RWD 410 User manual

Dual Color Multi-Channel Fiber
Photometry (410&470)
User Manual B

© 2021 Shenzhen RWD Life Science Co., Ltd, All rights reserved.
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Statement
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Assembly operation, expansion, adjustment, improvements and repairs carried out by RWD
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Relevant electrical equipment in line with national standards;
The instrument is operated according to the user manual.
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Components are disassembled, stretched or debugged;
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Product may not in accordance with the manual.

Contents
1. PRODUCT INTRODUCTION......................................................................................................................... 1
1.1. OVERVIEW.................................................................................................................................................... 1
1.2. SAFETY......................................................................................................................................................... 1
1.3. DESCRIPTION................................................................................................................................................ 1
1.4. PRODUCT FEATURES..................................................................................................................................... 2
1.5. ENVIRONMENT REQUIREMENTS................................................................................................................... 2
1.6. PRODUCT PARAMETERS................................................................................................................................ 3
1.7. PRODUCT LIST.............................................................................................................................................. 3
2- SYSTEM SAFETY............................................................................................................................................ 4
2.1. PRECAUTIONS............................................................................................................................................... 4
3- PRODUCT STRUCTUREAND SYSTEM CONNECTION......................................................................... 5
4- SOFTWARE INTRODUCTION...................................................................................................................... 7
4.1. ENVIRONMENTAL REQUIREMENTS OF THE SOFTWARE.................................................................................. 7
4.2. SUMMARY OF MAIN FUNCTIONS OF THE SOFTWARE...................................................................................... 7
4.2.1. Data collection..................................................................................................................................... 7
4.2.2. Data analysis....................................................................................................................................... 7
4.2.3. Overview of shortcut keys.................................................................................................................... 7
5- OPERATION EXAMPLE ................................................................................................................................ 8
6- DATA COLLECTION....................................................................................................................................... 9
6.1. INITIALIZATION ............................................................................................................................................ 9
6.2. LIGHT SETTING........................................................................................................................................... 10
6.3. CHANNEL/CAMERA PARAMETER SETTING...................................................................................................11
6.3.1. Adjusting the fiber end face............................................................................................................... 12
6.4. BEHAVIOR VIDEO........................................................................................................................................ 13
6.4.1. Output setting..................................................................................................................................... 14
6.5. MARKER PARAMETER SETTING................................................................................................................... 15
6.6. PREVIEW .................................................................................................................................................... 17

6.7. START DATA COLLECTION ........................................................................................................................... 18
7- DATAANALYSIS............................................................................................................................................ 20
7.1. GENERALANALYSIS.................................................................................................................................... 20
7.1.1. Overview............................................................................................................................................ 20
7.1.2. Data clip ............................................................................................................................................ 21
7.1.3. Pre-Processing................................................................................................................................... 22
7.1.4. Event analysis.................................................................................................................................... 28
7.1.5. Behavior video................................................................................................................................... 32
7.2. FLUORESCENT HEAT MAP ........................................................................................................................... 33
7.3. TRAJECTORY .............................................................................................................................................. 35
8- SYSTEM SETTING........................................................................................................................................ 36
8.1. TRACE COLOR............................................................................................................................................ 36
8.2. TRACKING SET ........................................................................................................................................... 36
8.3. LANGUAGES ............................................................................................................................................... 37
9- OTHER FUNCTIONS .................................................................................................................................... 38
9.1. FILE............................................................................................................................................................ 38
9.2. DEVICE....................................................................................................................................................... 38
9.3. HELP........................................................................................................................................................... 38
10- MAINTENANCE......................................................................................................................................... 39
11-TROUBLESHOOTING.............................................................................................................................. 40
12- WARRANTY................................................................................................................................................ 41

1
1. Product Introduction
1.1. Overview
Thank you for your choosing Dual Color Multi-Channel Fiber Photometry (410&470)
manufactured by RWD.
Before you assemble and use the product for the first time, read all the documents shipped with it
carefully, which can help you use the product in a more favourable way.
RWD Life Science Co., Ltd. is committed to continuously improving product functions and service
quality. RWD reserves the right to make changes to any product described in this manual and the
content of this manual without prior notice.
If you require the latest product information, please call or write to us, or visit our website
(http://www.rwdstco.com/). If there is any inconsistency between the actual use of the product and
this manual, or if you have any questions or suggestions, please contact us.
1.2. Safety
To avoid injuries to laboratory animals, operators and damage to the system during operation,
please read section 2 "System Safety" carefully.
If you have any questions or suggestions about product safety, please contact our customer service
support.
1.3. Description
This Dual Color Multi-Channel Fiber Photometry (410&470) is designed, developed and produced
by RWD Life Science Co., Ltd. It is a low fluorescence signal detection and the system based on
multi-mode fibers, can excite fluorescence stably for a long time and detect the weak change of
fluorescence signal. The system is a device to record the changes of neuronal activity in animal
population by sCMOS camera after the LED light source is used to excite fluorescence, which has
a wide range of applications in the study of brain loop function.
The purpose of optical fiber recording is to reflect the changes of nerve cell activity in real
time based on the fluorescence of detection probe. Gene-encoded neural probes are widely used in
the field of neuroscience to characterize neuronal activity. The system can be applied to the
fluorescence detection of various GFP-based neural probes, such as GCaMP, dLight, iGluSnFR,
etc. The neural probe was expressed in the neurons of the target brain region, and the excitation
light was introduced into the relevant brain region through the optical fiber to excite the generation
of fluorescence, for measuring fluorescence intensity.
The device is to be used only for animal experiments and pre-clinical
scientific research. It should never be used on humans.
The device should be operated and managed by trained professionals.

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1.4. Product Features
Two LED excitation light sources: 470 nm is used for the fluorescence excitation of green
calcium indicator, and 410 nm is used to eliminate the influence of motion noise.
Up to 9 channels of fluorescence signal recording can be supported for simultaneous
experiments.
Support 4 TTLsignal input and 4 TTL signal input.
Sequential /Continuous excitation output mode.
User-friendly UI for easy operation.
Up to 10,000 hours of service life.
1.5. Environment Requirements
Prepare the operating environment according to the table below to ensure operability and safety
of the system.
Description
Operating environment
Temperature range: 15 °C ~35 °C
Humidity range: 15%~80% (non-condensing)
Air pressure range: 57 kPa~106 kPa
Storage environment
Temperature: range -20 °C ~60 °C
Humidity range: 10%~80% (non-condensing)
Air pressure range: 50 kPa~106 kPa
Working power supply
AC power range: 90 V~264 V, 50/60 Hz, 2 A
DC power range: 12 Vdc, 6 A, 72 w
Voltage fluctuation of the power supply cannot exceed 10% of
the working voltage range.

3
1.6. Product Parameters
Parameter
Description
Dimension
315mm×290mm×111mm
Weight
3.9 kg
Frame rate of the fiber
photometry camera
10~300 fps
Exposure time range
Depends on the frame rate of the fiber photometry camera. If
the frame rate is 10 fps, the exposure time range can be set to
1~30 ms; if the frame rate is 300 fps, the exposure time range
can be set to 1~2 ms.
Frame rate of the behavior
camera
30 fps
LED power regulation
percentage
0~100%
Gain
1~100
1.7. Product List
Configuration
Items
Qty
Description
Standard
Host
1
Fiber photometry.
Standard
BNC cable
2
TTL input/output.
Standard
Power cord of
the host
1
Power supply.
Standard
Power adapter
1
Power supply.
Standard
USB 3.0 cable
1
Transmit the signals of the fiber photometry
camera.
Standard
USB 2.0 cable
1
Connect the device and the computer, downloads
programs, controls the main board, and transmits
marker signals.
Standard
Dongle
1
Used for software.
Standard
Behavior
camera set
1
Shoot behavior videos.
Standard
Optical fiber
1
Connect to the experimental subject's brain.
Standard
Computer
1
Used to install and operate software.
Standard
USB hub
1
USB expansion interface.
Standard
USB Disk
1
Backup software.

4
2-System Safety
Read safety instructions carefully. For safety purposes, pay attention to the following items.
Connect cables correctly.
Ensure that all cables are securely and firmly connected to the device.
Avoid having exposed electrical cables.
Do not touch any internal electronic devices or circuits.
Turn off the device in the case of suspected faults.
If the device has potential safety problems or cannot operate normally, contact
authorized technical support personnel.
Connect the device properly.
Connect the device properly to prevent operation difficulties and disconnection of the
device.
2.1. Precautions
Insert the power cord correctly to prevent poor power contact.
Avoid shaking, water, moisture, compression and fire.
Move and carry the device carefully to avoid damage to and dropping of the device.
Untrained personnel are not allowed to operate the device. Use reliable grounding devices.
Keep the optical fiber interface clean and cover it using a dust cap when the device is not in
use.
RWD is not responsible for any device failure caused by improper cleaning, maintenance and
operation.
If the equipment is not in use for a long time, it is recommended to turn off the host and
computer.
If you disassemble and assemble the device host without authorization from RWD, the
company will no longer provide any quality assurance or technical maintenance service
commitments for the device. If you have any technical problems, contact authorized personnel
or RWD for support.

5
3-Product Structure and System Connection
Figure 3-1
Figure 3-2
No.
Interface Name
Qty
Description
1
Power switch
1
Turns on/off the device.
2
Optical fiber interface
1
Connects the optical box and multi-mode fiber
bundles.
3
Focusing knob
1
Focus on fiber end faces.
4
USB 3.0 interface
1
1) Downloads software, controls the main board,
marks external enhancement.
2) Transmits camera videos.
5
Grounding interface
1
Reserved for electrophysiology device grounding.
6
12V DC power
interface
1
Powers the entire device.
7
USB 2.0 interface
1
Controls hardware communication.
8
Output BNC
connectors
4
Outputs 1-4 are signal channels.
9
Input BNC
connectors
4
Inputs 1-4 are channels of external signals.
9
5
4
8
6
7
1
2
3

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System connection
1）First, connect one end of the power adapter to the power supply and the other end to the 12V
power interface of the host.
2）Connect the USB cables to your computer
Note: be sure to use a USB 3.0 cable to connect the USB 3.0 interface on the host to the
USB 3.0 interface with power supply mark on the computer. The other USB cable is used to
connect the USB 2.0 interface on the host and any USB interface on the computer (or 3.0
USB Hub). The behavior camera must also be connected to the USB3.0 interface on the
computer (or 3.0 USB Hub).
3The fiber optics bundle is inserted into the optical interface for collection.
Figure 3-3

7
4-Software Introduction
4.1. Environmental requirements of the software
A 64-bit operating system of Windows 10 Home
4.2. Summary of main functions of the software
4.2.1. Data collection
1) Continuous collection and sequential collection
2) Synchronous collection of behavior video
3) Automatic marking, manual marking, ROI marking
4) Triggered collection, delayed collection and collection duration setting
4.2.2. Data analysis
1) Output △F/F, and Z-Score
2) Output the behavior track heat map of the experimental subject
3) Output data smoothing, baseline correction and motion correction results
4) Analyze the data event, generate the fluorescence heat map corresponding to the trail
and mean±sem
4.2.3. Overview of shortcut keys
Icon
Meaning
Reconnect (Refresh)
Minimize
Normal
Maximize
Close
Start/Stop Setting
Open Save Dir
Save/Change Save Dir
Auto Scale
Zoom In
Zoom Out
New Files
Save
Check the first 50 markers at once

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5-Operation Example
1) Ensure that behavior testing of an experimental animal is carried out in a dark room or a
laboratory with a stable environmental brightness. Otherwise, the signal recording
results can be affected.
2) Prepare an animal that has been injected with a virus and implanted with an optical fiber
needle. The virus must carry a GFP-based neural probe (such as GCaMP6), and animal
virus expression must last for at least two weeks.
3) Connect the system. For operation details, see section 3- “Product Structure and System
Connection”.
4) Press the power switch to turn on the device. Double-click the icon to start the
software on the desktop. Click Photometry and confirm that devices are properly
connected. The software automatically displays the main data collection tab.
5) Connect the fiber bundle. Shake the fiber end faces towards the light source, and turn
the focusing knob until the fiber end faces are clearly visible in the software.
6) Click Preview. Select 470 nm as the light source, use the optical power meter to
measure the output power, and adjust the optical power to an expected value (for
example, 20 uw) and measure. Change the light source to 410 nm and perform the same
operations. After the measurement is completed, set the light source mode to 410 nm &
470 nm.
7) Access the Channel Setting window. Set the sampling frame rate and select channels
(CH1 to CH9 for chosing). Shake the fiber end faces towards the light source or
increase the gain. Confirm the position of the fiber end faces, adjust the ROI size and
drag the ROI to a proper fiber end face. Set the gain to 1 and save the settings.
8) Point the behavior camera at the area where the animal resides, and adjust the behavior
camera until the video is clearly visible.
9) Set the channel of hardware marker as Input1 and set the signal output of the external
device to high level 3.3 to 5 V.
10) Before starting a formal experiment, keep the preview state for 5 to 10 minutes. The
warm-up process can stabilize the baseline.
11) Connect the optical fiber to the optical fiber needle on the animal head through a sleeve.
12) Adjust the power of the 410 nm light source to make the fluorescence value of 410 nm
close to that of 470 nm.
13) Click [Record] to collect data. After completing the experiment, click [Stop] to stop
data collection.
14) Switch to the Analysis tab to analyze the raw data collected. You can also view the
△F/F and Z-score data analysis results, fluorescence heat map, and behavior trajectory.
15) You can change other parameters, modes and settings according to specific
experimental needs.

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6-Data Collection
6.1. Initialization
Install the multi-channel fiber photometry software, and double-click the desktop software
icon to enter the operation interface.
Figure 6-1
Click [Photometry] or [Analysis] to enter the data collection or analysis interface.
When [Photometry] is selected, the software will automatically detect whether the Host, the
fiber photometry camera, and the behavior camera are connected normally. If the connection is
normal, it will automatically enter [Photometry]. If the interface in Figure 6-2 shows that the
connection status of the corresponding device is "disconnected", please click the refresh button
or conduct a self-check, and reconnect the device to ensure that the connection status is
"connected".
Figure 6-2

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If the connection of the Host, the fiber photometry camera, and the behavior video is identified as
normal, the system will automatically go to the main interface.
The system will enter the [Photometry] page by default, as shown in Figure 6-3. From left to right
are the parameter setting area, the trace area, and the behavior video area.
Figure 6-3
6.2. Light setting
Click to select the Light mode. The Excitation modes include 410nm, 470nm or 410nm
& 470nm. Drag the slider to set the light power proportion of the corresponding light.
Figure 6-4
When [Sequential] is checked, the sequential excitation mode will be used to output
excitation light in preview and collection. The output of excitation light will continue within the
duration, and stop at intervals. The output cycle will go according to the set Duration and
Interval. As shown in Figure 6-4, the duration is set to be 20 seconds, and the interval is set to
be 20 seconds. When the collection starts, the excitation light will be sent continuously for 20
seconds. After that there will be a 20-second interval, during which the software is still running,
Parameter
setting area
Trace area
Behavior video
area

11
but the excitation light will be turned off. At the end of the interval, the excitation light will be
sent for the next 20 seconds. Such a cycle repeats itself.
If it is not checked, the excitation light will be output continuously according to the selected
light mode.
6.3. Channel/Camera parameter setting
This interface displays the current camera frame rate/exposure duration/gain.
Figure 6-5
Offset Subtraction: If it is checked, the offset subtraction algorithm can be used in the process of
fluorescence raw data collection to reduce the background value of the camera and the influence of
the fiber spontaneous fluorescence on the data. When using low fluorescence fiber, it is not
recommended to turn on offset subtraction.
Click [More] in the upper right corner of Figure 6-5 to enter the channel setting window, as
shown in Figure 6-6. Click to select the camera frame rate. The higher the frame rate, the larger
the amount of data collected. 60-100fps is usually recommended.
Drag the slider to adjust the exposure duration and gain. It is recommended to use the
corresponding maximum exposure at the selected frame rate. In data collection, gain value of 1 is
recommended.
The software supports up to nine channels of signal collection. In [Channel Setting], desired
channel such as CH1/CH2 can be chosed. For each channel checked, a green ROI will be generated
in the camera view. By adjusting the gain and changing the brightness of the fiber end face, or by
shaking the fiber toward the light, the position of the fiber end face can be found in the view. Drag
the ROI to cover the corresponding fiber end face, and adjust the gain to the lowest value after
adjusting the ROI.
[ROI Diameter]: change the green ROI diameter to fit different fibers by adjusting the value in

12
the box or clicking and selecting the green ROI to adjust.
Figure 6-6
6.3.1. Adjusting the fiber end face
The fiber end face shall be clearly visible and located in the center of the view. If the fiber end face is
not visible, not clear, or deviated, please select the following methods to adjust.
1) Make sure that the device and the fiber are connected well. At this time, if the fiber end face
cannot be displayed normally, please aim the fiber end face at a strong light, such as
lamplight, flashlight, etc., after which the fiber end face will be visible.
2) If the fiber end face is not clear, the gain in Figure 6-6 can be increased.
3) Adjust the focusing knob until the fiber end face is clearly visible in the view.
Camera field
of view
Fiber end face

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6.4. Behavior video
Adjust the behavior camera and aim it at the observed animal. The video parameters can be set
and the behavior trace can be tracked in real time, as shown in Figure 6-7.
If "Behavior Recording" is checked, the behavior video file can be obtained synchronously in
the process of fluorescence data collection. If it is not checked, the behavior video will not be saved.
If "Behavior Tracking" is checked, the behavior-related file can be output synchronously during
behavior recording. If user has drawn the ROI and made the marking action, the automatic marking
data will be output synchronously.
Figure 6-7
Click [More] to enter the behavior camera setting area.
Click ROI to draw circle, rectangle or polygon. Up to nine ROIs can be drawn.

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Figure 6-8
Adjust the corresponding value of [Tracking Setting] to adjust the grayscale threshold of target
recognition, with a setting range: 0-255.
Check [Preview] to view the tracking status in real time.
6.4.1. Output setting
Click [Output Setting] to enter the output setting interface, where you can set the frequency and
pulse width of TTL signal at the corresponding output terminal, and apply the setting of TTL signal
to the output port selected by the corresponding fluorescence signal "automatic output" and behavior
video ROI "output". The same output port can be set for multiple ROIs.
Note: When the frequency is set to be "0", the signal will stay at a high output level according to the
set duration.
Figure 6-9
When the animal enters the ROI area, the automatic marker will be triggered and take effect in
the corresponding set [Channel], and marker information will be displayed and recorded on the
fluorescence trace.

15
6.5. Marker parameter setting
Figure 6-10
Check [Hardware]: turn on the external signal marker. During signal collection, if the system
recognizes that there is a signal input at the input port, it will identify the automatic marker signal
function on the fluorescent signal. It is necessary to connect the interface of the external device with
the input port of the fiber photometry system through the BNC line.
Check [Manual]: turn on the manual marker. During signal collection, if the system recognizes
that a manual marker action is triggered, the manual marker signal function will be identified on the
fluorescent signal. You can manually mark different events by tapping the keyboard or clicking any
marker icon below the trace.
Check [Auto Output]: turn on the automatic output marker. In signal collection, if the system
recognizes that the fluorescence signal of a certain channel meets the set △F/F trigger threshold, or
the collection has been started, it will output TTL signal to the external device.
Click [More] to open the [Marker/Output Setting] window. All the Hardware, Manual and
Auto Output parameters shall be set respectively.
[Hardware]
When the input port recognizes the external signal input with a high-level signal in the range of
3.3-5V, the software will automatically mark the event with the set marker color on the collection
trace at this time point. The event will take effect in the corresponding [Channel] (multiple channels
can be selected), and the marker information will be displayed and recorded on the data.
Figure 6-11

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[Manual]
Marker name and marker hotkeys can be customized. When the manual marker is triggered,
it will take effect in the corresponding [Channel].
Figure 6-12
When the marking event is triggered, the diagram of the event on the fluorescence signal trace is
as follows:
Figure 6-13