WPI SI-BF-100 User manual

SI-BF-100
Biofluorometer for Fluorescence Imaging
World Precision Instruments
INSTRUCTION MANUAL
Serial No._____________________
061413
www.wpiinc.com

SI-BF-100
World Precision Instruments iii
Copyright © 2013 by World Precision Instruments, Inc. All rights reserved. No part of this publication
may be reproduced or translated into any language, in any form, without prior written permission of
World Precision Instruments, Inc.
CONTENTS
ABOUT THIS MANUAL ..................................................................................................................... 1
INTRODUCTION.................................................................................................................................. 1
Warnings.......................................................................................................................................... 3
Parts List ........................................................................................................................................... 3
Unpacking ....................................................................................................................................... 3
INSTRUMENT DESCRIPTION.......................................................................................................... 4
Front Panel ...................................................................................................................................... 4
Back Panel....................................................................................................................................... 6
Setting Up the System ................................................................................................................. 6
Changing the PMT Filters........................................................................................................... 7
Adding an LED Module............................................................................................................... 9
Changing the Sample Averages of the Display................................................................11
Changing the Operating Mode ..............................................................................................11
Configuring the System.............................................................................................................12
Modifying the Sampling Frequency................................................................................12
Modifying the Gain Adjust .................................................................................................12
Modifying the Photomultiplier Gain Voltage...............................................................13
Modifying the Filter Frequency.........................................................................................13
Setting up LED Modules......................................................................................................14
OPERATING INSTRUCTIONS.........................................................................................................15
Changing the Display ................................................................................................................15
Parameters Display...............................................................................................................15
Ratio/Sample Averages Display.......................................................................................16
Turning On/Off the Modules...................................................................................................16
Configuration Parameters for Various Fluorophores......................................................17
MAINTENANCE..................................................................................................................................17
Monitoring the LED Output.....................................................................................................17
ACCESSORIES.....................................................................................................................................18
SPECIFICATIONS................................................................................................................................18
APPENDIX A: EXAMPLES OF CALCIUM MEASUREMENT..................................................19
Intracellular Calcium Concentration Measurement ........................................................19
Calcium Measurements with Fluo-4.....................................................................................19
Calcium Measurements with Indo-1 ....................................................................................21
Calcium Measurements with Fura-2.....................................................................................22
APPENDIX B: EXAMPLE OF ATPASE ACTIVITY MEASUREMENT.....................................23
INDEX ...................................................................................................................................................26
WARRANTY ........................................................................................................................................27

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SI-BF-100
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ABOUT THIS MANUAL
The following symbols are used in this guide:
This symbol indicates a CAUTION. Cautions warn against actions that can
cause damage to equipment. Please read these carefully.
This symbol indicates a WARNING. Warnings alert you to actions that can
cause personal injury or pose a physical threat. Please read these carefully.
NOTES and TIPS contain helpful information.
Fig. 1—The Biofluorometer is rack mountable.
INTRODUCTION
The SI-BF-100 is an LED-based fluorometer for life science applications. It is ideally
suited for detecting changes in calcium concentration and ATPase activity in cells.
With the capability of being configured with up to seven LED modules at a variety
of wavelengths, the SI-BF-100 can be utilized in many fluorometric applications in
neuroscience, muscle physiology and cell biology.
NOTE: LED modules, filters and probe configurations described in this manual are
examples of what can be accomplished with this incredibly flexible Biofluorometer.
For custom configurations, please contact WPI at [e-mail].
Until now, biofluorometers were complicated devices with powerful light sources,
filter wheels that selected the proper wavelengths of excitation light, multiple
photomultipliers tuned to detect emissions from bound and unbound indicators and
processors that compensated for fluctuations in light intensity, as well as motion
artifacts. Now, by using LED modules, the SI-BF-100 is designed to have greater
control over the fluorescence measurement cycle. The frequency, duration and
firing sequence of each LED can be programmed through controls on the front
panel of the SI-BF-100. Additionally, you can set the intensity of the LED output, the
sensitivity and filter frequency of the photomultiplier (PMT), the gain of the amplifier,
and the sampling average to optimize the emission signals that can be recorded.
The unit also has a feedback mechanism to monitor the actual light output of the
Biofluorometer and make required adjustments to the output readings.

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The Biofluorometer can be used for many applications, limited only by your
imagination. Some example applications are provided in the appendices:
• Specific information on calcium measurement with several examples is located
in. “Appendix A: Examples of Calcium Measurement” on page 19.
• Information on using NADH/NAD and TAMRA to measure ATPase activity can
be found in “Appendix B: Example of ATPase Activity Measurement” on page
23.
The table belows outlines the example applications used in this manual.
Excitation Wavelengths/Emission Filters for Biofluorescent Dyes and Molecules
Dye or
Molecule
Excited
Active
Molecule
Assayed
Excitation
Wavelength
(nm)
Emission Filter Wavelength
and Range for PMT1
Emission Filter Wavelength
and Range for PMT2
Center, Range
(nm) Filter Number
Center, Range
(nm) Filter Number
Fura-2 Calcium 340, 380 510, 84 84-081
FLUO-4/Fura
Red
Calcium 470 535, 43 67-017 650, 10 65-109
INDO-1 Calcium 360 405, 10 65-072 486, 10 65-085
NADH/TAMRA ATPase 360 472, 30 67-013 572, 28 84-084

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Warnings
CAUTION: The optical input feeds light into the photomultiplier, which is an
extremely sensitive light measurement device. To avoid damage to the
photomultiplier, it is imperative that the probe be attached to the optical
input or that the input be capped when the SI-BF-100 is powered on.
PHOTOMULTIPLIERS ARE EXPENSIVE TO REPLACE.
Parts List
After unpacking, verify that there is no visible damage to the system. Verify that all
items are included:
(1) SI-BF-100 Biofluorometer
(1) 12V Power adapter
(1) Species-specific probe (for example, calcium or ATPase)
(1) M3301 Manual Manipulator
(1) M10 Magnetic Stand
(1) Instruction Manual
Unpacking
Upon receipt of this instrument, make a thorough inspection of the contents and
check for possible damage. Missing cartons or obvious damage to cartons should be
noted on the delivery receipt before signing. Concealed damage should be reported
at once to the carrier and an inspection requested. Please read the section entitled
“Claims and Returns” on page 27 of this manual. Please contact WPI Customer Service
if any parts are missing at 941.371.1003 or [e-mail].
Returns: Do not return any goods to WPI without obtaining prior approval (RMA
# required) and instructions from WPI’s Returns Department. Goods returned
(unauthorized) by collect freight may be refused. If a return shipment is necessary,
use the original container, if possible. If the original container is not available, use a
suitable substitute that is rigid and of adequate size. Wrap the instrument in paper
or plastic surrounded with at least 100mm (four inches) of shock absorbing material.
For further details, please read the section entitled “Claims and Returns” on page
27 of this manual.

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INSTRUMENT DESCRIPTION
The SI-BF-100 works with the SI-H muscle tester systems like the SI-MKB.
Calcium Probe
SI-MKB Platform (sold separately) SI-BF-100 Biofluorometer
Fig. 2—The probe is mounted in a micromanipulator and positioned in the muscle tester
cuvette in close proximity to the tissue sample.
Front Panel
Probe
Excitation
Port
Probe
Emission
Port 1
Probe
Emission
Port 2
BNC Outputs
Display Control Buttons
For Future Use
Port
or
t 1
ort
2
t
N
N
NC Out
ut
NC Ou
NC Ou
p
LED Module
Status Indicator
For Future U
s
Power Button
Fig. 3—The Biofluorometer control unit is rack mountable.
Probe Excitation Port–Connect the excitation fiber of the probe to this port. All the
LED modules are connected inside the chassis to this port using fiber optic cables
and a combiner. When the LEDs are turned on, the light travels through this port to
the probe tip.
Probe Emission Ports–Connect the emission fiber(s) of the probe to these ports.
Fluorescent emissions from the tissue travel from the probe tip to these ports and
into their respective photomultipliers. Some applications (like calcium concentration

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measurement) only require one photomultiplier. Others (like the ATPase
measurement) require both photomultipliers.
BNC Outputs: The analog outputs can be connected to a data acquisition system.
They provide a normalized (relative) output based on the LED intensity and the PMT
(photomultiplier) gain factor. The output depends on the system application. For
example, for calcium measurement:
• Output A–Normalized output of the two 340nm LED modules
• Output B–Normalized output of the 380nm LED module
• Output A/B–Ratio of Output A to Output B
NOTE: The display shows the actual ratio of the emissions measured from the two
frequencies of light. To calculate the ratio based on the A/B BNC voltage output, use
the formula:
Ratio = A/B Voltage x 2
Gain Adjustment Factor
Display–Press the Display button to toggle between the system parameters and the
live ratio measurements. Press the Setup button to enter the Configuration mode
and change the system setup.
Fig. 4—The display (left side) shows the actual ratio measurement. The six control buttons
(right side) let you setup the configuration parameters, turn the LEDs off and on, and
manipulate the display.
Control Buttons–There are six control buttons.
• Display–Press this button to toggle the display between parameters and
ratio measurement.
• Setup–Press this button to enter the setup mode and change parameters.
Adjustable parameters include sampling frequency, gain adjustment,
photomultiplier gain voltage and filter frequency.
• Module Config–Press this button to configure any of the LED modules.
From this menu, you can enable/disable a module, set the current output,
set the delay before it illuminates and set the length of the period that the
LED will illuminate.
• LED On/Off–Press this button to turn all the enabled LED modules on or
off. When the enabled modules are on, the LED light flashes. When you are

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modifying the LED configuration the LED light illuminates without flashing
so you can see the measurement of the LED output on the display.
• Up/Down–Use the buttons to modify configuration setting or parameters.
Power Button–Use this toggle to turn the unit power off or on.
NOTE: The USB connection is reserved for future development.
Back Panel
LED Module Monitor Port
L
Power Supply Connector
L
L
P
Fig. 5—The back panel has a power supply connector and a connection to monitor the on/
off status of the LED modules.
Power Supply Connector–Connect the 12V power adapter to this port.
LED Module Monitor Port–This port gives you access to each LED module to trigger
activity based on a wavelength that is firing. It uses a 5.0V TTL pulse with high
indicating that the LED is firing and low indicating that the LED is off. The unit can
hold up to seven LED modules. Three modules are included with the SI-BF-100 that
is configured for calcium measurement.
Setting Up the System
1. Mount the M10 magnetic stand on the back of the muscle tester platform.
2. Mount the M3301 micromanipulator on the M10 stand.
3. Install the probe in the micromanipulator (Fig. 6).
Fig. 6—(Left) The probe is installed in the micromanipulator.
Fig. 7—(Right) The probe tip is positioned between the tissue holders next to the tissue.

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4. Position the probe so that it fits into the muscle tester cuvette between the
tissue mounts (Fig. 7).
5. If the probe has two fiber optic cables connected to the head, then plug the fiber
optic cable labeled Excitation into the port on the controller labeled LED Optical
Output. Plug the other fiber optic cable (labeled Emission) into the port labeled
Optical Input A. Some probes have two emission fibers. Plug the fiber optic
cable labeled Emission A into port labeled Optical Input A. Plug the fiber optic
cable labeled Emission B into port labeled Optical Input B.
Excitation
Fiber
Emission
Fiber
Fig. 8—Press the SMA connectors into place and tighten the nut to ensure that stray light
does not enter the fiber connection.
CAUTION: The optical input feeds light into the photomultiplier, which is an
extremely sensitive light measurement device. To avoid damage to the
photomultiplier, it is imperative that the probe be attached to the optical
input or that the input be capped when the SI-BF-100 is powered on.
PHOTOMULTIPLIERS ARE EXPENSIVE TO REPLACE.
Changing the PMT Filters
Each photomultiplier has a filter on the front of the SI-BF-100 unit. The filter limits
the emission light that gets sent to the photomultiplier to a single bandwidth of the
spectrum. The filter can be easily swapped out or removed. The filters are inside
the filter holders located behind the SMA connectors labeled Optical Input A and
Optical Input B.
1. Turn off the power to the SI-BF-100 and unplug the instrument.
CAUTION: The optical input feeds light into the photomultiplier, which is an
extremely sensitive light measurement device. To avoid damage to the
photomultiplier, it is imperative that the probe be attached to the optical
input or that the input be capped when the SI-BF-100 is powered on.
PHOTOMULTIPLIERS ARE EXPENSIVE TO REPLACE.