DORIC BFP How to use

BFP SYSTEM : Quick Starting Guide
BUNDLE IMAGING
FIBER PHOTOMETRY
'BFP' SYSTEM
QUICK STARTING GUIDE
© DORIC LENSES INC
357 rue Franquet - Québec, (Québec)
G1P 4N7, CANADA
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Date: 2020-02-28
Rev: 00
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BFP SYSTEM : Quick Starting Guide
1. DESCRIPTION
Bundle Imaging Fiber Photometry 'BFP' System consists of a fluorescence cube that images the
fluorescence from an optical fibre port to 1 or 2 image sensor. It includes a set of dichroic mirrors and
bandpass filters to separate excitation and emission spectrums from different fluorophores.
There is also 2 or 3 excitations ports for light coming from LED light sources. The LED source current is
adjusted by the LED driver 'LEDD'.
The BFP Driver 'BFPD' is the key component to synchronize the LED driver with camera acquisition in
an interleave mode. There is also 3 Digital Input or Output to synchronize the BFP system with other
equipment.
Since each camera, the BFP driver and the LED driver are connected to a computer via USB cables, a 4
ports USB 3.0 hub is included to connect only 1 USB cable to the computer..
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BFP SYSTEM : Quick Starting Guide
2. SOFTWARE INSTALLATION
2.1. BEFORE connecting anything to the computer, you need to install the camera driver that is on the
USB key supplied with the system.
Launch Cam33U_setup_4.7.0.1557.exe.
2.2. Install Doric Neuroscience Studio, using the version on the USB key supplied with the system;
Launch setup_DoricStudio5.4.0.15.exe
Or looks for the most recent release in our website
http //doriclenses.com/life-sciences/software/955-doric-neuroscience-studio.html
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BFP SYSTEM : Quick Starting Guide
3. CONFIGURATION HARDWARE
3.1. LED Driver 'LEDD'
LED driver is the current source that will deliver current to the excitation LEDs. Excitations LEDs could
be integrated to the BFMC (most common configuration), or separated unit for more flexibility.
See LEDD user Manual for complete instructions on how it works, but basic information is to connect it
to the 12V AC/DC power supply, make sure to have the SD card key in place, and turn switch ON (1).
Connect the M8 cables between LED driver output to corresponding excitation LED.
3.2. Bund e imaging F uorescence Mini Cube 'BFMC'
This is the central part of the BFP system. It contain set of dicrhroic mirrors and bandpass spectral
filters with optics to have uniform illumination on optical fibre port that is re-imaged to an image snesor.
In most common configuration the excitation LED are integrated within the BFMC. In such case, you
need to connect the M8 cable to LED driver. In another configuration where it is an external LED, there
is an optical fiber connector on excitation port and it is connected to LED source via an optical fibre
cable; ideally, 960 um core - NA0.63.
In most configuration, there is a CMOS camera included with the BFMC that is recognized with our
software, in such case, connect camera trigger cable (Hirose-to-BNC) between cameras and BFPD CAM
#1,2. In the configuration without camera, BFMC have C-Mount port where customer can install any
camera, but it will not be compatible with our software.
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BFP SYSTEM : Quick Starting Guide
3.3. BFP Driver 'BFPD'
The BFP Driver 'BFPD' synchronize the LED driver with camera acquisition in an interleave mode. There
is also 3 Digital Input or Output to synchronize the BFP system with other equipment.
Connect LED trigger cables between the LEDD IN (back-BNC) for channels #1,2,3 and corresponding
channels on BFPD EXC #1, 2, 3 (BNC).
3.4. USB hub
Use the jack Y-splitter to power the LED driver and the USB hub.
Connect the USB3 cables between cameras and the USB hub port 1 and 2.
Connect the USB2 cable between the LED driver and the USB hub port 3.
Connect the USB2 cable between the BFPD and the USB hub port 4.
**** it is important to have installed camera USB driver first as per step 2.1 efore to continue ***
Connect USB3 cable from USB Hub to PC and wait until all devices are recognized and all drivers are
installed
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BFP SYSTEM : Quick Starting Guide
4. SOFTWARE
4.1. Load configurations fi es
If a configuration already exist, you may load the configuration by clicking Load Configuration buttons in
the LED driver and the BFP driver tabs (2 different files).
4.2. Set LED driver
Turn ON the LED Driver, and set each channel to TTL by pushing the knob 2 times, then adjust the
current to the desired value by rotating the knob.
* You may also select the TTL mode and set the LED current using the software by clicking Add Channel
in the Configuration Tab as per figure below.
In order to set the excitation power, you need to monitor the optical power output from the optical fibre
cable that would connect to the sample (animal) with an optical power meter (i.e. Gentec-EO model
PRONTO-Si). Initial current adjustment could be made by setting the LED in continuous mode 'CW',
then calculate the average excitation power in experiment by divising by 2 if using 2 sources interleaved,
or by 3 for 3 sources interleaved.
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BFP SYSTEM : Quick Starting Guide
If you need to use very low current, < 100 mA, we recommend to set LED driver in low-power mode.
There is a minimum current threshold of 40 mA in normal mode, and 4 mA in low power mode. Below
this value, LED will turn OFF completely.
4.3. BFP Set New Configuration (Configuration Tab)
In the BFP Driver tab, go to Configuration tab, and click + NEW/EDIT button.
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BFP SYSTEM : Quick Starting Guide
4.3.1. Presets
The first selection you have to do is the preset for interleave control of the excitation LED and camera
trigger. There is few choice, and you can see a preview of the timing for each configuration.
CONFIG CAMERA LED CYCLES NOTE
#1 1 2 2 Excitation LED 1 and 2 are interleaved 50% each.
Camera 1 record excitation 1 and 2 (i.e. GCaMP with isosbestic).
#2 2 3 2 Excitation LED 2 and 3 are simultaneous and interleaved 50% with excitation 1.
Camera 1 record excitation 1 and 2+3 (i.e. GCaMP with isosbestic).
Camera 2 record excitation 2+3 only (i.e. RCaMP)..
* 2 cycles allow for longer exposure time, but have possible crosstalk between 2 and 3
#3 2 3 3 Excitation LED 1, 2 and 3 are interleaved 33% each.
Camera 1 record excitation 1 and 2 (i.e. GCaMP with isosbestic).
Camera 2 record excitation 3 only (i.e. RcaMP).
* shorter exposure time, but NO crosstalk between excitations
#4 1 1 1 Excitation LED 1 continuous. Camera 1 record excitation 1
* to record faster event without interleaving
* Other configuration will be available on customer request.
4.3.2. Sampling Rate
As you choose the sampling rate, please note that the camera will record at 2 or 3 times this rate when
using interleaving with 2 or 3 cycles.
4.3.3. Resolution, Binning
You can also define the Resolution and Binning which main interest is to reduce the size of the video file
if you are saving the video files for to perform your own processing.
4.3.4. Trigger
A straightfoward way to use the recording is to set the trigger source as Manual (default), then the
recording will start when you click LIVE or RECORD in Acquisition tab.
If you want to have the recording start when triggered by an external signal via a Digital Input, as trigger
source, there is 2 possibilities;
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BFP SYSTEM : Quick Starting Guide
A- Trigger Mode = Triggered
You should click LIVE or RECORD in the Acquisition Tab to have device ready to record, and recording
will start as soon as the selected DIO receive 'TTL-high' signal (5V). Recording will stop when you
manually click STOP in the software interface.
B- Trigger Mode = Gated
You should click LIVE or RECORD in the Acquisition Tab to have device ready to record, and recording
will start when the selected DIO receive 'TTL-high' signal (5V). Recording will continue as long as the
DIO is 'TTL-High', then data stream will interrupt when the DIO is 'TTL-low', but the time will continue,
and when the DIO is back to 'TTL-High', the recording will continue until you manually click STOP in the
software interface.
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BFP SYSTEM : Quick Starting Guide
4.4. Set Region Of Interest (ROI Tab)
You should now define the ROI that correspond to each optical fibre area where all pixels inside a ROI are
averaged to get an intensity for each optical fibre. Averaging on many pixels is a good way to reduce the
electronic noise, then improving the signal-to-noise ratio 'SNR'.
In order to be able to define the ROI, you need first to start a LIVE acquisition to see an image from the
camera(s). You may STOP it after a few seconds.
- Draw as many ROI as you need; we suggest to draw one ROI for each optical fibre, plus one outside to
monitor the background.
- A graph of the ROI average count vs time will appear and a row in right table for each ROI.
- You can rename each ROI by doubleclicking on its name in the table.
- With a configuration using 2 cameras, when a ROI is added, it is added on both cameras at the same
position. It is possible to keep them synchronized (Sync ROI between Cameras checkbox), but most of
the time, because of imperfect alignment between 2 cameras, it is preferred to move and resize ROI on
each camera.
- When ROI are defined, we suggest to save them using the 'Save' button in the ROI tab for faster setup
next day, and lock the editing to avoid moving them by mistake.
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BFP SYSTEM : Quick Starting Guide
4.5. Camera Settings (Configuration Tab)
When ready to start experiment, you can adjust camera exposure time and analog gain in the
configuration tab. First recommendation is to maximize exposure time, and use gain = 0 dB.
If the signal is too strong, we suggest to consider reducing the excitation power before to reduce
exposure time to reduce bleaching of the fluorophore.
If for the excitation power level you want to use, and maximum exposure time for sampling rate you want
does not give enough signal, you can increase the gain, it will also amplify electronic noise, then reducing
SNR, but it is a good way to increase sensitivity..
In our software, we use a resolution lower than maximum camera resolution with the camera field of
view centered. The camera field of view can be moved by unchecking the Auto-Center boxes in the
configuration tab and adjusting the sliders until the image is at the desired position. This feature is
practical to adjust camera 2 position relative to camera 1.
Since you can define independant set of ROI for each camera, it is not required to center both camera
field of view, but it improve visualisation and could facilitate post-processing.
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BFP SYSTEM : Quick Starting Guide
4.6. Recording and Data Saving (Acquisition Tab)
There is 3 operating mode for the system
4.6.1. LIVE :
This mode is acquiring images, but does not save the file on the computer. It allows to setup your
experiment, and test the system without saving useless data on your computer.
4.6.2. RECORD :
This is the mode you should use when doing single experiment recording. First step is to define the the
file name and target path in the Saving Options.
By default, we record the average values of all ROI, Digital I/O if any have been set, and the key event
that create a timestamp everytime user presses the F1, F2, F3 or F4 key. In some computers, it is
required to press Alt+F1 to have this functionalty working. In future, software will allow more
configurable Event keys. Video files are not saved by default. If Save Video is selected, then a separate
video file is created for each feed camera/excitation visible in the interface for the configuration that
have been set.
* Please note that if no ROI have been set, nothing will be recorded.
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BFP SYSTEM : Quick Starting Guide
4.6.3. Time Series :
Time Series allow to perform long term recording with long delay between recording sessions; i.e. 1
minute of recording every hour for 12 hours, as per example above. This will create a single file with all
the datas, NOT a file for each serie.
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BFP SYSTEM : Quick Starting Guide
4.7. View Options (View Tab)
By default, the view of the ROI graph is set to visualize 60 second window. It is possible to increase the
window up to 999 seconds (16 minutes). It is also possible to remove Autoscrolling option to view the
entire data and adjust the time scale manually.
By default, the video view is fitted to fill available space in your screen. If you prefer another zoom
factor, then uncheck the Fit in screen box and choose the desired Zoom factor.
When a zoom factor that requires to drag the display to see all image, you can choose to keep each
camera feed view synchronized by keeping the Synchronized FOV box checked. Or you may prefer to
uncheck it to be able to look at different areas on different video feed.
4.8. Additiona DIO (3)
To be documented.
More detai s on how to use the softare to come...
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BFP SYSTEM : Quick Starting Guide
5. EXPERIMENT
5.1. Focus Adjustment
It is possible to adjust focus of the BFMC by rotating fiber adapter.
First, you need to have an bundle optical fiber cable connected to fiber adapter, and start image
acquisition to adjust focus.
Loosen the counter-nut and rotate the fiber adpater until you get a clear image of the fiber bundle
image in focus.
* Be careful, you may need to rotate several turns to adjust focus. To avoid twisting the cable, you may
need to disconnect and re-connect optical fiber cable during this alignment process.
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Bundle patchcord image in focus (negative)