BD CARV II User manual

http://www.bdbiosciences.com/
Part No. 150096 Rev. 000
March 2005
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BD CARV II™
Confocal Imager
Installation and
User’s Guide

© 2005, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced,
transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any
form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior
written permission from BD Biosciences.
The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its
products and services at any time to incorporate the latest technological developments. Although this guide has been
prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions,
nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer
input on corrections and suggestions for improvement.
BD, the BD logo, and BD CARV II are trademarks of Becton, Dickinson and Company.
Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated.
AQM is a trademark of Kinetic Imaging.
DAPI Brightline, E-FFP Brightline, and Texas Red Brightline are trademarks of Semrock®.
IPLab is a trademark of Scanalytics.
Metamorph is a registered trademark of the Universal Imaging Corporation.
Microsoft and Windows are registered trademarks of Microsoft Corporation.
Texas Red is a registered trademark of Molecular Probes/Invitrogen.
X-Cite is a trademark of Electro-Optical Engineering, Inc. (EXFO).
All other company and product names might be trademarks of the respective companies with which they are
associated.
Patents
CARV II: 6,147,798 and patent pending
FCC Information
WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance
could void the user’s authority to operate the equipment.
Notice to Customers
BD Biosciences delivers software and workstations that are intended for running the instruments supplied by
BD Biosciences. It is the responsibility of the buyer/user to ensure that all added electronic files including software
and transport media are virus free. If the workstation is used for Internet access or purposes other than those specified
by BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software.
BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation.
BD Biosciences is not liable for any claims related to or resulting from buyer/user's failure to install and maintain
virus protection.
History
Revision Date Change Made
000 03/05 Initial release

iii
Contents
Chapter 1: BD CARV II Hardware 5
BD CARV II Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
BD CARV II Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
BD CARV II Light Path . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Chapter 2: BD CARV II Installation 13
BD CARV II Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Filter Wheel Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Filter Descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Changing the Filter Wheels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Changing Filters and Dichroics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Binocular Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Touch Pad Controller Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Light Source (EXFO X-Cite 120) Assembly . . . . . . . . . . . . . . . . . . . . . . . . . 28
CARV II Attachment to Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Camera Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Camera Focus Adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Focusing the Pinholes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

iv BD CARV II Installation and User’s Guide
Chapter 3: BD CARV II Touch Pad Controller 33
Touch Pad Controller Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Touch Pad Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Main CARV II Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Secondary CARV II Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Eight-Position Filter Wheel Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Dichroic Wheel Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Intensity Iris Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
FRAP Iris Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Presets Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Calibrating the Prism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Calibrating the Confocal Disk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Calibrating the Filter Wheels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Chapter 4: BD CARV II Command Sets 51
Software Communications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Command Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Command Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index 1

5
1
BD CARV II Hardware
The following topics are covered in this chapter:
• BD CARV II Description
• BD CARV II Specifications
• BD CARV II Light Path

6BD CARV II Installation and User’s Guide
BD CARV II Description
The BD Confocal and Real-time Vision (CARV) II™ is a fully-automated, full
spectrum spinning disk confocal with florescence recovery after photobleaching
(FRAP) capabilities. It can be attached to most major inverted epi-fluorescence
microscopes, and comes with its own variable intensity 120W Hg/metal halide
light source, which allows for full spectrum (360nm-700nm) confocal imaging.
Synchronous automation of an eight-position excitation wheel, a five-position
dichroic wheel, and an eight-position emission wheel allows for fast multi-
wavelength confocal imaging. This eliminates the use of multi-band pass filter
sets and provides maximum light throughput and fast sequential imaging.
BD CARV II can be used with most high-end interline or frame transfer charged
couple device (CCD) cameras, as well as with such imaging software as IPLab
(Scanalytics, Fairfax, VA), Metamorph (Universal Imaging Corporation,
Downington, PA), and AQM (Kinetic Imaging, Nottingham, England).

Chapter 1: BD CARV II Hardware 7
In addition to the main unit, which houses all of the optics and motors, the
CARV II also has a touch pad controller, which controls virtually all the input/
output for the system.
The light source is an EXFO X-Cite 120W Hg/metal halide lamp which delivers
light to the CARV II via a 1.5 M EXFO liquid light guide.

8BD CARV II Installation and User’s Guide
BD CARV II Specifications
The following table details the specifications for the BD CARV II system.
Table 1: BD CARV II Specifications
Confocal Scanner Nipkow spinning disk (pinholes)
Disk Scan Rate 1000 scans per second
Pinhole Diameter 70 µm
Spectral Transmission 360 nm – 700 nm
Z-Resolution 0.5 µm (PSF); 100X PlanApo 1.4NA
Illumination Source 120W Hg-Metal Halide (1200hr)
Internal Excitation Changer automated eight position wheel (25 mm)
Internal Dichroic Changer automated five position wheel (25.7x36 mm)
Internal Emission Changer automated eight position wheel (25 mm)
Filter Sets Provided DAPI, E-GFP, TEXAS RED; (Semrock Bright line
series)
Operation Mode automated confocal, wide field, bright field
Observation direct confocal binocular viewing or camera port
Detector Compatibility CCD camera: Sensicam EM, QE (Cooke);
Coolsnap HQ, Cascade 512B (Photometrics);
ORCA ER, AG (Hamamatsu) and more
Microscope Compatibility most inverted fluorescence microscopes with
100% camera port
Florescence Recovery After
Photobleaching (FRAP)
aperture control–touch pad or RS232
Control touch pad or RS232
Software Drivers IPLab (Scanalytics); Metamorph (Molecular
Devices/Universal Imaging Corporation) RS232
command set also available

Chapter 1: BD CARV II Hardware 9
Figure 1-1 Complete System Mounted on an Olympus IX 71 Microscope with a Hamamatsu Ocra ER
Camera
Size 11(w)x 15.5 (L) x 6 (h) inches
27.94(w) x 39.37 (L) x 15.24 cm
Weight 14.5 lbs /6.6Kg
Power 100-240 VAC, 1.2 A, 50/60 Hz

10 BD CARV II Installation and User’s Guide
BD CARV II Light Path
The following diagram illustrates the light path as it travels through the BD
CARV II and interacts with the various components of the system.
Figure 1-2 CARV II Confocal Imager Light Path
Arc Lamp: The light source is an EXFO X-Cite 120W Hg/metal halide lamp
which delivers light to the CARV II via a 1.5 M EXFO liquid light guide. The
liquid light guide delivery system produces even illumination and eliminates the
need for arc lamp alignment. The lamp has typical Hg arc spectral characteristics
and has a life of 1200 hours.
Shutter: The illumination shutter can be controlled via the touch pad controller
or via RS232 commands (software control).

Chapter 1: BD CARV II Hardware 11
Illumination Intensity Iris: This iris allows the user to vary the illumination
intensity via the touch pad controller or via RS232 commands (software control).
Excitation Filter Wheel: The quick release excitation filter wheel has eight filter
positions and accepts commercially available 25 mm filters. Filter positions can
be controlled via the touch pad controller or via RS232 commands (software
control).
Dichroic Filter Wheel: The quick release dichroic wheel has five positions and
accepts commercially available 25.7x36 mm dichroic mirrors. Dichroic positions
can be controlled via the touch pad controller or via RS232 commands (software
control).
Emission Filter Wheel: The quick release emission filter wheel has eight filter
positions and accepts commercially available 25 mm filters. Filter positions can
be controlled via the touch pad controller or via RS232 commands (software
control).
Spinning Disk: The Nipkow spinning disk can be moved in and out of the light
path, allowing both confocal images and wide-field images to be obtained. The
spinning disk can be controlled via the touch pad controller or via RS232
commands (software control).
FRAP Slit: The adjustable FRAP slit sits on the image plane and is moved into
position when the spinning disk is moved out. The FRAP disk is controlled via
the touch pad controller or via RS232 commands (software control).
Eyepiece: The binocular eyepiece is used to observe both wide field and confocal
images in real time.
Prism: The prism is used to divert the image either to the eyepiece or camera and
is controlled via the touch pad controller or via RS232 commands (software
control).
Camera: A wide range of high-end CCD cameras can be attached to the confocal
unit to obtain confocal, wide-field, or dark-field images.

12 BD CARV II Installation and User’s Guide

13
2
BD CARV II Installation
The following topics are addressed in this chapter:
• BD CARV II Components
• Filter Wheel Assembly
-Filter Descriptions
-Changing Filters and Dichroics
• Binocular Assembly
• Touch Pad Controller Assembly
• Light Source (EXFO X-Cite 120) Assembly
• CARV II Attachment to Microscope
• Camera Attachment
• Camera Focus Adjustment

14 BD CARV II Installation and User’s Guide
BD CARV II Components
The following table lists all of the components of the BD CARV II:
Part Picture Part Description
1
Confocal head with two filter housing covers and camera
tube attached.
2
Eight-position excitation filter wheel with DAPI, GFP,
and TEXAS RED exciter filters in position 1, 2 and 3.
(See Filter Descriptions on page 19 for more
information.)
3
Eight-position emission filter wheel with DAPI, GFP, and
TEXAS RED exciter filters in position 1, 2 and 3. (See
Filter Descriptions on page 19 for more information.)
4
Five-position dichroic wheel with DAPI, GFP, and
TEXAS RED dichroic mirrors and a mirror at positions
1, 2, 3 and 4 respectively. (See Filter Descriptions on
page 19 for more information.)
5
Microscope specific adapter

Chapter 2: BD CARV II Installation 15
6
Binocular attachment
7
Two eyepieces x10
8
Camera tube shroud
9
Touch pad
10
CARV II power supply
Part Picture Part Description

16 BD CARV II Installation and User’s Guide
11
Country-specific CARV II power supply AC cord
12
Interconnect cable
13
DB 9 RS232 cable
14
BNC camera sync cable
15
EXFO X-Cite 120 power supply
Part Picture Part Description

Chapter 2: BD CARV II Installation 17
16
EFXO X-Cite 120 Hg/metal halide lamp
17
1.5M liquid light guide
18
Country specific X-Cite 120 power supply AC cord
19
3mm T handle wrench
20
Hex keys (1.5 mm & 0.05 mm)
Part Picture Part Description

18 BD CARV II Installation and User’s Guide
Filter Wheel Assembly
CARV II accommodates three filter wheels: an eight-position excitation wheel, a
five-position dichroic wheel, and an eight-position emission wheel. The user is
provided with three single filter sets which come mounted on the filter wheels:
DAPI, E-GFP, and Texas Red (see Filter Descriptions on page 19). In addition, the
fourth position on the dichroic wheel has a mounted mirror to be used in
conjunction with the FRAP capability.
The topics that are discussed in the following sections are:
• Filter Descriptions
• Changing the Filter Wheels
• Changing Filters and Dichroics
21
Filter ring spanner wrench
22
Bubble level
Part Picture Part Description

Chapter 2: BD CARV II Installation 19
Filter Descriptions
Three filters are included in the CARV II system:
• DAPI BrightLine™ FF409 Fluorescence
• E-FFP BrightLine™ FF495 Fluorescence
• Texas Red®BrightLine™ FF593 Fluorescence
NOTICE Graphs provided by Semrock®.
DAPI BrightLine™ FF409 Fluorescence
Optimized for these fluorophores:
DAPI, Alexa Fluor 350, AMCA, BFP, Hoechst, LysoSensor Blue, Marina Blue,
Pacific Blue, and sgBFP

20 BD CARV II Installation and User’s Guide
E-FFP BrightLine™ FF495 Fluorescence
Optimized for these fluorophores:
GFP, Cy2™, CyQuant, DiO, EGFP, sgGFP™, wtGFP (non-UV excitation),
YOYO-1, YO-PRO-1
Texas Red®BrightLine™ FF593 Fluorescence
Optimized for these fluorophores:
Texas Red®, 5-ROX, Alexa Fluor 568 & 594, Calcium Crimson, Cy3.5™,
HcRed, and MitoTracker Red