fi}{N}d' at the RZ processor acquisition rate. These data are the sine waves used to modulate the light driver channels. For n light drivers, there will be n channels of light driver waveforms. These are not saved by default to save data space.

Note

{Fi} are the first two characters and {N} is the last character for that Fiber Photometry gizmo name in the experiment tree. By default, the first Fiber Photometry gizmo added to the experiment tree is 'FibPho1', so the name will be 'Fi1d'. If this was renamed to 'PhotometryX', the name would be 'PhXd'.

Store Driver Parameters - These data are saved under the store name '{Fi}{N}i'. They contain information about each light driver's parameters. A new timestamp containing these parameters is saved when the Light Drivers are enabled and whenever a setting is changed during Run-Time.

Store Sensor Signals - These data are saved under the store name '{Fi}{N}r'. They are the raw photosensor signal(s). These are saved at the RZ processor acquisition rate. They are saved by default and are helpful to keep in case debugging must be done on already saved data.