DORIC Fiber Photometry System User manual

Fiber Photometry System
Getting Started Guide
Version 2.2.1

Contents
1 Fiber Photometry Systems at a Glance 3
1.1 Systems Overview .................................................... 3
1.2 Fiber Photometry Console ............................................... 5
1.3 Integrated Connectorized Fluorescence Mini Cube ................................ 6
1.4 Light Sources ....................................................... 7
1.5 Rotary Joints ....................................................... 10
1.6 Low Autofluorescence Patch Cords .......................................... 10
1.7 Photodetectors ..................................................... 11
2 Getting Started 14
2.1 Connecting the Fiber Photometry System ...................................... 14
2.2 Configuring the Fiber Photometry Console ..................................... 17
2.3 Using the Fiber Photometry Analysis Module .................................... 25
3 Support 26
3.1 Contact us ........................................................ 26
2

1
Fiber Photometry Systems at a Glance
1.1 Systems Overview
In neuroscience, Fiber photometry denotes a method whereby chronically implanted optical fibers deliver excitation
light to neurons tagged with a fluorescent calcium indicator and collect their overall activity-induced fluorescence. Fiber
photometry sums up the activity-induced fluorescence of all neurons expressing the indicator(s).
In addition to systems custom-designed for your needs, we offer systems for typical modular fiber photometry mea-
surement setups. The Locked-in or Sequential Detection for GCaMP Isosbestic and Functional Excitations system measures
the 405 nm (isosbestic point) excited GCaMP fluorescence, and the 465 nm excited calcium-dependent GCaMP fluo-
rescence, on a single photodetector. The Separated Two Fluorophores Fluorescence contains all the items necessary to
perform photometry measurements of two independent colors in freely-moving animals for GFP-like and RFP-like fluo-
rophores.
A typical set-up for freely behaving animals contains of the following elements (Fig. 1.1).
• A Fiber photometry console to synchronize output control and the acquisition of data.
• An Integrated fluorescence mini cube where beam splitters combine the excitation wavelengths and separate the
emission wavelengths. This Fluorescence Mini Cube comes with the following.
–One or more Built-in fluorescence detector heads capable of sensing low-intensity light. The fluorescence emis-
sion can be collected with one photodetector and subsequently demodulated or, after spectral separation,
collected with respective photodetectors.
–One or more Built-in LED optical heads that provide light for the experiment.
• An optional Fiber-optic rotary joint can be used to allow free movement of the experimental subject.
• An Optical cannula, with connecting Fiber-optic patch cords, to deliver light to the subject.
• A Low Autofluorescence Patch Cord to allow light collection with minimal fluorescence noise.
The following section describes these standard elements and their purpose.
Chapter 1. Fiber Photometry Systems at a Glance 3

ilFMC.jpg
Figure 1.1: Typical Modular Fiber Photometry Measurement Setup
Chapter 1. Fiber Photometry Systems at a Glance 4

1.2 Fiber Photometry Console
This FPGA based data acquisition unit synchronizes the output control and the acquisition of the input data. This device
seamlessly integrates with Doric Neuroscience Studio software, which provides the user-interface for multi-channel
photometry experiments.
The software interface enables control over the CW excitation light pulses, square or sinusoidal waveform of an ex-
ternal source (i.e. LED driver) with 4 digital input/outputs and 4 analog voltage outputs. The software interface displays
real-time data of up to 4 detectors input signals and performs data acquisition. The other new functionalities are being
developed and users can upload them as they become available.
The Fiber Photometry Console inputs and outputs are shown in figures 1.2 and 1.3.
Figure 1.2: Front view of the Fiber Photometry Console: Inputs and outputs
• The LCD Screen displays console information.
• The Digital/IO ports sends 0-4.75 V TTL pulses.
• The HDMI port acquires digital signals and digital communication SPI and LVDS via a custom pinout HDMI con-
nector.
• The Analog-out ports send a variable ±4.75 V analog signal signal.
• The Analog-in ports acquire analog signals up to ±10 V.
• The Power On/Off opens and closes the device.
Chapter 1. Fiber Photometry Systems at a Glance 5

Figure 1.3: Back view: Maintenance and Power Supply
• The 12V port connects to the 12 VDC Power supply.
• The USB port allows a USB-B connection to a computer.
• The Service port is a USB-B port through which the firmware of the device can be updated.
• The USB-3 charging port is currently disabled.
1.3 Integrated Connectorized Fluorescence Mini Cube
Figure 1.4: 4-port Fluorescence Mini Cube
The integrated Fluorescence Mini Cube is an optical assembly that allows the combination of multiple excitation and detec-
tion signals. The cubes are classified by their number of ports, with 3, 4, 5, 6 and 7 port cubes available. The ports are
each classified according to their usage, and are also qualified by a wavelength band corresponding to the bandwidth of
optical filters within the cube.
•Eand IE (Fig. 1.4) represent entry ports for fluorescence and isosbestic point excitation light. Each port of this type
comes with a Built-in LED Optical Head that contains the following elements.
–The Intensity Adjustment Ring (Fig. 1.5a) allows additional adjustment of the overall output intensity of
light.
–The M8 connector port (Fig. 1.5a) is used to connect the optical head to the LED Driver using a Male-Female
M8 Cable.
Chapter 1. Fiber Photometry Systems at a Glance 6

–The Heat-sink Fins (Fig. 1.5a) are used to evacuate heat from the light source, allowing stable output power.
Ensure the fins are not blocked to allow proper cooling.
•F(Fig. 1.4) represents ports for fluorescence emission light. Each port of this type comes with a Built-in Doric
Fluorescence Detector Head.
–The M5 Connector (Fig. 1.5b) allows the Fluorescence Detector Head to be connected to the Fluorescence
Detector Amplifier using an M5 male/male connection cable.
–For extremely low light level applications, the fluorescence ports (F, F1, etc.) can have a Built-in Photomultiplier
Tube rather than a Built-in Fluorescence Detector Head.
•O(Fig. 1.4) represents optogenetic activation or silencing ports. These are always FC receptacles to allow connec-
tion to laser or fluorescence light sources.
•S(Fig. 1.4) represents the exit port to the sample. These are always FC receptacles to allow connection to an ex-
perimental subject.
(a) Built-in LED Optical Head (b) Built-in Fluorescence Detector Head
Figure 1.5: ilFMC Built-in components
1.4 Light Sources
1.4.1 LED Driver
Doric LED Drivers can be used as a stand-alone device or controlled via USB port. Each channel connects to a single
Connectorized LED which can be controlled manually or via the Doric Neuroscience Studio Software.
The LEDs drivers can be used as a stand alone device. During stand-alone operation, it is possible to change the operating
mode (CW or external analog mode) and the current sent to the Connectorized LED. These changes can be done directly
on the device with the control knobs and the LCD display.
Connecting the LED drivers to a computer provides the user with more options. Doric Neuroscience Studio Software
allows the access to more operating modes like CW, external TTL, external analog, internal TTL and internal Complex
modes. Doric Neuroscience Studio Software enables the creation of different sequences of light source activation. It
also provides the possibility to let these sequences be triggered or paused by an external signal. If more power is needed,
it is possible to overdrive the LED driver with the software. Our LED driver has a live pulse capability allowing the
visualization of the signal modulation on the input BNC in a scope-like manner.
• The LCD display (Fig. 1.7) allows easy operation and monitoring. For each channel, the LCD displays the type of
light source (LED), the operating mode, the center wavelength in nm and the current setting.
Chapter 1. Fiber Photometry Systems at a Glance 7

Figure 1.6: LED Drivers; 1-, 2- and 4-channel
Figure 1.7: Front view of a 4-channel LED Driver
• The Power key must be properly inserted into the key switch to enable operation of the light source(s) connected
to the driver. Note that, despite its similar shape, the power key is not a standard micro SD card such as those used
in some digital cameras. Do not attach the Key to a key fob or similar holder; this may prevent proper insertion of
the Power key.
• The M8 connector is used to link the driver and LED.
• The Interlock Connector Plug (Fig. 1.8) allows the user to connect the driver to an interlock system. It is recom-
mended to connect the interlock plug to a laboratory interlock system. This is critical when using LEDs in the UV
or Infrared spectrum, as they are invisible to the naked eye.
• The Input BNC allows the control of the LED driving current of the corresponding source with an analog or TTL
signal.
• The Output BNC are used to monitor the driving current of the corresponding light source.
• The 12 VDC power input connects the driver to its 12 VDC power supply.
• The USB-B Connector allows the driver to be connected to a computer using a USB-A/USB-B cable.
Chapter 1. Fiber Photometry Systems at a Glance 8

Figure 1.8: Rear view of a 4-channel LED Driver
• The On/Off switch (Fig. 1.9) turns on/off the driver.
Figure 1.9: Side view of a 4-channel LED Driver
Chapter 1. Fiber Photometry Systems at a Glance 9

1.5 Rotary Joints
The various cables and fibers used to connect a Fiber Photometry system to an animal can be problematic when used
with a freely-moving animal subject. Rigid cables can limit animal freedom and comfort, while excessive torque applied
to the cables by animal movement can break them. A rotary joint allows the cables and fibers to move with the animal,
without effect on signal quality. When used for fiber photometry, Pigtailed Rotary Joints are favored.
1.5.1 Pigtailed 1x1 Fiber-optic Rotary Joint
The pigtailed variant of the 1x1 Fiber-optic Rotary Joint (Fig. 1.10) includes FC connectorized fiber-optic patch cords
pigtailed on both sides of the rotary joint. This involves pre-alignment of the optical fibers, which reduces intensity
variation in rotation normally observed. They have been designed for applications where optical power variation must
be minimized, such as fiber photometry. These rotary joints are optimized for use with 400 µm and 200 µm diameter
core optical fibers, with an NA of 0.57.
Figure 1.10: Pigtailed 1x1 Fiber-optic Rotary Joint
1.6 Low Autofluorescence Patch Cords
All optical fibers posses a certain innate fluorescence due to their chemical composition. Often called Autofluorescence,
this light can interfere in photometry measurements by overwhelming them. The Doric Lenses Low Autofluorescence (LAF)
Patch Cords have minimal autofluorescence and are thus ideal for fiber photometry. All LAF patch cords are identified by
the red Coupling Nut on the connector.
Chapter 1. Fiber Photometry Systems at a Glance 10