Fluid Imaging Technologies FlowCAM Manuale utente

FlowCAM®Manual

Version 3.0

September 2011

Fluid Imaging Technologies, Inc.

65 Forest Falls Drive ∙ Yarmouth, Maine 04096 USA

Tel: 207.846.6100 ∙ Fax: 207.846.6110

[email protected] ∙ www.fluidimaging.com

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Preface ........................................................................................................................................................................................5

FlowCAM Safety........................................................................................................................................................................6

Standard Factory Limited Warranty.......................................................................................................................................7

SECTION ONE-INTRODUCTION.........................................................................................................................................8

The Scope of this Manual..........................................................................................................................................................8

SECTION TWO-MODES OF OPERATION.......................................................................................................................13

Understanding AutoImage Mode...........................................................................................................................................13

Understanding Trigger Mode (Fluorescence & Scatter)......................................................................................................16

SECTION THREE-INSTRUMENT SETUP ........................................................................................................................19

Instrument Setup .....................................................................................................................................................................19

Model C50 & C51 Peristaltic Pump.......................................................................................................................................23

Model C70 & C71 Syringe Pump...........................................................................................................................................27

SECTION FOUR-VISUALSPREADSHEET........................................................................................................................38

Main Window...........................................................................................................................................................................48

File Menu..............................................................................................................................................................................48

Edit Menu .............................................................................................................................................................................52

Analyze Menu.......................................................................................................................................................................53

Setup Menu...........................................................................................................................................................................54

Context….........................................................................................................................................................................54

Setup Menu… Pump…....................................................................................................................................................70

Setup Menu… Setup and Focus… (F6) ...........................................................................................................................72

AutoImage Mode (No Save)… (F7).................................................................................................................................77

Trigger Mode Setup… (F8)..............................................................................................................................................78

Trigger Mode (No Save)… (F9).......................................................................................................................................78

File Processing Mode (No Save)…..................................................................................................................................78

Magnification…...............................................................................................................................................................78

Tools Menu...........................................................................................................................................................................79

Preferences Menu..................................................................................................................................................................81

Help Menu ............................................................................................................................................................................84

View Window...........................................................................................................................................................................87

Tool Bar................................................................................................................................................................................88

Status Bar..............................................................................................................................................................................89

View Window… File Menu..................................................................................................................................................90

View Window… Edit Menu .................................................................................................................................................91

View Window… Sort Menu .................................................................................................................................................92

View Window… Filter Menu ...............................................................................................................................................93

View Window… Statistics Menu..........................................................................................................................................97

View Window… Show Menu...............................................................................................................................................98

Right Click Popup Menu ....................................................................................................................................................100

Keyboard Commands..........................................................................................................................................................100

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Library Window ....................................................................................................................................................................101

Library Window… File Menu ............................................................................................................................................102

Library Window… Edit Menu............................................................................................................................................104

Library Window… Sort Menu............................................................................................................................................105

Library Window… Show Menu .........................................................................................................................................106

Right Click Popup Menu ....................................................................................................................................................106

Keyboard Commands..........................................................................................................................................................106

Classification Window...........................................................................................................................................................107

Classification Window… File Menu ..................................................................................................................................108

Classification Window… Edit Menu..................................................................................................................................110

Classification Window… Classes Menu.............................................................................................................................111

Classification Window… Operation Menu.........................................................................................................................113

Classification Window… Sort Menu..................................................................................................................................113

Classification Window… Filter ..........................................................................................................................................113

Classification Window… Statistics Menu ..........................................................................................................................114

Classification Window… Show Menu................................................................................................................................114

Right Click Popup Menu ....................................................................................................................................................114

Keyboard Commands..........................................................................................................................................................115

Appendix I: Flow Cells and Tubing Specifications.............................................................................................................116

Appendix II: Glossary of Particle Properties & Other Terms...........................................................................................117

Appendix III: How VisualSpreadsheet Determines Parts/Million and Particles/mL......................................................120

Appendix IV: FlowCAM Trigger Mode Guide...................................................................................................................122

Appendix V: FlowCAM Calibration Verification Procedure............................................................................................125

FlowCAM Size & Count Calibration Verification Report.................................................................................................129

Appendix VI: FlowCAM Quick Start Guide.......................................................................................................................130

Appendix VII: Standard FlowCAM Hardware Configurations........................................................................................133

Appendix VIII: Frequently Asked Questions (FAQ)..........................................................................................................136

Index .......................................................................................................................................................................................141

Notes........................................................................................................................................................................................145

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Thank You

Fluid Imaging Technologies, Inc. would like to thank you for making the important decision to purchase

the FlowCAM®system. We are committed to manufacturing quality products and providing

unsurpassed customer service.

FlowCAM®and VisualSpreadsheet®are registered trademarks of Fluid Imaging Technologies, Inc.

Microsoft Windows and Microsoft Excel are trademarks of Microsoft Corporation. All other trademarks

used within this manual are hereby acknowledged.

Fluid Imaging Technologies customers may reproduce this manual in printed form provided the content

remains unchanged. Reproduction or republication of any portion of this manual for any other purpose

is prohibited without the expressed written consent of Fluid Imaging Technologies, Inc.

Manual Information

FlowCAM®Manual. Version 3.0, September 2011. VisualSpreadsheet®3.0, Portable, Open Benchtop,

Benchtop Models. Manual edited by Benjamin Spaulding, Laboratory Manager.

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·Before operating the system, read the manual carefully to prevent damage to

humans, animals, integrated devices, and connected devices. Always follow local

safety rules.

·Do not look directly into the laser beam. Do not look at any laser light directly or

through optical lenses. Wear appropriate safety glasses to prevent laser light from

entering the eye, even reflections from any surface. When changing optical

components, always disable (via VisualSpreadsheet) the laser.

·Beware of electrical shock hazard. To prevent the risk of electrical shock or fire,

always disconnect the AC/DC power before disconnecting cables or wiring

connectors. Electrical units should not be operated in hazardous environments.

Do not touch any damaged, non-isolated electrical parts.

·This instrument may be harmed by mishandling the flow chamber and/or removing

the photomultiplier tube from its mounting bracket and/or exposing it to direct

room light (while the unit is plugged in and has power). Improper software

settings will not cause damage.

·There are no user serviceable parts inside this unit. Do not attempt any repairs

yourself. Doing so will void your Standard Factory Limited Warranty. Should

your FlowCAM require service, please contact Fluid Imaging Technologies, Inc.

for additional instructions. This product must not be disposed of in normal

household waste. Contact Fluid Imaging Technologies for proper disposal

instructions.

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Standard Factory Limited Warranty

Fluid Imaging Technologies, Inc. (“Seller”) warrants that the FlowCAM®product (“Product”)

purchased by you (“Customer”) shall be free from material defects in workmanship and material for a

period of one (1) year from the date of shipment by the Seller (the “Limited Warranty”); provided,

however, that the Limited Warranty does not cover any consumables (flow cells, flow cell holders, or

tubing) or third party manufactured/customer purchased items incorporated in the Product. For Service

and Spare/Replacement parts, the Seller also warrants the services we perform and the spare and

replacement parts we install, for a period of one (1) year from the date of performance of such services

and the date of installation of the spare or replacement parts, respectively. Customer understands and

agrees that the Limited Warranty shall only apply if Customer has used the Product in accordance with

all specifications, documentation, and other information provided to Customer by Seller.

Customer’s sole and exclusive remedy and Seller’s entire liability under the Limited Warranty shall be

(i) at Seller’s option, repair or replacement of the Product or any defective Product components

(including any labor or other services related thereto), and (ii) all shipping costs related to the repair or

replacement of the Product or any defective Product components for both on-site and off-site repairs and

replacements.

Any Product or Product components returned to Seller must have prior approval and must reference a

Return Material Authorization (RMA) number issued by Seller. In the event Seller determines that the

entire Product must be returned to Seller for repair or replacement, such Product must be shipped in its

original shipping container to assure adequate protection during transit. If the original shipping

container is not available, a new shipping container may be purchased from Fluid Imaging

Technologies, Inc. at an additional cost.

The foregoing Limited Warranty is in lieu of all other warranties, written or oral, express or implied,

including, but not limited to, a warranty of merchantability, non-infringement, title or fitness for a

particular purpose. In no event shall seller be liable for any direct, indirect, consequential, punitive,

incidental or any other damages of any kind whatsoever, arising out of or relating to the product, any

product components, any specifications, documentation or other information provided to Customer in

connection with the product, or the limited warrant set forth herein, even if Seller has been advised of, or

otherwise should have been aware of, the possibility of such damages, and regardless of the legal theory

or basis for such claim.

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Introduction

The Scope of this Manual

Throughout the pages of this manual, you will learn how to set up and operate your FlowCAM®. This

manual is intended to be used with both the Benchtop and Portable Series of FlowCAM models. Minor

differences exist between the models and as such, will not affect the usefulness of this manual.

Additionally, this manual is written to address and describe the configuration of a model VS-IV

FlowCAM (AutoImaging, two Fluorescence Trigger channels, and one Scatter Trigger channel)

equipped with a syringe pump. It must be recognized that some configurations of the FlowCAM system

may not contain fluorescence, light scatter triggering/measurement capability, or a different type of

pump. Please disregard any sections of this manual which do not directly apply to your particular

instrument.

Benchtop B3 Series FlowCAM

Benchtop B2 Series FlowCAM Portable Series FlowCAM

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The FlowCAM®

The FlowCAM®is an integrated system for rapidly analyzing particles in a moving fluid. The

instrument combines selective capabilities of flow cytometry, microscopy, and fluorescence detection.

The FlowCAM automatically counts, images, and analyzes the particles or cells in a sample or a

continuous flow. Best of all, a FlowCAM can be customized to accommodate almost any environment

or application.

Originally developed for oceanographic investigations of organisms and particles in seawater, the

FlowCAM provides the user with the capability to rapidly evaluate particulate matter in fluids. The

instrument and software provides the user with tools to quickly and efficiently meet challenges that

previously required multiple instruments and many hours of tedious work to complete. Your FlowCAM

has the following features and capabilities:

·High-Speed Digital Imaging

·Particle Size, Count, and Shape

·Real-Time Bulk and Individual Particle Analysis

·The Combined Benefits of Multiple Instruments

·Compact and Durable Packaging

·Ability to Image Particles 2 µm to 3 mm in Diameter

·Fluorescence Detection Providing Additional Selectivity

·Scatter Detection for Low Particle Concentrations

Basic Overview of the FlowCAM

In the FlowCAM system, sample is drawn into the flow chamber by a pump. Using the laser in Trigger

Mode, the photomultiplier tubes (PMTs) and scatter detector monitor the fluorescence and light scatter

of the passing particles. When a particle passing through the laser fan has sufficient fluorescence or

laser light scatter, the camera is triggered to take an image of the field of view. The fluorescence

value(s) and/or scatter detection value are then saved by VisualSpreadsheet (in addition to all other

particle properties). The computer, digital signal processor, and trigger circuitry work together to

initiate, retrieve and process images of the field of view. Groups of pixels that represent particles are

then “segmented” out of each raw image and saved as a separate collage image (along with all

associated parameter measurements). The process described above is similar when using AutoImage

Mode, except the camera is set to capture raw images at a user defined interval.

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Advanced Overview of the FlowCAM

The FlowCAM architecture can be divided into three distinct systems:

·Optics

·Fluidics

·Electronics

Optics

Figure 1 is a block diagram of the FlowCAM architecture. The following

section explains the operation of the components illustrated when using laser

dependent Fluorescence and/or Scatter Trigger modes.

As the particles are drawn into the flow chamber they pass through a “laser

fan” that has been generated by the laser and a lens. Fluorescence which

results from this illumination is passed back through the objective and the

partial mirror. This light then passes through a system of optical filters and

mirrors. The shorter wavelengths of green to orange are reflected by a mirror

to the 500-600 nm fluorescence detector. Longer wavelength light passes

through another filter to the 600-700 nm fluorescence detector. Finally, a scatter detector is located at

the end of the optical path (in the same cube as the flash LED) to detect any disturbances in the laser

light.

If either the fluorescence or scatter detector receives a strong enough signal, indicating the presence of a

particle of interest, the flash LED is turned on for a very short interval. This light is then imaged with

the objective onto the camera. A patented depth of focus enhancer stretches out the focus of the

objective, thus enhancing the resolution of particle images.

Fluidics

Sample fluid is drawn through the Flow chamber for analysis by a syringe

pump and then deposited into an outflow collection. FlowCAM’s unique

flow chamber is a key component to this system. Unlike conventional flow

cytometers, the FlowCAM does not use a sheath fluid for hydrodynamic

focusing (laminar flow). Instead, it uses a flow chamber with a variable size

cross section. A complete list of the standard flow cell dimensions

recommended for each objective is available in Appendix I.

The appropriate flow chamber size is chosen by considering the following

interdependent factors:

·Objective Magnification

·Size and Size Range of Particles Under Study

·Desired Flow Rate

·Viscosity of the Fluid

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