Optical Connections
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Connection diagram for a 3-color fiber photometry setup. The RZ10x is configured with 6 LEDs, 3 Photosensors, and 1 Power Meter |
A general connection scheme for a 3-color fiber photometry setup is shown in the above diagram. RZ10x deluxe models have six LED light driver outputs and four sensor inputs organized into two banks. RZ10 base models have a single bank of three LED outputs and two sensor inputs.
The above RZ10x is configured with six Lux LEDs (405 nm, 465 nm, 560 nm), three Lux Photosensors, and one Lux Power Meter. For the 3-color setup, the Lux LEDs output light through a series of filters and dichroic mirrors ('fluorescent ports') that send excitation light to the subject and receive fluorescence back. The fluorescence signals are then sent to two Lux Photosensors on the RZ10x sensor inputs.
The RZ10x can also be configured with M8 output connectors to drive external LEDs, or BNC inputs to receive external photosensor signals. These can be interchanged by the user.
Lux Pods
Important
Please turn off the RZ10x before adding or removing any connections (optical cables or electrical cables) to Lux Pods on the front banks.
LED_{x} - This is a Lux LED of a specified wavelength x. Common wavelengths used in fiber photometry include 405 nm (autofluorescence detection, isosbestic control), 465 nm (GCaMP, dLight), 560 nm (TDtomato, mCherry, RCaMP). Please see the Lux LED webpage for a list of all available wavelengths.
M8 - This is an M8 connector that is commonly used for external LEDs. Standalone LEDs from Thor Labs and Doric both use M8 connectors for power.
PS1 or PS2 - This is the Lux photosensor.
PM1 - This is the Lux power meter.
BNC - This is a BNC (coaxial) connector that can be used to drive an external LED driver or receive the output of an external photoreceiver. This connector enables the 'DAC Out' or 'ADC In' checkbox, depending on if the BNC is for the Driver or Sensor hardware slots. Enable this checkbox only if you are using the BNC connector outside of the Fiber Photometry gizmo. It will be available on the 'DAC' and 'ADC' tabs of the RZ10x gizmo, respectively.
Fluorescent Ports - these are the series of filters and dichroic mirrors that send excitation light to the subject and receive fluorescence back. Many labs will use Doric Mini Cubes as their light filters instead of creating their own optical benchtop, but both options are feasible. These need to be configured specifically for the wavelengths of light sources and fluorescent signals that are expected. Be sure to route the appropriate light wavelengths to the correct bandpass filter ports.
For example: with a 465 nm GCaMP and 405 nm isosbestic setup that uses a four-port Doric Minicube, the 465 light will route to E1, the 405 light to AE, the subject will be connected to Sample, and the output to the photosensor will be the F1 port.
Fiber optic patch cords - TDT sells a fiber optic patch cable kit with our recommended cables. This includes: a 200 µm core diameter cables for the LED to fluorescent port/ Minicube connection; a 600 µm core diameter cable for the fluorescent port/ Minicube to PS1 or PS2 connection; a 400 µm core diameter cable to serve as the Subject cable* when connected to the Lux PM1 power meter. All cables should have a black jacket to prevent ambient light interference. TDT also recommends that customers order low auto-fluorescent specific subject cables from either Doric or Thorlabs.
Note
Fiber optic cables used in photometry have FC connectors, which have a 'notch-and-key' system. Make sure the key on the male FC connector is fully aligned with the notch on the female connector and that the cable is screwed all the way in. You will get a reduction in light power output if they are not aligned.
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| Checking the FC Connection - Example of Notch and Key |
Note
* For accurate power measurements for your setup, the core diameter of the PM1 cable should match the core diameter of the Subject cable that you are using in your experiments (typically either 200 µm or 400 µm).
Output Attenuation
For customers who want to use larger core diameter cables, such as 400 µm, but need to drive power levels low (less than 40 µW), TDT sells an 85% attenuation coupler to reduce the amount of light going to the subject. This is included in the Lux cable kit. The attenuation coupler connects as follows: LED Patch Cable Attenuation Coupler Patch Cable Fluorescent Port/ Minicube.
For setups with external LED drivers (especially Doric) and a TDT RZ5P, it was common to use patch cords with attenuation filters (1%, 5%, or 10%) to reduce the power output of the excitation light sources before light reaches the fluorescent ports. This is because Fiber photometry is a low light power application, and it was often difficult to drive the LEDs with low enough currents to reach target power levels. The RZ10x and iX6 have superior output signal quality and can adjust the max current output range to allow for very low current outputs, so using attenuating patch cables is not necessary. Also, never connect an attenuating fiber to the photoreceiver; this will severely diminish fluorescent output.
Using a 3rd Party Photosensor
This would be connected to a LUX BNC connector in place of the PS1 or PS2. For Newport photoreceivers, the gain should always be set to DC Low. This provides the widest bandwidth of light detection and detects signal clipping easier. Here is a link to the photoreceiver frequency response plots. If your photoreceiver has a 1x, 10x, 100x option, typically 10x will provide the clearest output response.