Options Manual. COULTER EPICS ALTRA Flow Cytometer COULTER EPICS ALTRA HyPerSort System Flow Cytometer. PN AA (August 2010)

Transcription

1 COULTER EPICS ALTRA Flow Cytomete COULTER EPICS ALTRA HyPeSot System Flow Cytomete Options Manual (August 2010) Beckman Coulte, Inc. 250 S. Kaeme Blvd. Bea, CA 92821

2 WARNINGS AND PRECAUTIONS READ ALL PRODUCT MANUALS AND CONSULT WITH BECKMAN COULTER-TRAINED PERSONNEL BEFORE ATTEMPTING TO OPERATE INSTRUMENT. DO NOT ATTEMPT TO PERFORM ANY PROCEDURE BEFORE CAREFULLY READING ALL INSTRUCTIONS. ALWAYS FOLLOW PRODUCT LABELING AND MANUFACTURER S RECOMMENDATIONS. IF IN DOUBT AS TO HOW TO PROCEED IN ANY SITUATION, CONTACT YOUR BECKMAN COULTER REPRESENTATIVE. HAZARDS AND OPERATIONAL PRECAUTIONS AND LIMITATIONS WARNINGS, CAUTIONS, and IMPORTANTS alet you as follows: WARNING - Can cause injuy. CAUTION - Can cause damage to the instument. IMPORTANT - Can cause misleading esults. BECKMAN COULTER, INC. URGES ITS CUSTOMERS TO COMPLY WITH ALL NATIONAL HEALTH AND SAFETY STANDARDS SUCH AS THE USE OF BARRIER PROTECTION. THIS MAY INCLUDE, BUT IT IS NOT LIMITED TO, PROTECTIVE EYEWEAR, GLOVES, AND SUITABLE LABORATORY ATTIRE WHEN OPERATING OR MAINTAINING THIS OR ANY OTHER AUTOMATED LABORATORY ANALYZER. WARNING Risk of opeato injuy if: All doos, coves and panels ae not closed and secued in place pio to and duing instument opeation. The integity of safety intelocks and sensos is compomised. Instument alams and eo messages ae not acknowledged and acted upon. You contact moving pats. You mishandle boken pats. Doos, coves and panels ae not opened, closed, emoved and/o eplaced with cae. Impope tools ae used fo toubleshooting. To avoid injuy: Keep doos, coves and panels closed and secued in place while the instument is in use. Take full advantage of the safety featues of the instument. Do not defeat safety intelocks and sensos. Acknowledge and act upon instument alams and eo messages. Keep away fom moving pats. Repot any boken pats to you Beckman Coulte Repesentative. Open/emove and close/eplace doos, coves and panels with cae. Use the pope tools when toubleshooting. CAUTION System integity might be compomised and opeational failues might occu if: This equipment is used in a manne othe than specified. Opeate the instument as instucted in the Poduct Manuals. You intoduce softwae that is not authoized by Beckman Coulte into you compute. Only opeate you system s compute with softwae authoized by Beckman Coulte. You install softwae that is not an oiginal copyighted vesion. Only use softwae that is an oiginal copyighted vesion to pevent vius contamination. IMPORTANT If you puchased this poduct fom anyone othe than Beckman Coulte o an authoized Beckman Coulte distibuto, and, if it is not pesently unde a Beckman Coulte sevice maintenance ageement, Beckman Coulte cannot guaantee that the poduct is fitted with the most cuent mandatoy engineeing evisions o that you will eceive the most cuent infomation bulletins concening the poduct. If you puchased this poduct fom a thid paty and would like futhe infomation concening this topic, call you Beckman Coulte Repesentative.

3 REVISION STATUS Initial Release 2/00 Softwae Vesion 1.0 Issue AA, 8/10 Softwae Vesion 1.0. Updates wee made to the company copoate addess. Note: Changes that ae pat of the most ecent evision ae indicated in text by a ba in the magin of the amended page. This document applies to the latest softwae listed and highe vesions. When a subsequent softwae vesion changes the infomation in this document, a new issue will be eleased to the Beckman Coulte website. Fo labeling updates, go to and download the most ecent manual o system help fo you instument. iii

4 REVISION STATUS iv

5 CONTENTS LEGAL NOTICES REVISION STATUS, iii CONTENTS, v INTRODUCTION, xi HOW TO USE YOUR COULTER EPICS ALTRA FLOW CYTOMETER MANUALS, xi ABOUT THE OPTIONS MANUAL, xi HyPeSot System (HPSS) Option, xii CONVENTIONS, xii 1 SAFETY PRECAUTIONS, BIOLOGICAL HAZARDS, LASER SAFETY PRECAUTIONS, 1-1 Radiation Hazads, AIR-COOLED LASERS, GENERAL INFORMATION, 2-1 Installation Requiements, 2-1 Floo Loading and Space, 2-1 Heat and Ventilation, 2-1 Opeation, 2-1 Aligning the Mios, 2-1 Mio Alignment Pocedue, 2-2 Cleaning and Othe Maintenance, AIR-COOLED ARGON LASERS, 2-17 Electical Powe, 2-17 Statup, 2-17 Shutdown, RED HeNe LASERS, 2-19 Electical Powe, 2-19 Contols and Indicatos, 2-19 Lase Head, 2-19 Statup, 2-20 Shutdown, GREEN HeNe LASERS, 2-22 Electical Powe, 2-22 Contols and Indicatos, 2-22 Powe Supply, 2-22 Statup, 2-24 Shutdown, v

6 CONTENTS 2.5 HeCd LASERS, 2-26 Electical Powe, 2-26 Contols and Indicatos, 2-26 Powe Supply, 2-26 Lase Head, 2-28 Statup, 2-29 Pocedue, 2-29 Shutdown, WATER-COOLED LASERS, GENERAL INFORMATION, 3-1 Installation Requiements, 3-1 Heat and Ventilation, 3-1 Opeation, 3-1 Changing Lase Wavelength, 3-1 Aligning the Mios, 3-2 Mio Alignment Pocedue, 3-2 Cleaning and Othe Maintenance, ENTERPRISE II SERIES LASERS, 3-14 Floo Loading and Space, 3-14 Electical Powe, 3-14 Cooling Wate, 3-15 Contols and Indicatos, 3-15 Powe Supply, 3-15 Remote Contol Module, 3-16 Lase Head, 3-17 Statup, 3-18 Set Lase Powe, 3-19 Shutdown, SERIES LASERS, 3-20 Floo Loading and Space, 3-20 Electical Powe, 3-21 Cooling Wate, 3-21 Contols and Indicatos, 3-21 Powe Supply, 3-21 Remote Contol Module, 3-22 Lase Head, 3-23 Statup, 3-24 Set Apetue, 3-27 Set Lase Powe, 3-27 Tune Lase fo Maximum Powe, 3-27 Shutdown, vi

7 CONTENTS 3.4 SPECTRUM LASER, 3-28 Floo Loading and Space, 3-29 Electical Powe, 3-29 Cooling Wate, 3-29 Contols and Indicatos, 3-29 Powe Supply, 3-29 Remote Contol Module, 3-31 Lase Head, 3-32 Statup, 3-33 Set Lase Powe, 3-34 Tune Lase fo Maximum Powe, 3-35 Shutdown, STORAGE OPTIONS, GENERAL INFORMATION, 4-1 Magnetic Stoage Media Cae, Zip DRIVE, RECORDABLE CD-ROM DRIVE, 4-1 Easy CD Ceato, 4-2 Easy CD-Ceato Installation Pocedues, jaz DRIVE, Autoclone SORTING OPTION, INTRODUCTION, INSTALLATION REQUIREMENTS, 5-1 Installation Calibation Pocedue, STARTUP, SORT MATRIX, SORTING, CALIBRATION, 5-21 Fine Tune Calibation, 5-22 Adjust Autoclone Soting Option Speed, 5-26 Noisy Movement, 5-26 Missing Wells, GATED AMP OPTION, WHAT THIS CHAPTER EXPLAINS, 6-1 -vii

8 CONTENTS 6.2 FEATURES OF THE GATED AMP, 6-1 Time-Domain Gating, 6-1 Lowe Beam Lockout, 6-1 Dual Lase Applications, 6-1 Example: DAPI/FITC fo Viable DNA and Immunofluoescence, 6-1 Example: Calcium Flux with Indo-1 and Two-Colo Cell-Suface Makes, 6-1 Example: Hoechst/Chomomycin fo Dual-Colo Chomosome Soting, 6-2 Example: Simultaneous Fou-Colo Clone Studies, IMAGING OPTICS ALIGNMENT, SET UP GATED AMP SCREENS, 6-6 Gated Amp Assignment Sceen, 6-6 Gated Amp Contol Sceen, 6-8 Gated Amp Contol Sceen Settings, 6-10 Fluoescence Compensation Combinations, 6-10 Softwae Selectable Amplifie Speed/Sensitivity, CHECK ALIGNMENT, ADJUST WINDOW WIDTH, LARGE PARTICLE SORTING OPTION, DESCRIPTION, COMPONENTS, INSTALL THE LARGE PARTICLE SORTING OPTION, 7-1 Pocedue, REPLACE THE SAMPLE TUBING ASSEMBLY, 7-12 Pocedue, RUN SAMPLES, 7-17 Install Lage Paticle Soting option, 7-17 Select the Flow Cell Tip, 7-17 Select Settings, 7-17 Run Sample, FORWARD FLUORESCENCE DETECTOR OPTION, DESCRIPTION, INSTALL THE FORWARD FLUORESCENCE DETECTOR OPTION, 8-1 INDEX, INDEX-1 TRADEMARKS -viii

9 CONTENTS ILLUSTRATIONS 1.1 Agon Lase, Manufactue s Waning Labels, Red Helium-Neon (HeNe) Lase, Manufactue s Waning Labels, Geen HeNe Lase, Manufactue s Waning Label, Helium Cadmium (HeCd) Lase, Manufactue s Waning Labels, Coheent Innova Entepise II Seies Lase, Manufactue s Waning Labels, Coheent Innova 300 Seies Lase, Manufactue s Waning Labels, Coheent Innova 70 Spectum Lase, Manufactue s Waning Labels, Cytomete Sensing Aea, Lase Waning Labels, Fowad Fluoescence Detecto Option Sensing Aea, Lase Waning Labels, Cytomete Sensing Compatment s Cove, Lase Waning Labels, Cytomete Oute Cove, Lase Classification Label, Cytomete Rea and Left Cove Lase Waning Labels and Lase Cetification Label, Lase Aangement - Ai-Cooled Lase, Example, Lase Head - Red HeNe Lase, Powe Supply - Geen HeNe Lase When Used As a Thid Lase, Lase Head - Geen HeNe Lase, Powe Supply - HeCd Lases, Lase Head - HeCd Lases, Lase Aangement - Wate-Cooled Lases in a Thee Lase Configuation, Powe Supply - Entepise II Seies Lases, Remote Contol Module - Entepise II Seies Lases, Lase Head - Entepise II Seies, Powe Supply Seies Lases, Remote Contol Module Seies Lases, Lase Head Seies Lases, Powe Supply - Spectum Lase, Remote Contol Module - Spectum Lase, Lase Head - Spectum Lase, Gated Amp Assignment Sceen, Gated Amp Contol Sceen, 6-8 -ix

10 CONTENTS TABLES 2.1 Powe Supply Contol Settings - HeCd Lases, Powe Supply Indicatos - HeCd Lases, Cooling Wate Specifications - Entepise II Seies Lases, Powe Supply Contols/Indicatos - Entepise II Seies Lases, Remote Contol Module - Entepise II Seies Lases, Cooling Wate Specifications Seies Lases, Remote Contol Module Seies Lases, Cooling Wate Specifications - Spectum Lase, Powe Supply and Remote Contol Module Contols and Indicatos - Spectum Lase, Typical Settings fo Lage Paticle Soting, x

11 INTRODUCTION This intoductoy section contains the following topics: How to use you COULTER EPICS ALTRA and ALTRA HyPeSot System flow cytomete manuals About the Options manual Conventions. HOW TO USE YOUR COULTER EPICS ALTRA FLOW CYTOMETER MANUALS Use the Refeence manual fo in-depth infomation about: What the instument does The methods it uses Its specifications Infomation about installation. Safety. Use the Opeato s Guide fo the day-to-day unning of you instument: Read the Contols and Indicatos chapte to become familia with the diffeent pats of you system. Read the Softwae chapte to become familia with how to wok with the softwae. Then go though the detailed step-by-step pocedues of daily statup, unning contols and samples, eviewing data, soting and shutdown. Appendices include infomation on defining potocols, managing data, setting up pism, atio and time paametes, woking with metafiles, setting display colos, using panelyze mode, and using the time of flight and pulse pile up featues. Use the Special Pocedues and Toubleshooting manual fo weekly cleaning and eplacement/adjustment pocedues and toubleshooting infomation. Use the Options manual fo infomation on how to wok with any of the options on you instument. Use the FlowCente Multimedia Wokstation Getting Stated manual fo infomation how to inteface with the Micosoft Windows 98 softwae. See the Documentation page on the back cove of this manual fo the contents of each manual. It can help you to detemine quickly in what manual is the infomation you need. ABOUT THE OPTIONS MANUAL This manual includes instuctions fo the basic opeation of the options available with the ALTRA flow cytomete. Additional manuals may be supplied with the option by the oiginal manufactue of the option. Refe to the manufactue s manual fo complete opeating instuctions and the use of advanced featues. Some pocedues in the ALTRA flow cytomete manuals may change when an option is installed. See the chapte in this manual coveing the option fo these changes. Some options such as Pintes ae not contained in this manual. Refe to the manufactue s manual fo opeating instuctions. -xi

12 INTRODUCTION CONVENTIONS s s s s s s s s s Chapte 1, Safety Pecautions Contains biological and lase safety infomation. Chapte 2, Ai-Cooled Lases Contains desciptions and basic opeating instuctions fo the available ai-cooled lases: Agon, ed HeNe, geen HeNe, and HeCd lases. Chapte 3, Wate-Cooled Lases Contains desciptions and basic opeating instuctions fo the available wate-cooled lases: Coheent Innova Entepise II, 300 Seies and Spectum lases. Chapte 4, Stoage Options Contains desciptions of the diffeent stoage options available and any pocedues needed such as installation o achiving. Chapte 5, Autoclone Soting Option Descibes how to pefom soting with the Autoclone soting option. Chapte 6, Gated Amp Option Descibes the featues of the Gated Amp option. Chapte 7, Lage Paticle Soting Option Descibes the featues of the Lage Paticle Soting option. Chapte 8, Fowad Fluoescence Detecto Option Descibes the featues of the Fowad Fluoescence Detecto option. Index Use the Index to easily locate specific infomation in this manual. HyPeSot System (HPSS) Option Infomation fo the COULTER EPICS ALTRA HyPeSot System flow cytomete is included in the ALTRA flow cytomete Refeence, Opeato s Guide, and Special Pocedues and Toubleshooting manuals. CONVENTIONS This manual uses the following conventions: Italics indicate sceen messages. Bold indicates a menu item. Couie font indicates text you have to type using the keyboad. ì indicates a key (such as Û). -xii

13 INTRODUCTION CONVENTIONS ì+ì indicates that the two keys listed (such as Þ+Ê) ae linked fo a specific function and must be pessed in this sequence: 1. Pess down on the fist key listed and while continuing to pess it, pess down on the second key listed. 2. Release both keys at the same time. ì ì indicates to pess and elease the fist key listed then pess and elease the next key listed. Fo example, y Û. Sceen tt Analysis Select the Sceen item on the Analysis menu. [OKAY] É though Ô Use the mouse to click on the sceen button labeled [OKAY]. Special function keys. -xiii

14 INTRODUCTION CONVENTIONS -xiv

15 1SAFETY PRECAUTIONS BIOLOGICAL HAZARDS The aea aound the system may contain mateials that ceate a biological hazad to laboatoy pesonnel. Pocess samples with the appopiate pecautions to pevent exposue to biohazadous mateial. The extent of biohazad containment depends on the degee of isk involved, accoding to the type and classification of the sample. Povide fo decontamination of the instument and associated laboatoy equipment, supplies and waste. Decontaminate immediately afte examining potentially biohazadous samples. Fo help in establishing pocedues and facilities fo biohazad contol, efe to the guidelines pesented in the following publications. Always obtain the latest edition of these publications. Biohazads Safety Guide National Institutes of Health, Centes fo Disease Contol. Acquied Immune Deficiency Syndome (AIDS): Pecautions fo clinical laboatoy staffs. Mobidity and Motality Weekly Repot 31(43): , Classification of Etiologic Agents on the Basis of Hazad, 3d ed., Centes fo Disease Contol, U.S. Public Health Sevice, June National Committee fo Clinical Laboatoy Standads. Potection of laboatoy wokes fom infectious disease tansmitted by blood, body fluids and tissue; Tentative guideline. NCCLS Document M29-T. Villanova, PA.: NCCLS; National Committee fo Clinical Laboatoy Standads. Clinical applications of flow cytomety: quality assuance of peipheal blood lymphocytes; Tentative Guideline. NCCLS Document H42-T. Villanova, PA.: NCCLS; pp. 12, 13, 37, MMWR, Vol. 46, No. RR-2, Revised guidelines fo pefoming CD4+ T-cell deteminations in pesons with Human Immunodeficiency Vius (HIV): Laboatoy Safety Centes fo Disease Contol; pp Jan 10, LASER SAFETY PRECAUTIONS The lase is a unique light souce that shows chaacteistics diffeent fom conventional light souces. The safe use of the lase depends upon familiaity with the instument and with the popeties of coheent, intense beams of light. The beam can cause eye damage o blindness and instument damage. Thee is enough powe fom the lase to ignite substances placed in the beam path, even at some distance. The beam might also cause damage if contacted indiectly fom eflective sufaces (specula eflection). Fo these easons follow the pecautions that follow. WARNING Use of contols o adjustments o pefomance of pocedues othe than those specified heein may esult in hazadous adiation exposue. 1. Caefully ead the safety instuctions in the manual fo the lase. 2. Do not use the lases in the pesence of flammables o explosives; these include volatile substances such as alcohol, solvents, and ethe. 3. Limit access to the instument. Keep the instument out of the hands of inexpeienced and untained pesonnel. 1-1

16 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS 4. Neve look diectly into the lase light souce o at scatteed lase light fom any eflective suface. Neve look down the beam into the souce. 5. As a pecaution against accidental exposue to the output beam o to its eflection, those pefoming sevice o maintenance pocedues on the system should wea safety glasses as equied by the wavelength being used. 6. The intensity of the beam can cause flesh buns. Avoid diect exposue and specula eflections. 7. Post waning signs in the aea of the lase beam to alet those pesent. 8. Advise all those using the lase of these pecautions. Radiation Hazads In its design and manufactue of the ALTRA flow cytomete, Beckman Coulte Inc. has complied with the equiements govening the use and application of a lase as stipulated in egulatoy documents issued by the: U.S. Depatment of Health and Human Sevices Cente fo Devices and Radiological Health (CDRH). In compliance with these egulatoy documents, evey measue has been taken to ensue the health and safety of uses and laboatoy pesonnel fom the possible danges of lase use. WARNING Risk of pesonal injuy. This instument contains components dangeous to the opeato. If any attempt has been made to defeat a safety featue, o if this instument does not pefom as listed in this manual, disconnect powe and call you Beckman Coulte Repesentative. CDRH-appoved waning labels ae placed nea o on those coves that when emoved might expose lase adiation. Only emove those coves when pefoming sevice and maintenance on the system as instucted in the COULTER EPICS ALTRA Flow Cytomete Special Pocedues and Toubleshooting manual. See Figues 1.1 though 1.12 fo the labels and thei locations on the lases: Figue 1.1, Agon Lase, Manufactue s Waning Labels Figue 1.2, Red Helium-Neon (HeNe) Lase, Manufactue s Waning Labels. Figue 1.3, Geen HeNe Lase, Manufactue s Waning Label. Figue 1.4, Helium Cadmium (HeCd) Lase, Manufactue s Waning Labels Figue 1.5, Coheent Innova Entepise II Seies Lase, Manufactue s Waning Labels Figue 1.6, Coheent Innova 300 Seies Lase, Manufactue s Waning Labels Figue 1.7, Coheent Innova 70 Spectum Lase, Manufactue s Waning Labels Figue 1.8, Cytomete Sensing Aea, Lase Waning Labels. Figue 1.9, Fowad Fluoescence Detecto Option Sensing Aea, Lase Waning Labels Figue 1.10, Cytomete Sensing Compatment s Cove, Lase Waning Labels Figue 1.11, Cytomete Oute Cove, Lase Classification Label. Figue 1.12, Cytomete Rea and Left Cove Lase Waning Labels and Lase Cetification Label 1-2

17 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS 1 When pefoming sevice o maintenance on the system with the coves open and the intelock defeated, obseve the following: WARNING Reflections of the lase beam fom a shiny object, such as a scewdive, o diect viewing of the lase beam can seveely damage you eyes. Pay attention to the waning labels in the sampling compatment. Do not wea any jewely that might eflect the lase beam. Do not obseve the intesection of the lase beam and the flow chambe diectly. To obseve this point, use the video camea and gaphics sceen at the cytomete. Wea lase safety glasses that potect against the wavelengths in use. Replace the coves befoe esuming nomal opeation. Figue 1.1 Agon Lase, Manufactue s Waning Labels LASER RADIATION WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM Cyonics a division of uniphase 163 BAYPOINTE PKWY SAN JOSE, CA MODEL SERIAL NO. MANUFACTURED THIS LASER PRODUCT COMPLIES WITH 21 CFR 1040 AS APPLICABLE LASER RADIATION-AVOID DIRECT EXPOSURE TO BEAM ARGON :.50 WATT CW. CLASS IIIb LASER PRODUCT LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE A 1-3

18 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS Figue 1.2 Red Helium-Neon (HeNe) Lase, Manufactue s Waning Labels LASER APERTURE CAUTION-LASER LIGHT WHEN OPEN OPEN SHUTTER CLOSE LASER LIGHT AVOID EXPOSURE TO BEAM CLASS IIIb LASER PRODUCT (CDRH) CLASS 3B LASER PRODUCT (IEC 825-1:1993) 30mW MAXIMUM AT nm MELLES GRIOT 05 - LHP MFG S/N CARLSBAD CALIFORNIA THIS LASER PRODUCT COMPLIES WITH DHHS/CDRH RADIATION PERFORMANCE STANDARDS 21 CFR SUB-CHAPTER J AND IEC :1993 CE Lase shutte C LASER LIGHT AVOID EXPOSURE TO BEAM CLASS IIIb LASER PRODUCT (CDRH) CLASS 3B LASER PRODUCT (IEC 825-1:1993) 30mW MAXIMUM AT nm MELLES GRIOT MFG S/N 05 - LHP CARLSBAD CALIFORNIA THIS LASER PRODUCT COMPLIES WITH DHHS/CDRH RADIATION PERFORMANCE STANDARDS 21 CFR SUB-CHAPTER J AND IEC :1993 CE Figue 1.3 Geen HeNe Lase, Manufactue s Waning Label AVOID EXPOSURE - Laselight is emitted fom this apetue. OPEN SHUTTER CLOSE 05 - LGR MFG S/N THIS LASER PRODUCT COMPLIES WITH DHHS/CDRH RADIATION PERFORMANCE STANDARDS 21 CFD SUB-CHAPTER J AND IEC :1993 CE Lase shutte LASER LIGHT AVOID EXPOSURE TO BEAM CLASS IIIb LASER PRODUCT (CDRH) CLASS 3B LASER PRODUCT (IEC 825-1:1993) 5mW MAXIMUM AT nm LASER LIGHT AVOID EXPOSURE TO BEAM CLASS IIIb LASER PRODUCT (CDRH) CLASS 3B LASER PRODUCT (IEC 825-1:1993) 5mW MAXIMUM AT nm MELLES GRIOT 05 - LGR MFG S/N CARLSBAD CALIFORNIA THIS LASER PRODUCT COMPLIES WITH DHHS/CDRH RADIATION PERFORMANCE STANDARDS 21 CFR SUB-CHAPTER J AND IEC :1993 CE C 1-4

19 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS 1 Figue 1.4 Helium Cadmium (HeCd) Lase, Manufactue s Waning Labels INVISIBLE LASER RADIATION-AVOID DIRECT EXPOSURE TO BEAM LESS THAN 50.0 MW HeCd CLASS lllb LASER PRODUCT DANGER LASER LIGHT WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM INVISIBLE RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE A Figue 1.5 Coheent Innova Entepise II Seies Lase, Manufactue s Waning Labels DANGEROUS VOLTAGE UNDER THIS COVER VISIBLE AND INVISIBLE LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE VISIBLE AND INVISIBLE LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION A *PRACTICAL LIMIT ARGON ION LASER 2 WATTS MAX.* CW CLASS IV LASER PRODUCT 1-5

20 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS Figue 1.6 Coheent Innova 300 Seies Lase, Manufactue s Waning Labels VISIBLE AND INVISIBLE LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION. ARGON ION LASER 12 WATTS MAX.* CW *PRACTICAL LIMIT CLASS IV LASER PRODUCT VISIBLE AND INVISIBLE LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE A Figue 1.7 Coheent Innova 70 Spectum Lase, Manufactue s Waning Labels VISIBLE AND INVISIBLE LASER RADIATION IS EMITTED FROM THIS APERTURE AVOID EXPOSURE VISIBLE LASER LIGHT- AVOID EXPOSURE TO DIRECTOR SCATTERED LIGHT A ARGON AND KRYPTON MIXED GAS LASER 8 WATTS MAX. CW/MULTILINE OUTPUT. PRACTICAL LIMT CLASS IV LASER PRODUCT 1-6

21 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS 1 Figue 1.8 Cytomete Sensing Aea, Lase Waning Labels VISIBLE AND/OR INVISIBLE LASER RADIATION WHEN OPEN AND INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION DANGER Intelock defeat ba LASER RADIATION AVOID EXPOSURE TO BEAM CLASS 3B LASER PRODUCT DANGER LASER RADIATION AVOID EXPOSURE TO BEAM CLASS 3B LASER PRODUCT CLASS 1 LASER PRODUCT AVOID EXPOSURE VISIBLE AND/OR INVISIBLE LASER RADIATION IS EMITTED FROM THIS APERTURE CLASS 1 LASER PRODUCT C 1-7

22 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS Figue 1.9 Fowad Fluoescence Detecto Option Sensing Aea, Lase Waning Labels VISIBLE AND/OR INVISIBLE LASER RADIATION WHEN OPEN AND INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION DANGER Intelock defeat ba LASER RADIATION AVOID EXPOSURE TO BEAM CLASS 3B LASER PRODUCT DANGER LASER RADIATION AVOID EXPOSURE TO BEAM CLASS 3B LASER PRODUCT AVOID EXPOSURE VISIBLE AND/OR INVISIBLE LASER RADIATION IS EMITTED FROM THIS APERTURE CLASS 1 LASER PRODUCT C 1-8

23 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS 1 Figue 1.10 Cytomete Sensing Compatment s Cove, Lase Waning Labels VISIBLE AND/OR INVISIBLE LASER RADIATION WHEN OPEN AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION Latch LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION CLASS 4 LASER PRODUCT VISIBLE AND/OR INVISIBLE LASER RADIATION WHEN OPEN LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION CLASS 4 LASER PRODUCT AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION C Figue 1.11 Cytomete Oute Cove, Lase Classification Label A CLASS 1 LASER PRODUCT 1-9

24 SAFETY PRECAUTIONS LASER SAFETY PRECAUTIONS Figue 1.12 Cytomete Rea and Left Cove Lase Waning Labels and Lase Cetification Label VISIBLE AND/OR INVISIBLE LASER RADIATION WHEN OPEN LASER RADIATION AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION CLASS 4 LASER PRODUCT AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION THIS LASER PRODUCT CONFORMS TO THE PROVISIONS OF 21 CFR. SUBCHAPTER J, SECTIONS AND C Manufactued: DATE Beckman Coulte, Inc. 250 S. Kaeme Blvd. Bea, CA by: 1-10

25 2AIR-COOLED LASERS GENERAL INFORMATION The following ai-cooled lases ae available as options on the ALTRA flow cytomete: Cyonics Ai-cooled Agon lase (heeafte called the ai-cooled Agon lase) Melles Giot o Uniphase Red Helium Neon (HeNe) lase (heeafte called the Red HeNe lase) Melles Giot Geen Helium Neon (HeNe) lase (heeafte called the Geen HeNe lase) Omnichome Helium-Cadium (HeCd) lase (heeafte called the HeCd lase). The infomation unde this section (Heading 2.1, GENERAL INFORMATION) is geneic and applies to all the ai-cooled lases listed above. Infomation specific to each lase listed above is included in a sepaate section in this chapte. Installation Requiements Do not unpack o install the option youself. The option must be unpacked and installed by a Beckman Coulte Repesentative. Cetain intenal adjustments of the lases and the instument may be equied at the time of installation. Refe to the Installation chapte in the ALTRA flow cytomete Refeence manual fo othe installation equiements. Floo Loading and Space The dimensions and weight ae the same as the standad ALTRA flow cytomete. Refe to the ALTRA flow cytomete Refeence manual. Heat and Ventilation The ALTRA flow cytomete dissipates appoximately 2,400 W (8,191 Btu/hou) of heat. Depending upon the type and powe of the associated lases, the total heat dissipation can incease consideably. Be sue to conside this to ensue that the oom whee the ALTRA flow cytomete is located is popely ventilated and can emove the geneated heat. Fo example: a typical ai-cooled 15 mw agon lase can dissipate an additional 2,000 W (6,824 Btu/hou) of heat. An ai-cooled Omnichome Model 74 HeCd lase dissipates about 100 W (341 Btu/hou) of heat. See the lase manufactue s specifications fo details. The system must be opeated in an ai-conditioned space. Keep oom tempeatue between 18 C and 29 C (64 F and 85 F). Opeation This chapte contains the minimum infomation necessay fo opeation with the ALTRA flow cytomete. Fo complete opeating instuctions, efe to the specific lase manufactue s manual. Aligning the Mios To align the mios you need to be awae of the aangement of the lases, the path the lase beam follows, and the filte configuation. One example is shown in the mio alignment pocedue that follows. 2-1

26 AIR-COOLED LASERS GENERAL INFORMATION With the addition of lases, the oientation of the lases ae changed. All lases point towad the ight side of the instument into an optical ail suppoting thee dichoic alignment mios, one fo each lase (see Figue 2.1). The dichoic alignment mios eflect the lase light into the sensing aea. Figue 2.1 Lase Aangement - Ai-Cooled Lase, Example Optical ail Wate-cooled Agon lase o HeCd lase HeNe (Lase 2) Ai-cooled Agon (Lase 1) Top view A Mio Alignment Pocedue Align the mios when changing to a diffeent lase o application equiing a diffeent lase. WARNING Risk of pesonal injuy. Diect eye contact with the output beam fom any lase may cause seious damage and possible blindness. Wea lase safety glasses that potect against the wavelengths being used. Read the infomation unde Heading 1.2, LASER SAFETY PRECAUTIONS befoe aligning the mio. WARNING Risk of pesonal injuy. If opeating a lase with ultaviolet (UV) wavelengths, note that the beam is invisible to the eye and dangeous to the skin. Wea lase safety glasses that potect against the UV wavelengths of the lase. Special Fluoescent cads can be used to spot-check the beam position. 2-2

27 AIR-COOLED LASERS GENERAL INFORMATION 2 1 Without tuning on the lases, stat up the system as diected in Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 Open the coves to the sensing aea. a. Lift up the font cove. b. Pull up the intelock od. a b c. Squeeze the intelock cove elease button. c DANGER d d. Remove the intelock cove. 3 Remove the two scews and open the intenal lase cove. 4 Defeat the mechanical intelock by inseting the intelock defeat ba: a. Pull the intelock od up. b. Inset the defeat ba. c. Push the intelock od down. 2-3

28 AIR-COOLED LASERS GENERAL INFORMATION 5 Gain access to the flow cell: a. Pull the deflection plates staight out. b. Loosen the thumbscew holding the beam-shaping lens assembly in place. c. Remove the beam-shaping lens assembly. H.V. ON Thumbscew Beam-shaping lens assembly Deflection plates 6 Remove the flow cell tip. H.V. ON 2-4

29 AIR-COOLED LASERS GENERAL INFORMATION 2 7 Tun the INTERLOCK OVERRIDE keyswitch on the Cytomete to defeat the electical intelock. Tun key clockwise to opeate lases LASER ON/OFF INTERLOCK OVERRIDE INTERLOCK OPEN RESET MAIN POWER 8 Remove the fowad scatte senso. Do not disconnect wies. Let the fowad scatte senso hang outside the doo to the sot collection aea. 9 Remove the pneumatic shutte. Do not disconnect the tubing. 10 Install the shot lase tagets in place of the fowad scatte senso and the pneumatic shutte. The scews to fasten the tagets should be down. 11 At the touch sceen, tun Lase 1 o Lase 2 (HeNe lase) On. Refe to Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2-5

30 AIR-COOLED LASERS GENERAL INFORMATION 12 Step 13 shows how to adjust the mio. Adjust the mio (eflecting the beam you ae aligning) to guide the beam though the tagets as shown. Note: Always align the lase closest to the flow cell fist. Then align each one futhe away fom the flow cell, in ode. Top View Ai-cooled HeCd o HeNe Agon Wate-cooled (Lase 2) (Lase 1) lase Lase tagets Lase beams 633 mio eflects 633 nm and tansmits 320 to 365 nm and 488 nm 488 mio eflects 488 nm and tansmits 320 to 365 nm Beam educe UV mio eflects 325 to 365 nm 2-6

31 AIR-COOLED LASERS GENERAL INFORMATION 2 13 To adjust the mio: a. Loosen the height setscew and adjust height so beam stikes cente of mio. b. Loosen the fine adjustment and ail setscews. Move the stage along the optical ail so beam stikes middle of mio. Tighten the fine adjustment setscew. c. Tighten the ail setscew so the mio and post do not ock on the ail. Keep the setscew loose enough so that the fine adjustment knob can still move the mio and post along the ail. d. Loosen height setscew and otate mio so beam goes to fist taget. Tighten height setscew. e. Use the hoizontal and vetical otation knobs to guide the beam though the fist taget. Look fo lase light to appea on the second taget. f. If the light on the second taget is a wide cicle of light to the left side of the hole (away fom the PMTs), use the fine adjustment knob to move the post and mio futhe back along the ail. Repeat step e. g. If the light on the second taget is a tight point, adjust vetical and hoizontal otation to get beam though second taget. h. Tighten the ail setscew. Vetical otation knob Fine adjustment knob Fine adjustment setscew Rail setscew Hoizontal otation knob Height setscew 14 Close the shutte on the lase. 2-7

32 AIR-COOLED LASERS GENERAL INFORMATION 15 To align a second ai-cooled lase, epeat steps 11 to 14 fo that lase. 16 Close the shutte on that lase. Tun the flathead scew above that lase apetue to close the lase shutte. 17 Follow steps 17 to 28 to align the HeCd lase. a. Loosen the height setscew and emove the beam educe that is positioned between the lase head and the dichoic alignment mio. b. Tun the HeCd lase on as diected unde Statup in Heading 2.2, AIR-COOLED ARGON LASERS. Beam educe Height setscew 2-8

33 AIR-COOLED LASERS GENERAL INFORMATION 2 18 Adjust the UV mio (eflecting the beam) to guide the beam though the tagets. (You need to place a fluoescent taget, such as a white business cad, in font of the beam to detect lase action.) Repeat steps 12 and 13 with the UV mio. HeNe (Lase 2) Top View Ai-cooled Agon (Lase 1) HeCd o Wate-cooled lase Lase tagets Lase beams 633 mio eflects 633 nm and tansmits 320 to 365 nm and 488 nm 488 mio eflects 488 nm and tansmits 320 to 365 nm Beam educe UV mio eflects 325 to 365 nm 19 Replace the beam educe/expande and adjust it so the beam goes though the beam educe tagets. You can look fom the ea of the system and see the beam as it goes though the beam educe. Remove the beam educe tagets. Adjust the focus so the beam is about 1/8 in. in diamete as it leaves the expande. 20 Close the shutte on the HeCd lase. 2-9

34 AIR-COOLED LASERS GENERAL INFORMATION 21 Reinstall the flow cell tip and adjust it fo pope back eflection. Apetue Lase beam Reflected spot 22 Move the flow cell Z-axis to cente the lase beams on the channel of the quatz cuvette. 23 Reinstall the beam shape. Adjust the beam shape until the beam spot is centeed on the hole in the taget located in the fowad scatte position. 24 Remove the tagets and eplace the fowad scatte senso and pneumatic shutte. 25 Pefom a full optical alignment as descibed in Chapte 3, REPLACEMENT/ADJUSTMENT Pocedues, in the Special Pocedues and Toubleshooting manual. Leave all coves and doos open. 2-10

35 AIR-COOLED LASERS GENERAL INFORMATION 2 26 Run beads. 27 Open the doo to the detecto compatment and set up the filtes as shown below. PMT4 PMT3 PMT2 PMT BP 381 BP PMT5 550 DL 430 DL 325 DL Close the doo to the detecto compatment. 2-11

36 AIR-COOLED LASERS GENERAL INFORMATION 29 Check Red HeNe lase alignment: a. Remove any ND filte fom the fowad scatte detecto. b. Close the shutte on the ai-cooled Agon lase. c. Open the shutte on the Red HeNe lase. d. At the Camea sceen touch sceen, set up a scope display to show: Fowad scatte peak on the lowe tace. PMT5 peak fluoescence pulse on the uppe tace. e. Adjust beam-shaping lens assembly hoizontally fo maximum pulse heights. Adjust gains and PMT voltages as necessay. f. Close the shutte on the Red HeNe lase. 2-12

37 AIR-COOLED LASERS GENERAL INFORMATION 2 30 Check 488 ai cooled alignment co-linea with the Red HeNe lase: a. Open the shutte on the ai-cooled Agon lase. b. Adjust the 488 mio hoizontal otation fo maximum fowad scatte pulse height. c. Open the shutte on the Red HeNe lase. d. At the Camea sceen touch sceen, set up a scope display to show: PMT5 peak fluoescence on the lowe tace. PMT2 peak fluoescence on the uppe tace. e. Adjust the 488 mio vetical otation so the PMT2 pulses on the uppe tace ae ight above the PMT5 pulses on the lowe tace. (Do NOT eadjust the beam-shaping lens assembly.) f. Close the shuttes on the ai-cooled Agon and HeNe lases. 2-13

38 AIR-COOLED LASERS GENERAL INFORMATION 31 Pepae to check HeCd lase alignment: a. Loosen the height setscew and emove the beam educe (take cae not to distub any of the settings). b. Open the shutte on the HeCd lase. c. See the filte configuation below. PMT4 PMT3 PMT2 PMT BP 381 BP PMT5 550 DL 430 DL 325 DL

39 AIR-COOLED LASERS GENERAL INFORMATION 2 32 Check the HeCd alignment: a. At the Camea sceen touch sceen, set up a scope display to show: PMT2 peak fluoescence on the lowe tace. PMT5 peak fluoescence on the uppe tace. b. Adjust the UV mio hoizontal otation to maximize PMT2 pulse height. c. Replace the beam educe, and adjust it fo maximum PMT2 pulse height. You may need to adjust the focus. DO NOT eadjust the UV mio. d. At the Camea sceen touch sceen, set up a scope display to show: PMT5 peak fluoescence on the lowe tace. PMT2 peak fluoescence on the uppe tace. e. Open the shutte on the Agon ai-cooled lase. f. The signal fom the Agon lase is on the bottom tace. Adjust the beam educe so the signal fom the HeCd lase on the uppe tace lines up with signal on the bottom tace. g. Pess the ON/OFF button on the emote contol box of the HeCd lase. h. Replace all coves. 2-15

40 AIR-COOLED LASERS GENERAL INFORMATION 33 At the Cytomete, tun the INTERLOCK OVERRIDE keyswitch to enable the intelocks. Lases 1 and 2 (ai-cooled Agon and HeNe lases) tun off when the coves ae eplaced. Tun lases 1 and 2 On at the touch sceen. Tun key counte clockwise LASER ON/OFF INTERLOCK OVERRIDE INTERLOCK OPEN RESET MAIN POWER 34 Restoe the oiginal filte configuation and esume nomal opeation. Cleaning and Othe Maintenance Tun the ai-cooled lase on fo about 1 hou at least once a week if the lase is not to be used fo moe than 2 weeks. Refe to the manufactue s manual fo the cleaning and maintenance pocedues involving the lase. 2-16

41 AIR-COOLED LASERS AIR-COOLED ARGON LASERS AIR-COOLED ARGON LASERS Ai-cooled Agon lases pemit a wide ange of applications fom basic immunofluoescence to moe complex kinetics expeiments. Its small size makes the ai-cooled Agon lase easy to combine in multilase configuations. When used alone o with the ai-cooled Red HeNe lase, the ai-cooled Agon lase can be mounted fo staight though opeation and ease of alignment. Due to low output powe, this lase is not ecommended fo sense-in-ai opeation. The ai-cooled Agon lase is suitable fo sense-in-quatz applications equiing 488 nm excitation and may equie TEM 00 mode. Electical Powe Opeates with household cuent (standad 110 V outlet) Output powe: 15 mw of 488-nm output (99% wavelength puity). Statup 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to Agon Lase On. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 On Off CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-17

42 AIR-COOLED LASERS AIR-COOLED ARGON LASERS Shutdown 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to Agon Lase Off. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 Off Off CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-18

43 AIR-COOLED LASERS RED HeNe LASERS RED HeNe LASERS Used togethe, the Red HeNe and the ai-cooled Agon lase enable the ALTRA flow cytomete to wok with a geate ange of pobes. Due to the Red HeNe s small size, the ai-cooled Agon can still be mounted in the staight-though configuation. This gives the ALTRA flow cytomete the unique featue of having only one beam-steeing dichoic fo its two-lase configuation. The Red HeNe lase is suitable fo sense-in-quatz applications equiing 633-nm excitation and may equie TEM 00 mode. Due to low output powe, this lase is not ecommended fo sense-in-ai opeation. Featues include: Economical ai-cooled opeation Opeation in TEM 00 mode Compact design No maintenance o daily adjustment. Electical Powe Opeates with household cuent (standad 110 V outlet) 10 mw of 633-nm output Opeating voltage: 3,100 ±200 Vdc Opeating cuent: 6.5 ±0.2 ma Contols and Indicatos All contol is though the Cytomete. Lase Head The lase (see Figue 2.2) is mounted inside the Cytomete. Figue 2.2 Lase Head - Red HeNe Lase Lase shutte AVOID EXPOSURE THIS APERTURE IS EMITTED FROM LASER RADIATION 2-19

44 AIR-COOLED LASERS RED HeNe LASERS Statup 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to HeNe Lase On. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 On On CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-20

45 AIR-COOLED LASERS RED HeNe LASERS 2 Shutdown 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to HeNe Lase Off. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 On Off CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-21

46 AIR-COOLED LASERS GREEN HeNe LASERS 2.4 GREEN HeNe LASERS Though only a 1.5 mw lase, the Geen HeNe can poduce data that is indistinguishable fom lage ai-cooled o wate-cooled lases. The Geen HeNe uses the same mounting hadwae as the Red HeNe. The mounting hadwae must be puchased sepaately. The Geen HeNe s 544-nm line can impove signal-to-noise atios by educing auto-fluoescence and aman scatte. Featues include: Compact design Opeation in TEM 00 mode No maintenance o daily adjustments. Electical Powe Opeates on household cuent (standad 110 V outlet) 1.5 mw of 544-nm output powe Opeating cuent: 6.5 ±0.2 ma Opeating voltage: 3,000 ±100 Vdc. Contols and Indicatos If the Geen HeNe lase is used as the second lase, on/off is though the Cytomete. It is contolled via the Cytomete contol sceen unde the HeNe lase button. If used as a thid lase, on/off is though the powe supply. Powe Supply When the Geen HeNe is used as the thid lase, the keyswitch on the powe supply (see Figue 2.3) tuns the lase on/off. 2-22

47 AIR-COOLED LASERS GREEN HeNe LASERS 2 Figue 2.3 Powe Supply - Geen HeNe Lase When Used As a Thid Lase A If the lase is used as the second lase, the powe supply is mounted inside the Cytomete. If the lase is used as the thid lase, the powe supply is extenal to the Cytomete. The lase head (see Figue 2.4) should always be mounted on the optical bench inside the Cytomete. Figue 2.4 Lase Head - Geen HeNe Lase Lase shutte 05 - LHP MFG 0 NOVEMBER 1994 S/N 27478T THIS LASER PRODUCT COMPLIES WITH DHHS/CDRH RADIATION PERFORMANCE STANDARDS 21 CFD SUB-CHAPTER J A 2-23

48 AIR-COOLED LASERS GREEN HeNe LASERS Statup 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to HeNe Lase On. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 On On CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-24

49 AIR-COOLED LASERS GREEN HeNe LASERS 2 Shutdown 1 At the ALTRA CYTOMETER touch sceen, select Contol. ALTRA CYTOMETER High Volt Int Gain Peak Gain FS PMT PMT PMT PMT PMT Data Rate 0 Sot Left Right #/Sec 0 0 Options % 0 0 Sample Flow 50 Contol Count 0 0 Dec. Inc. Limit 0 0 Sevice Fl Comp. None Discim. Sot Contol Camea Gaph Scope 2 At the CONTROL SCREEN touch sceen, in the box unde the Lase heading, toggle to HeNe Lase Off. Mix mode On/Off Beepe Off Lase Agon Lase HeNe Lase Data Rate 0 Sot Left Right #/Sec 0 0 % 0 0 Count 0 0 Limit 0 0 On Off CONTROL SCREEN Wavelength nm Sample Flow 50 Dec Inc Requested Powe Powe mw* Cuent Amps *Actual powe output Backflush Delay 0.5 Sec Push 1.0 Sec Backflush 2.0 Sec Powe up Valves Shutdown Valves Option Sceen Valves Sceen MAIN Sceen 2-25

50 AIR-COOLED LASERS HeCd LASERS 2.5 HeCd LASERS The HeCd lase kit has been specifically designed fo intacellula Calcium studies. It is not ecommended fo DNA applications due to the multi-mode beam pofile. Because of the inheent popeties of the HeCd lase, it may not be suitable fo applications equiing high esolution, such as chomosome o DNA ploidy analysis. The HeCd lase option compises thee poducts: The Optical Upgade kit includes the mios and optical ail fo diecting the lase beams into the sensing aea. Mounting feet. An ai-cooled Omnichome M helium-cadmium Lase. This manual biefly coves installation equiements, opeation, sevice and maintenance, and pecautions. Fo moe infomation, efe to the Omnichome Helium-Cadmium Lase Instuction Manual. The HeCd lase emits only in the UV ange at 325 nm. The guaanteed powe is 20 mw at this wavelength, but is typically between 20 and 30 mw. The beam is multimode (not a single point of light but a combination of seveal nodes of light). Because of the lasing paametes, the lase powe is not adjustable. The HeCd lase does not include a powe mete. Fo applications that equie tacking of the lase powe, you can puchase a powe mete fo use in the UV ange fom the lase manufactue o othe optical supply companies. The powe output is constant thoughout the day afte the initial wam-up peiod. Ove the lifetime of the lase, the powe output gadually deceases. Electical Powe The HeCd lase is available fo opeation fom eithe: Vac o 220/240 Vac, 6 A, Hz. The box on top of the powe supply is used fo 220/240 Vac opeation. The powe outlet can be the same as used fo an optional Pinte. Do not use the cicuit used by the ALTRA flow cytomete. Refe to the Installation chapte in the ALTRA flow cytomete Refeence manual fo additional electical equiements. Contols and Indicatos Powe Supply The Powe Supply has a Lase Contol knob, an Emegency ON/OFF keyswitch and a ow of LED indicatos (see Figue 2.5). 2-26

51 AIR-COOLED LASERS HeCd LASERS 2 Figue 2.5 Powe Supply - HeCd Lases Lase contol knob LED indicatos Emegency ON/OFF keyswitch B A The Lase Contol knob selects one of thee settings as shown in Table 2.1: Table 2.1 Powe Supply Contol Settings - HeCd Lases Setting When to Use Desciption OPERATE Fo nomal opeation. Lasing usually stats 2 o 3 minutes afte setting to OPERATE. In some cases lasing may stat immediately. STANDBY When the lase will not be used fo >2 hous, but <4 hous. Lasing stops about 5 minutes afte setting to STANDBY. Wam-up time is educed when going fom STANDBY to OPERATE. SHUTDOWN To shutdown the lase. Initiates a 3-minute shutdown sequence. Lasing stops about 2.5 min afte setting to SHUTDOWN. Powe is off afte 3 minutes. Note: The 3-minute shutdown sequence sweeps cadmium vapo still in plasma tube to a special esevoi nea the cathode. Without the 3-minute shutdown, cadmium vapo condenses on the inne sufaces of the plasma tube which can educe the lase life. 2-27

52 AIR-COOLED LASERS HeCd LASERS The LED indicatos show the lase states descibed in Table 2.2. Table 2.2 Powe Supply Indicatos - HeCd Lases LED Indicato When Illuminated Toubleshooting POWER ON When powe is on None. Cd Heate When in OPERATE mode Cadmium in lase is heated to supply cadmium vapo fo lasing action. If you obseve constant 1 second dim/bight pulses, call you Sevice Repesentative. The dim/bight cycles indicate a possible poblem. He Heate TEMP LOCKOUT When in OPERATE mode and system is adjusting helium pessue Biefly afte shutdown, and anytime lase is too wam to be stated Lase Head The lase emission POWER indicato lights anytime lasing is possible (see Figue 2.6). The lase beam is invisible. Avoid the beam path when the emission indicato is lit. Figue 2.6 Lase Head - HeCd Lases Nomally illuminates fo about 20 minutes afte statup and occasionally flickes on and off thoughout the day. If on continuously thoughout the day, call you Sevice Repesentative. Afte shutdown, the system must cool down fo 5 to 10 minutes befoe estating. When illuminated, estating is pevented until the lase cools down. A Emission POWER indicato POWER B Lase shutte (Closed) C A 2-28

53 AIR-COOLED LASERS HeCd LASERS 2 Statup Unlike the othe ai-cooled lases on the ALTRA flow cytomete, the HeCd lase is contolled fom its powe supply. Except fo the INTERLOCK OVERRIDE keyswitch, settings at the wokstation o the Cytomete do not affect the HeCd lase. Note: When the lase will not be used fo moe than 2 weeks, stat the lase and let it un fo about 1 hou evey week. Pocedue 1 Stat the ALTRA flow cytomete as diected in the Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 At the HeCd lase powe supply, tun the Emegency ON/OFF keyswitch to ON. 3 Tun the Lase Contol knob to OPERATE. STANDBY OPERATE SHUTDOWN OFF ON EMERGENCY ONLY POWER ON Cd HEATER B He HEATER TEMP LOCKOUT Omnichome MODEL100 4 Afte 2 to 3 minutes, the lase emits the lase beam. Let the lase wam up fo 15 minutes befoe unning samples. 2-29

54 AIR-COOLED LASERS HeCd LASERS Shutdown The nomal shutdown pocedue uses the SHUTDOWN setting on the Lase Contol knob (efe to Table 2.1). This timed shutdown continues plasma dischage until all cadmium plasma in the boe condenses in a special aea nea the cathode. The Emegency ON/OFF keyswitch is fo emegency puposes only as diected by the following CAUTION message. CAUTION Risk of damage to the instument. If you stop lasing immediately by using the ON/OFF keyswitch, cadmium vapo condenses on the inne sufaces of the plasma tube and this can educe the lase life. The ON/OFF keyswitch immediately stops lasing by stopping plasma dischage in the lase plasma tube. Do not use the ON/OFF keyswitch fo shutdown of the lase EXCEPT in an emegency. 1 Tun the Lase Contol knob to SHUTDOWN. STANDBY OPERATE SHUTDOWN OFF ON EMERGENCY ONLY POWER ON Cd HEATER B He HEATER TEMP LOCKOUT Omnichome MODEL100 2 Afte about 2.5 minutes, the lase stops lasing. Afte about 3 minutes, powe is off to the lase. 3 Shut down the est of the system as descibed in Chapte 8, SHUTDOWN, in the ALTRA flow cytomete Opeato's Guide. 2-30

55 3WATER-COOLED LASERS GENERAL INFORMATION The following wate-cooled lases ae available as options to the ALTRA flow cytomete: Coheent Innova Entepise II ion lase system (heeafte efeed to as the Entepise II seies lases) Coheent Innova 300 Seies ion lase system (heeafte efeed to as the 300 seies lases) Coheent Innova 70 Spectum ion lase system (heeafte efeed to as the Spectum lase). The infomation unde this section (Heading 3.1, GENERAL INFORMATION) is geneic and applies to all the wate-cooled lases listed above. Infomation specific to each lase listed above is included in a sepaate section in this chapte. Installation Requiements Do not unpack o install the option youself. The option must be unpacked and installed by a Beckman Coulte Repesentative. Cetain intenal adjustments of the lases and the instument may be equied at the time of installation. Refe to the Installation chapte in the ALTRA flow cytomete Refeence manual fo othe installation equiements. Heat and Ventilation The ALTRA flow cytomete dissipates appoximately 2,400 W (8,191 Btu/hou) of heat. Depending upon the type and powe of the associated lases, the total heat dissipation can incease consideably. Be sue to conside this to ensue that the oom whee the ALTRA flow cytomete is located is popely ventilated and can emove the geneated heat. Fo example: an wate-cooled Entepise II seies lase dissipates about 7,500 W (25,590 Btu/hou) of heat. Note that the Entepise II lase is a special case whee the heat can be emoved into the suounding oom with an optional chille o down the dain with the cooling wate. See the lase manufactue s specifications fo details. The system must be opeated in an ai-conditioned space. Keep oom tempeatue between 18 C and 29 C (64 F and 85 F). Refe to the Installation chapte in the ALTRA flow cytomete Refeence manual fo othe installation equiements. Opeation The wate-cooled lases have numeous advanced opeating featues. The infomation in this chapte explains the minimum necessay fo opeation with the ALTRA flow cytomete. Fo complete opeating instuctions, efe to the specific lase manufactue s manual. Changing Lase Wavelength Refe to the lase manual to change the lase wavelength. Remembe to also set the wavelength at the emote contol box to maintain powe mete calibation. 3-1

56 WATER-COOLED LASERS GENERAL INFORMATION When woking with jet-in-ai flow cells, othe changes may be necessay. Contact you Beckman Coulte Repesentative fo moe infomation. Aligning the Mios To align the mios you need to be awae of the aangement of the lases, the path the lase beam follows and the filte configuation. This configuation changes depending on the mix of ai-cooled vs. wate-cooled lases installed on you instument. With the addition of lases, the oientation of the lases ae changed. All lases point towad the ight side of the instument into an optical ail suppoting thee dichoic alignment mios, one fo each lase (see Figue 3.1). The dichoic alignment mios eflect the lase light into the sensing aea. Figue 3.1 Lase Aangement - Wate-Cooled Lases in a Thee Lase Configuation Optical ail Wate-cooled Agon lase o HeCd lase HeNe (Lase 2) Ai-cooled Agon (Lase 1) Top view A The wate-cooled lases can be set up fo eithe 488-nm opeation, o UV opeation (efe to the lase manufactue s manual to switch the lase back and foth). Fo 488-nm opeation, switch the dichoic mio fom the ai-cooled Agon lase to the wate-cooled lase. The ai-cooled Agon lase is not used when the wate-cooled lase is opeated at 488 nm. Mio Alignment Pocedue Align the mios when changing to a diffeent lase o application equiing a diffeent lase. WARNING Risk of pesonal injuy. Diect eye contact with the output beam fom any lase will cause seious damage and possible blindness. Wea lase safety glasses that potect against the wavelengths being used. Read the infomation unde Heading 1.2, LASER SAFETY PRECAUTIONS befoe aligning the mio. 3-2

57 WATER-COOLED LASERS GENERAL INFORMATION 3 WARNING Risk of pesonal injuy. If opeating a lase with ultaviolet (UV) wavelengths, note that the beam is invisible to the eye and dangeous to the skin. Wea lase safety glasses that potect against the UV wavelengths of the lase. Special Fluoescent cads can be used to spot-check the beam position. 1 Without tuning on the lases, stat up the system as diected in Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 Open the coves to the sensing aea. a. Lift up the font cove. b. Pull up the intelock od. a b c. Squeeze the intelock cove elease button. c DANGER d d. Remove the intelock cove. 3 Remove the two scews and open the intenal lase cove. 4 Defeat the mechanical intelock by inseting the intelock defeat ba: a. Pull the intelock od up. b. Inset the defeat ba. c. Push the intelock od down. 3-3

58 WATER-COOLED LASERS GENERAL INFORMATION WARNING Risk of electical shock. The high voltage caied in the deflection plates can shock. Do NOT touch the deflection plates if the H.V. ON button is lit. If the H.V. ON button is lit, pess the button to tun the high voltage off. 5 Check the H.V. ON button. If it is lit, pess the button to tun off the high voltage to the deflection plates. 6 Gain access to the flow cell: a. Pull the deflection plates staight out. b. Loosen the thumbscew holding the beam-shaping lens assembly in place. c. Remove the beam-shaping lens assembly. H.V. ON Thumbscew Beam-shaping lens assembly Deflection plates 7 Remove the flow cell. 3-4

59 WATER-COOLED LASERS GENERAL INFORMATION 3 8 Tun the INTERLOCK OVERRIDE keyswitch on the Cytomete to defeat the electical intelock. Tun key clockwise to opeate lases LASER ON/OFF INTERLOCK OVERRIDE INTERLOCK OPEN RESET MAIN POWER 9 Remove the fowad scatte senso. Do not disconnect wies. Let the fowad scatte senso hang outside the doo to the sot collection aea. 10 Remove the pneumatic shutte. Do not disconnect the tubing. 11 Install the shot lase tagets in place of the fowad scatte senso and the pneumatic shutte. Scews to fasten tagets should be down. 12 Tun on the wate-cooled lases on you instument. Allow 30 minutes of wam up time fo the lases. 13 Close the shutte on one of the wate-cooled lases while aligning the second lase. 3-5

60 WATER-COOLED LASERS GENERAL INFORMATION 14 Align the wate-cooled lase by adjusting the dichoic alignment mio eflecting the lase beam to guide it though the tagets. Note: If the lase is 488 nm wavelength this will be a 488 nm mio. HeNe (Lase 2) Top View Agon (Lase 1) Wate-cooled agon lase Lase tagets Lase beams 633 mio eflects 633 nm and tansmits 320 to 365 nm and 488 nm 488 mio eflects 488 nm and tansmits 320 to 365 nm Beam educe UV mio eflects 325 to 365 nm 3-6

61 WATER-COOLED LASERS GENERAL INFORMATION 3 15 To align the mio: a. Loosen the height setscew and adjust height so the beam stikes the cente of mio. b. Loosen the fine adjustment and ail setscews. Move the stage along the optical ail so the beam stikes the middle of the mio. Tighten the fine adjustment setscew. c. Tighten the ail setscew so the mio and post do not ock on the ail. Keep the setscew loose enough so that the fine adjustment knob can still move the mio and post along the ail. d. Loosen the height setscew and otate the mio so the beam goes to the fist taget. Tighten the height setscew. e. Use the hoizontal and vetical otation knobs to guide the beam though the fist taget. Look fo lase light to appea on the second taget. f. If the light on the second taget is a wide cicle of light, use the fine adjustment knob to move the post and mio futhe back along the ail. Repeat step e. g. If the light on the second taget is a tight point, adjust the vetical and hoizontal otation to get the beam though the second taget. h. Tighten the ail setscew. Vetical otation knob Fine adjustment knob Fine adjustment setscew Rail setscew Hoizontal otation knob Height setscew 16 Close the shutte on the lase. 3-7

62 WATER-COOLED LASERS GENERAL INFORMATION 17 Open the shutte on a second wate-cooled lase, if pesent, to pefom an alignment on that one. To align a second wate-cooled lase, epeat steps 14 and Align a wate-cooled lase with a beam educe if you ae using the ai-cooled and wate-cooled lases togethe. The beam educe is used to educe the diamete of the wate-cooled lase beam to equal that of the ai-cooled beam. Beam educe Height setscew 3-8

63 WATER-COOLED LASERS GENERAL INFORMATION 3 19 The example uses a wate-cooled UV lase line and the ai-cooled Agon lase. a. Loosen the height setscew and emove the beam educe. b. Tun the wate-cooled lase on as diected in the Statup instuctions fo the appopiate lase listed in this chapte. c. Adjust the UV mio (eflecting the beam) to guide the beam though the tagets (see step 14). (You need to place a fluoescent taget, such as a white business cad, in font of the beam to detect the lase action.) Repeat steps 14 and 15 with the UV mio. d. Replace the beam educe and adjust it so the beam goes though the tagets. You can look fom the ea of the system and see the beam as it goes though the beam educe. Adjust the focus so the beam as it leaves the expande is about an eighth of an inch in diamete. Just befoe the 633 mio, the wate-cooled lase beam is about 1/16 in. in diamete. e. Close the shutte on the wate-cooled lase. f. Remove the tagets and eplace the fowad scatte senso and pneumatic shutte. g. Pefom a full optical alignment as descibed in the Special Pocedues and Toubleshooting manual. Leave all coves and doos open. h. Run beads. 3-9

64 WATER-COOLED LASERS GENERAL INFORMATION 20 Open the doo to the detecto compatment and set up the filtes as shown below. PMT4 PMT3 PMT2 PMT BP 381 BP PMT5 550 DL 430 DL 325 DL Continue the alignment. Close the doo to the detecto compatment. 22 Tun on the ai-cooled Agon lase at the Cytomete contol sceen and close the shutte. 23 Open the shutte on the wate-cooled lase. 3-10

65 WATER-COOLED LASERS GENERAL INFORMATION 3 24 At the Camea sceen touch sceen, set up a scope display to show: Fowad scatte peak fluoescence on the lowe tace. PMT2 peak fluoescence on the uppe tace. 25 Adjust beam-shaping lens assembly hoizontally fo maximum pulse heights. 26 Adjust gains and PMT voltages as necessay. 27 Close the shutte on the wate-cooled lase. 28 Open the shutte on the ai-cooled Agon lase. 29 Adjust the 488 mio hoizontal otation fo maximum fowad scatte pulse height. 30 Open the shutte on the wate-cooled lase. 3-11

66 WATER-COOLED LASERS GENERAL INFORMATION 31 At the Camea sceen touch sceen, set up a scope display to show: PMT5 peak fluoescence on the lowe tace (fom the ai-cooled Agon lase). PMT2 peak fluoescence on the uppe tace (fom the UV wate-cooled lase). 32 Adjust the UV mio vetical otation so the PMT2 pulses on the uppe tace ae ight above the PMT5 pulses on the lowe tace. Note: DO NOT eadjust the beam-shaping lens assembly. 33 Close the shuttes on the ai-cooled Agon and wate-cooled lases. 34 Adjust the UV mio hoizontal otation to maximize PMT2 pulse height. 35 Replace the beam educe, and adjust it fo maximum PMT2 pulse height. You many need to adjust the focus. Note: DO NOT eadjust the UV mio. 3-12

67 WATER-COOLED LASERS GENERAL INFORMATION 3 36 The signal fom the UV wate-cooled lase is on the uppe tace. Adjust the beam educe so the signal fom the wate-cooled lase on the uppe tace lines up with signal on the bottom tace. 37 Pess the ON/OFF button on the emote contol box of the wate-cooled lase. 38 Replace all coves. 39 At the Cytomete, tun the INTERLOCK OVVERRIDE keyswitch to enable the intelocks. Lases 1 and 2 (ai-cooled Agon and HeNe lases) tun off when the coves ae eplaced. Tun lases 1 and 2 On at the touch sceen. Tun key counte clockwise LASER ON/OFF INTERLOCK OVERRIDE INTERLOCK OPEN RESET MAIN POWER 40 Restoe the oiginal filte configuation and esume nomal opeation. Cleaning and Othe Maintenance Statup the wate-cooled lase fo about 1 hou at least once a week if the lase will not be used fo moe than 2 weeks. Refe to the manufactue s manual fo the cleaning and maintenance pocedues involving the lase. 3-13

68 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS 3.2 ENTERPRISE II SERIES LASERS The Entepise II Seies lase option contains thee poducts: ENTERPRISE Lase with dual 488 nm and UV capability. ENTERPRISE Optics Kit t Beam steeing optics t ND filte assembly (splits 488 nm and UV lase lines). ENTERPRISE Monitoing Hadwae The manufactue s manual is the Coheent Innova Entepise II Opeato's manual. The manufactue s manual biefly coves installation equiements, opeation, sevice, maintenance and pecautions. Read the manufactue s manual befoe opeating the lase. Floo Loading and Space The total system weight including this lase head and powe supply is: 567 kg (1,260 lb). The dimensions of the lase head and powe supply ae: Component Width Height Length Lase Head 18.5 cm (7.3 in.) 17.3 cm (6.8 in.) 76.2 cm (30.0 in.) Lase Powe Supply 43.9 cm (17.3 in.) 19.3 cm (7.6 in.) 50.3 cm (19.8 in.) Electical Powe Input powe: t Single-phase with gound, 50 o 60 Hz t 208 to 240 Vac ±10%, 7.0 Κva maximum. Maximum input cuent vs. Input voltage: t 34 A 208 V (ms) t 31 A 230 V (ms) t 29 A 240 V (ms). 3-14

69 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS 3 Cooling Wate CAUTION Risk of damage to instument. If the wate tempeatue falls below the ambient dew point, wate can condense inside the powe supply and can cause catastophic damage. Do not let the wate tempeatue fall below the ambient dew point. Table 3.1 Cooling Wate Specifications - Entepise II Seies Lases Item Contols and Indicatos Specification Flow ate, minimum 8 L (2 U.S. gal.) pe minute Flow ate, maximum 16 L (4.0 U.S. gal.) pe minute Incoming wate pessue 1.4 to 4.2 Kg/cm 2 (20 to 60 psi) Incoming wate tempeatue 10 to 45 C (50 to 110 F Heat load to cooling wate 4.5 kw (256 Btu/minute) Heat load to ai at powe supply 300 W maximum Powe Supply The powe supply has a voltage ange of 190 to 260 Vac, single-phase 50/60 Hz input. The powe supply is ai-cooled, high-fequency switching and has font panel LEDs (see Table 3.2 and Figue 3.2). Table 3.2 Powe Supply Contols/Indicatos - Entepise II Seies Lases Contol/Indicatos MAIN POWER indicato LASER EMISSION indicato CURRENT LIMIT indicato SYSTEM FAULT indicato HEAD FAULT indicato INTERLOCK FAULT indicato LASER POWER keyswitch Desciption When lit, indicates the powe is on. When lit, indicates the system is unning. When lit, indicates that the maximum allowable cuent has been eached. When flashing, indicates the need to eplace powe supply, emote contol module, o RS-232 device. When lit, indicates a heat exchange fault. When flashing, indicates the powe supply is ove tempeatue. When lit, indicates the powe supply is defective. When flashing, indicates system is taking an automatic gas fill. When lit, indicates an open intelock fault exists. When flashing, indicates an intelock fault occued but the fault is no longe pesent. Key contol fo tuning the powe supply on/off. 3-15

70 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS Figue 3.2 Powe Supply - Entepise II Seies Lases A Remote Contol Module The emote contol module povides complete system contol. All system infomation including opeating status, contol settings, and diagnostics, appeas on a 4-line, 20-chaacte, back-lit LCD display (see Figue 3.3 and Table 3.3). Figue 3.3 Remote Contol Module - Entepise II Seies Lases A 3-16

71 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS 3 Table 3.3 Remote Contol Module - Entepise II Seies Lases Contol POWER ON/OFF SELECT EXIT SCROLL Desciption Inceases o deceases the pimay and seconday powe output. Tuns the lase on and off. Allows access to the Main Menu and selects an item fom the menu. Retuns to the pevious menu. Scolls to the next o pevious menu options o changes a value in a paamete selection menu. Lase Head The Entepise II Seies lase head featues (see Figue 3.4): A wate-cooled design A sealed-mio, Innova plasma tube An integal SupeInva esonato Automated mio alignment. Figue 3.4 Lase Head - Entepise II Seies LASER EMISSION indicato A Apetue contol 3-17

72 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS Statup Note: Stat the wate-cooled Agon lase and leave it poweed on fo about 1 hou at least once a week if the lase is not to be used fo moe than 2 weeks. 1 Stat the ALTRA flow cytomete as diected in the Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 Tun on the cooling wate fo the lase. 3 Tun the keyswitch on the powe supply to ON. 4 Tun the Main powe switch ON. 3-18

73 WATER-COOLED LASERS ENTERPRISE II SERIES LASERS 3 Set Lase Powe 1 Use the aow keys on the emote contol module to set the powe (50 to 200 mw at 488 nm). Shutdown 1 Pess the ON/OFF button on the emote contol module. 2 Tun the keyswitch on the powe supply to OFF. 3-19

74 WATER-COOLED LASERS 300 SERIES LASERS 3 Let cooling wate continue to flow until at least 5 minutes afte the lase is tuned off. 4 Follow the diections unde Chapte 8, SHUTDOWN, in the ALTRA flow cytomete Opeato's Guide to shut down the est of the system SERIES LASERS The 300 Seies lase option contains thee poducts: The Optical Upgade kit which includes the mios and optical ail fo diecting the lase beams into the sensing aea Mounting feet A 300 Seies lase (5-W all lines) with ultaviolet (UV) capability. The manufactue s manual is the Coheent Innova 300 Seies Ion Lase Opeato's manual. The manufactue s manual biefly coves installation equiements, opeation, sevice, maintenance and pecautions. Read the manufactue s manual befoe opeating the lase. Floo Loading and Space The total system weight including this lase head and powe supply is: 576 kg (1,280 lb). The dimensions of the system ae Component Width Height Length Lase Head 15.0 cm (5.9 in.) 14.7 cm (5.8 in.) cm (48.3 in.) Lase Powe Supply 43.2 cm (17.0 in.) 17.8 cm (7.0 in.) 53.4 cm (21.0 in.) 3-20

75 WATER-COOLED LASERS 300 SERIES LASERS 3 Electical Powe The 300 Seies lases equie: Input powe: 3-phase with gound, no neutal Voltage: 208 Vac ±10%, 50 o 60 Hz Cuent: t Vac t Vac t Vac. The electical sevice fo the lase should have a main powe disconnect switch located in the same oom as the lase. Refe to the Installation chapte in the ALTRA flow cytomete Refeence manual fo additional electical equiements. Cooling Wate CAUTION Risk of damage to instument. If the wate tempeatue falls below the ambient dew point, wate can condense inside the powe supply and can cause catastophic damage. Do not let the wate tempeatue fall below the ambient dew point. Table 3.4 Cooling Wate Specifications Seies Lases Item Flow ate, minimum Flow ate, maximum Maximum static pessue Contols and Indicatos Specification 8.5 L (2.2 U.S. gal.) pe minute 11.6 L (3.0 U.S. gal.) pe minute 90 psi Pessue dop (inlet - outlet) 22 psi to 40 psi Wate tempeatue 10 to 35 C (50 to 95 F) Resistivity 5.0 kw-cm to 2.0 MW-cm ph 6 to 8 Hadness <100 mg/l (5.9 gains/gal.) o 100 ppm of calcium Powe Supply The only contols on the powe supply ae the ON/OFF keyswitch and a seies of LED indicatos (see Figue 3.5). All indicatos light when the keyswitch is in the ON position. Intelocks on the powe supply ae descibed in the manufactue s manual. 3-21

76 WATER-COOLED LASERS 300 SERIES LASERS Figue 3.5 Powe Supply Seies Lases OFF HK POWER +15V -15V +15V CPU CPU ±15V CPU ON Remote Contol Module The emote contol module (see Figue 3.6) can only be used when the keyswitch on the powe supply is in the ON position. See Figue 3.6 and Table 3.5 fo the contols and thei functions. Figue 3.6 Remote Contol Module Seies Lases A 3-22

77 WATER-COOLED LASERS 300 SERIES LASERS 3 Table 3.5 Remote Contol Module Seies Lases Contol Desciption ON/OFF Tuns lase on/off. Scoll Aows With lase in light egulation, scoll aows incease/decease lase powe. With lase in cuent egulation, scoll aows incease/decease lase cuent. Afte pessing MENU, scoll aows toggle though submenus, o settings on submenu. MENU Pessing at default display to display submenu titles. Pess at a Menu display to display a pevious menu level, o etun to default display. SELECT Afte pessing MENU, pess SELECT to select submenu on display. SET Pessing SET at default display initiates a 15-second PoweTack calibation outine. On a submenu display of vaious settings, pess to choose a paticula setting. Refe to lase manual fo moe instuctions. PWR TRK Pess to activate PoweTack mode. PT ON appeas on display. If PoweTack active, PWR TRK LED lights. Refe to manufactue s manual fo moe instuctions. LIGHT Pess to toggle between light and cuent egulation. Light egulation is active when LIGHT LED lights. Display also indicates egulation mode. MEMORY Refe to manufactue s manual fo instuctions. Lase Head Unlike the othe two lases, the 300 Seies lases (see Figue 3.7) ae contolled fom a emote contol box. Settings at the wokstation o the Cytomete do not affect the 300 Seies lases. Intelocks on the lase head ae descibed in the manufactue s manual. 3-23

78 WATER-COOLED LASERS 300 SERIES LASERS Figue 3.7 Lase Head Seies Lases Apetue contol Lase Shutte Lase emission indicato Vetical seach tab Hoizontal otation high eflecto High eflecto assembly Vetical otation high eflecto A Statup Note: Stat the wate-cooled Agon lase on fo about 1 hou at least once a week if the lase will not be used fo moe than 2 weeks. 1 Stat the ALTRA flow cytomete as diected in the Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 Tun on the cooling wate fo the lase. 3-24

79 WATER-COOLED LASERS 300 SERIES LASERS 3 3 At the lase powe supply, tun the keyswitch to ON. All LEDs on the font panel of the powe supply light. If they do not all light, efe to the lase manufactue s manual. COHERENT INNOVA 300 HK POWER +15V -15V +15V CPU CPU ±15V CPU OFF ON 4 At the emote contol box, pess the ON/OFF button. COHERENT INNOVA 300 LASER EMISSION MEMORY MENU SELECT SET ON/OFF TUNE PWR TRK LIGHT 5 Afte a 30-second stat delay, the lase emits the lase beam. 6 At the emote contol box, pess the LIGHT button to put the lase in light egulation. In light egulation, the lase system inceases o deceases the cuent to achieve the powe setting. 3-25

80 WATER-COOLED LASERS 300 SERIES LASERS 7 If PT ON does not display on the emote contol box, pess the PWR TRK button. IMPORTANT Risk of unpedictable esults. Auto Light Cal uses bief lase output oscillations to calibate the PoweTack featue. These oscillations can cause unpedictable esults fom the ALTRA flow cytomete. Only use Auto Light Cal when tuning the lase on. Be sue Auto Light Cal is off when unning samples. 8 Tun Auto Light Cal on: a. Pess the MENU button. b. Pess the scoll aows until Auto Light Cal appeas on the display. c. Pess the SELECT button until Yes appeas. d. Pess the SET button. e. Pess the MENU button. 9 Let the lase wam up fo about 5 minutes. 10 Tun Auto Light Cal off. 3-26

81 WATER-COOLED LASERS 300 SERIES LASERS 3 Set Apetue The 300 Seies lases must opeate in TEM 00 mode. The TEM mode depends on the specific plasma tube, lase powe, wavelength and apetue setting. Refe to the lase manufactue s manual to detemine the TEM mode. Geneally, apetue settings between 4 and 8 give TEM 00 mode at about 500 mw at 488 nm. You can often see if the lase is in some othe TEM mode by looking at peak fluoescence pulses of fluoosphees. If the peak pulse has two peaks o a shoulde, the lase may not be in TEM 00 mode. Set Lase Powe Pess the up o down Scoll Aows on the emote contol box (efe to Figue 3.6) to set the lase powe. At 488 nm, the suggested opeating powe is between 200 mw and 500 mw. Tune Lase fo Maximum Powe Because of the PoweTack featue, the 300 Seies lases do not need tuning on a daily basis. Afte statup, the PoweTack featue keeps the lase tuned until the fist time you close the lase shutte o change the powe. Afte that, PoweTack does not actively tune the lase until the next statup. Tune the lase once a week accoding to the following pocedue: 1 Pess the TUNE button on the emote contol module. 2 Adjust the hoizontal high eflecto contol on the lase head fo maximum powe as indicated by the lase powe ba gaph on the emote contol box. 3 Adjust the vetical high eflecto contol fo maximum powe. 4 Pess the TUNE button on the emote contol module. The lase etuns to its pevious settings. 3-27

82 WATER-COOLED LASERS SPECTRUM LASER Shutdown 1 Pess the ON/OFF button on the emote contol module. COHERENT INNOVA 300 LASER EMISSION MEMORY MENU SELECT SET ON/OFF TUNE PWR TRK LIGHT 2 Tun the keyswitch on the powe supply to the OFF position. COHERENT INNOVA 300 OFF HK POWER +15V -15V +15V CPU CPU ±15V CPU ON 3 Let cooling wate continue to flow until at least 5 minutes afte the lase is tuned off. 4 Follow the diections unde Chapte 8, SHUTDOWN, in the ALTRA flow cytomete Opeato's Guide to shut down the est of the system. 3.4 SPECTRUM LASER The Spectum lase has these featues: Mixed gas Agon/Kypton lase Multiwavelength opeation including a 647 nm lase line 3-28

83 WATER-COOLED LASERS SPECTRUM LASER 3 The Spectum lase option contains two poducts: I-70 Spectum lase (2.5 W multiline, 250 W, 647 nm) Mounting hadwae. The manufactue s manual is the Coheent Innova I-70 Spectum Opeato's manual. The manufactue s manual biefly coves installation equiements, opeation, sevice, maintenance and pecautions. Read the manufactue s manual befoe opeating the lase. Floo Loading and Space The total system weight including this lase head and powe supply is: 567 kg (1,260 lb). The dimensions of the system ae: Component Width Height Length Lase Head 18.5 cm (7.3 in.) 16.5 cm (6.5 in.) cm (44.8 in.) Lase Powe Supply 44.7 cm (17.6 in.) 22.4 cm (8.8 in.) 36.6 cm (14.4 in.) Electical Powe The Spectum lase equies: Input powe: 3-phase with gound Voltage: 208 ±10% Vac 50 o 60 Hz 40 A pe 208 Vac. Cooling Wate CAUTION Risk of damage to instument. If the wate tempeatue falls below the ambient dew point, wate can condense inside the powe supply and can cause catastophic damage. Do not let the wate tempeatue fall below the ambient dew point. Table 3.6 Cooling Wate Specifications - Spectum Lase Item Flow ate Incoming wate pessue Incoming wate tempeatue Specification 8.5 L (2.2 U.S. gal.) pe minute 1.41 to 4.23 Kg/cm 2 (20 to 60 psi) 10 to 35 C (50 to 95 F) Contols and Indicatos Powe Supply The powe supply can be opeated fom the powe supply (see Figue 3.8) o the emote contol module (see Figue 3.9). The emote contol module is connected to the powe supply by a cable and povides you with full contol of the lase fom a convenient location. It suppots: 3-29

84 WATER-COOLED LASERS SPECTRUM LASER Use selectable light and cuent egulation though the emote contol module (see Figue 3.9 and Table 3.7). Continuous tube pessue monitoing to alet you to the need fo a gas fill (see Figue 3.9 and Table 3.7). Continuous wate flow monitoing - if the wate flow dops below the minimum levels, the powe supply shuts off to potect the system (see Figues 3.8 and 3.9). Safety intelocks to pevent lase opeation in an unsafe condition (see Figue 3.9 and Table 3.7). Figue 3.8 Powe Supply - Spectum Lase A Table 3.7 Powe Supply and Remote Contol Module Contols and Indicatos - Spectum Lase Contol/Indicato Location Desciption LASER EMISSION indicato Powe supply Illuminates when the powe supply is tuned ON. LASER indicato Remote contol module WATER FLOW indicato Powe supply Red LED that lights continuously when wate flow dops below WATER indicato Remote contol module 2.2 gal./minute and flashes when lase tube pessue is too low. SYSTEM ON/OFF pushbutton Powe supply Enegizes powe supply. SYSTEM ON/OFF keyswitch Remote contol module 3-30

85 WATER-COOLED LASERS SPECTRUM LASER 3 Table 3.7 Powe Supply and Remote Contol Module Contols and Indicatos - Spectum Lase Contol/Indicato Location Desciption INTERLOCK indicato FAULT indicato Regulation mode switch POWER contol START pushbutton Analog eadout Remote contol module Remote contol module Remote contol module Remote contol module Remote contol module Remote contol module LED on indicates an open intelock. Check the lase head cove, powe supply cove, o fuse cove. LED on indicates: No ac powe, o Tube unde pessue, o Tube ove cuent, o ± 15 V powe supply failue. Fou-position switch fo selecting cuent contol (CUR) o UV o one of thee light egulation anges (LO, MED, HI). Adjusts tube cuent fom minimum to maximum when in cuent egulation (CUR) mode. Initiates the stat sequence Analog eadout can be set to show eithe LIGHT POWER, TUBE CURRENT, o TUBE VOLTAGE. Remote Contol Module The emote contol module (see Figue 3.9 and Table 3.7) houses: The status indicatos A main powe keyswitch An analog eadout showing the tube cuent, tube voltage and output powe. Figue 3.9 Remote Contol Module - Spectum Lase A 3-31

86 WATER-COOLED LASERS SPECTRUM LASER Lase Head Figue 3.10 Lase Head - Spectum Lase Vetical scanning lase Beam shutte A 3-32

87 WATER-COOLED LASERS SPECTRUM LASER 3 Statup Note: Stat the wate-cooled Agon lase on fo about 1 hou at least once a week if the lase will not be used fo moe than 2 weeks. 1 Stat the ALTRA flow cytomete as diected in the Chapte 3, STARTUP, in the ALTRA flow cytomete Opeato's Guide. 2 Tun on the cooling wate fo the lase. 3 At the lase powe supply, tun the keyswitch to ON. The WATER FLOW LED lights momentaily while the wate flow is veified. 4 Pess the ON button. The LASER EMISSION indicato lights. 5 Afte a 30-second stat delay, the lase emits the lase beam. 3-33

88 WATER-COOLED LASERS SPECTRUM LASER 6 If the beam shutte is not aleady open, set the beam shutte contol knob so that the white line is paallel to the tube boe to open the shutte. 7 Set the egulation mode to CUR and tun the POWER knob to full powe. Set Lase Powe 1 Adjust the POWER knob on the emote contol module to the desied powe level. 3-34

89 WATER-COOLED LASERS SPECTRUM LASER 3 Tune Lase fo Maximum Powe 1 Adjust the vetical and hoizontal adjustment knobs to tune the lase powe to Maximum on the emote contol module. Shutdown 1 Tun the intelock key on the emote contol module counteclockwise to tun off the lase. 2 Wait 5 minutes afte tuning off the lase befoe tuning off the wate. 3-35

90 WATER-COOLED LASERS SPECTRUM LASER 3-36

91 4STORAGE OPTIONS GENERAL INFORMATION The following stoage media ae available as options fo the ALTRA flow cytomete: Iomega Zip Dive (heeafte efeed to as the Zip dive). Yamaha Recodable CD-ROM (heeafte efeed to as the CD-ROM). Iomega jaz intenal dive (heeafte efeed to as the jaz dive). The infomation unde this section (Heading 4.1, GENERAL INFORMATION) is geneic and applies to all the stoage media listed above. Infomation specific to each stoage media listed above is included in a sepaate section in this chapte. Refe to the manufactue s manual fo each option fo opeating instuctions. Magnetic Stoage Media Cae Despite quality-contol standads, some magnetic stoage media become defective. To help pevent data loss, adhee to the following guidelines: Always keep backup copies of you data on sepaate diskettes o othe magnetic stoage media. Do not touch ecoding sufaces. Keep media away fom magnetic fields, such as those geneated aound adios and phones. Do not bend diskettes, CD-ROMs o othe stoage media. Always stoe magnetic stoage media at oom tempeatue. 4.2 Zip DRIVE The Zip dive is an intenal IDE stoage device. A state set of stoage media is included in the kit and additional disk packs ae available though Beckman Coulte, o at most compute supply stoes. The Iomega Zip Dive kit includes: Iomega Zip Dive 1 pack of media (250 MB each) 4.3 RECORDABLE CD-ROM DRIVE The Yamaha Recodable CD-ROM is an intenal one-time ecodable, many-times eadable, media allowing you to achive and etieve Histogam and Listmode files to CD-ROM media, fo long-tem stoage. It is intended fo use with the FlowCente multimedia wokstation. The CD-ROM kit contains: The Yamaha intenal CD-ROM Dive 10 Pak of CD-ROM media (650 MB each) We ecommend that you puchase an Uninteuptable Powe Supply locally fo the Wokstation and Recodable CD-ROM dive. Any powe inteuptions while you ae ecoding to a CD endes that CD-ROM disk completely useless. 4-1

92 STORAGE OPTIONS jaz DRIVE The Recodable CD-ROM Dive includes two diffeent softwae packages to ceate CD-ROMs. The softwae packages ae Adaptec Easy CD-Ceato Deluxe Edition and Adaptec Diec-CD. Easy CD Ceato Easy CD-Ceato ceates CD-ROMs that can be ead in Windows 98, Windows 95, Windows 3.1, Windows NT, and MS-DOS opeating system, thus allowing fo full coss-platfom compatibility. Files achived to CD-ROM using Easy CD-Ceato howeve can be ead in XL System II o Elite Softwae. Easy CD-Ceato also allows fo ecoding of audio, video and gaphics pesentations to CD-ROM as well as Multi-Session ecoding. CD-Ceato also featues a CD Copie pogam, that duplicates CD-ROMs. Note: It is a violation of copyight laws to duplicate any softwae fom CD-ROMs which is copyight potected. The custome assumes full esponsibility fo any such action. Beckman Coulte assumes no liability whatsoeve. Easy CD-Ceato Installation Pocedues 1. Locate the Easy CD-Ceato softwae pogam kit, and inset the CD-ROM into the dive. 2. The installation sceen appeas. Use the mouse cuso and click on EASY CD-Ceato to install the softwae, following instuctions on the sceen. 3. Afte installation, fom the Windows 98 desktop, click on START, PROGRAMS, EASY CD-CREATOR and select EASY CD-CREATOR DELUXE, to stat the ecoding pogam. 4. Place a blank CD-ROM media disk into the caddy supplied with the ecodable CD-ROM dive. 5. It is ecommended that you utilize the EASY CD-CREATOR WIZARD to duplicate a CD-ROM diskette. 6. Follow the on-sceen instuctions to ecod a test file (any file) onto the blank CD-ROM media. Files ecoded in ISO-9660 fomat can be ead by any opeating system, fo example: Windows 98, Windows 95, Windows 3.1, Windows NT, Macintosh, and MS-DOS. Files ecoded in Joliet fomat, allow fo long file names in Windows 98 and Windows 95, and ae eadable in Windows 3.1 and MS-DOS. 7. Once the test file has been ecoded, emove the CD-ROM fom the caddy and inset into the CD-ROM dive pesent in the wokstation. 8. Open up Windows Exploe and locate the CD-ROM dive in the wokstation, and click on it twice. Veify that the test file ecoded is pesent on the CD-ROM disk. Fo additional infomation on the featues of Adaptec Easy CD-Ceato, please efe to the Adaptec s Use Guide. 4.4 jaz DRIVE The jaz dive is an intenal stoage dive. It povides 2-GB of stoage pe disk. 4-2

93 5Autoclone SORTING OPTION INTRODUCTION The EPICS ALTRA Autoclone soting option is a single-cell deposition system only. It pefoms two functions. 1. It is a pogammable single-cell deposition system. It sots a use-defined numbe of cells pe well into a multiwell micocultue plate. Automates hybidoma sceening fo eseach and development of monoclonal antibodies, steile soting of bacteial cultues, and sceening fo high-density monoclonal makes. The system accepts standad 6, 12, 24, 48, 60, o 96 well micocultue plates fo automated cell deposition. 2. The second function, Sot Matix, automates the pocess of detemining the optimum Dops Delay setting. A holde fo micoscope slides is used when pefoming a Sot Matix. It depends upon the ALTRA host system fo pogamming instuctions, data accumulation, and electical powe. When the Autoclone soting option is not in use, the ea cove and disable knob must be in place. 5.2 INSTALLATION REQUIREMENTS The installation equiements fo the ALTRA system emain unchanged when the Autoclone system is installed. Refe to the Installation chapte in the Refeence manual fo ALTRA system installation equiements. Do not unpack o install the Autoclone system youself. The system must be unpacked and installed by you Beckman Coulte Repesentative. Installation Calibation Pocedue Afte initial installation o einstallation of the ALTRA softwae, pefom this abbeviated calibation pocedue: 5-1

94 Autoclone SORTING OPTION INSTALLATION REQUIREMENTS IMPORTANT Risk of electic shock. The voltage on the deflection plates can shock you. DO NOT touch the deflection plates if the H.V. ON button is lit. If the button is lit, pess the button to tun the deflection plates off. 1 If the H.V. ON button is lit, pess the button to tun off the deflection plates. 2 Remove the ea plate coveing the Autoclone am by tuning the disable knob counteclockwise. 5-2

95 Autoclone SORTING OPTION INSTALLATION REQUIREMENTS 5 3 At the cytomete MAIN touch sceen, select in ode: Options Autoclone Contol 4 At the Autoclone sceen, select: Adjust Sceen 5 At the Autoclone Adjustment sceen, select: Calibate 6 Afte calibation, etact the Autoclone am. Select: Autoclone Contol 7 At the Autoclone Contol sceen, select: Retact Sceen 5-3

96 Autoclone SORTING OPTION STARTUP 8 Follow the instuctions on the Retact sceen and pess: OK 9 Replace the Autoclone cove. 5.3 STARTUP 1 If the HPSS option is NOT installed, check the pessue gauges on the compesso. If the system vacuum o pessue is outside limits, adjust them as descibed in the Special Pocedues and Toubleshooting manual SYSTEM VACUUM Extenal compesso SYSTEM PRESSURE 5-4

97 Autoclone SORTING OPTION STARTUP 5 IMPORTANT Risk of electic shock. The voltage on the deflection plates can shock you. DO NOT touch the deflection plates if the H.V. ON button is lit. If the H.V. ON button is lit, pess the H.V. ON button to tun the deflection plates off. 2 If the H.V. ON button is lit, pess the H.V. ON button to tun off the deflection plates. 3 Remove the ea plate coveing the Autoclone soting option am by tuning the disable knob counteclockwise. 5-5

98 Autoclone SORTING OPTION STARTUP 4 At the cytomete MAIN touch sceen, select in ode: Options Autoclone Contol Load. The Autoclone soting option am extends out to accept the plate holde and pefom autocalibation. 5 Install the multiwell plate holde with cutout aea facing font. Fo sot matix, install the slide adapte in the plate holde with the micoscope slide to the left. Fo soting, slide a multiwell plate onto plate holde so that well A1 is to the left. Slide/plate holde 6 Bend the guides on the plate holde so that the multiwell plate fits snug. 5-6

99 Autoclone SORTING OPTION STARTUP 5 WARNING Risk of electic shock. The voltage on the deflection plates can shock you. DO NOT touch the deflection plates if the H.V. ON button is lit. If the H.V. ON button is lit, pess the H.V. ON button to tun the deflection plates off. 7 Enable sheath and setup sot steams as shown in Chapte 7, SORTING, in the Opeato s Guide. Sot steam Slide adapte Slide/ plate holde 8 Tun the STREAM knob until the left sot steam is flowing staight down. 9 Toggle deflection high voltage off and on to check the steam position. With high voltage on, the left sot steam should look the same as the sheath steam does when high voltage is off. 5-7

100 Autoclone SORTING OPTION SORT MATRIX 5.4 SORT MATRIX The Autoclone system pefoms an automated Sot Matix to help detemine coect Dops Delay. 1 Use the camea cusos to find the appoximate Dops Delay. Refe to Chapte 7, SORTING, in the Opeato s Guide. 2 Ensue the Sot Contol sceen and Sot Settings box contain coect values. 3 Run a sample of Flow-Check fluoosphees, o equivalent, and place a sot egion aound the fluoosphees. 5-8

101 Autoclone SORTING OPTION SORT MATRIX 5 4 Set up the sot equation. Sceen tt Sot. Region name fom the egion list. 5 Id label in the Autoclone Equation box 6 Type a name fo the sot equation and pess Û. 5-9

102 Autoclone SORTING OPTION SORT MATRIX 7 Select Sot Matix. Sceen tt Autoclone. Select Plate. Up o down aow until Sot Matix is highlighted. Okay. 8 Ente Slide Chaacteistics. Oifice Diamete. Type 76 o 100 and pess Û. 9 Ente Sot Settings The Sot Settings ae eceived fom the Sot sceen, howeve a Dops Delay Step value needs to be enteed. Dop Dely. To accept the cuent value pess Û. Type a Dops Delay Step value and pess Û. 5-10

103 Autoclone SORTING OPTION SORT MATRIX 5 10 Examples of Dops Delay Step values: A Dops Delay Step of 1 pogams the system to sot a ange of 4 dop delays highe and lowe than the Dop Dely in the sot settings box. 11 Pogam the slide. Id label fom equation list. 5-11

104 Autoclone SORTING OPTION SORT MATRIX 12 The Id label automatically displays above the slide next to Eq:. 13 Type the numbe to be soted and pess Û. 14 Type a desciption and pess Û. 5-12

105 Autoclone SORTING OPTION SORT MATRIX 5 15 Inset a micoscope slide in the slide adapte. Install the slide adapte in the plate holde with the micoscope slide to the left. Slide Slide adapte Plate holde IMPORTANT Risk of electic shock. The voltage on the deflection plates can shock you. DO NOT touch the deflection plates if the H.V. ON button is lit. If the H.V. ON button is lit, pess the H.V. ON button to tun the deflection plates off. 16 If the H.V. ON button is lit, pess the H.V. ON button to tun off the deflection plates. 17 Activate. 5-13

106 Autoclone SORTING OPTION SORT MATRIX 18 Examine the slide with a fluoescence micoscope to detemine the coect dop delay value. Sot Settings to toggle to Send. Dop Dely. Type the coect dop delay and pess Û. 19 Save the Autoclone and sot settings with the potocol. Sceen tt Potocol. File tt Save. To use the same name fo the potocol pess Û, OR Type a name fo the potocol and pess Û. 5-14

107 Autoclone SORTING OPTION SORTING SORTING 1 Pefom Statup, Heading 5.3, to pepae the Autoclone system fo soting. 2 Run a sot matix, efe to Heading 5.4, SORT MATRIX, to detemine the coect dop delay fo soting. 3 Run a sample and ceate o edit the Gate and Sot egions. Refe to Chapte 5, RUN SAMPLES in the Opeato s Guide, fo instuctions on ceating and editing egions. 4 Set up the Autoclone equation. Sceen tt Sot. Region name fom the egion list. 5-15

108 Autoclone SORTING OPTION SORTING 5 Id label in the Autoclone Equation box. 6 Type a name fo the equation and pess Û. 7 Select a multiwell plate. Sceen tt Autoclone. Select Plate. Up o down aow until Sot Matix is highlighted. Okay. 5-16

109 Autoclone SORTING OPTION SORTING 5 8 Ente Plate Chaacteistics. Oifice Diamete. 9 Type 76 o 100 and pess Û. 10 Well Timeout Type the numbe of seconds to wait fo soted paticles befoe moving to the next well position and pess Û. 11 Liquid in well Type in the amount of media in the wells and pess Û. 5-17

110 Autoclone SORTING OPTION SORTING 12 Sot Settings The sot settings ae the same values as in the sot sceen. The sot settings ae saved with the potocol afte pefoming a Sot Matix. See Heading 5.4, SORT MATRIX. Fo the Autoclone soting option, use the AccuSot Mode (Dops 7, Coincidence Abot = ON). 13 Pogam the plate with Autoclone equations. Id numbe fom the pogam equation box. 5-18

111 Autoclone SORTING OPTION SORTING 5 14 Well that you want to sot into. Type the numbe of cells to be soted and pess Û. Type a desciption and pess Û. The Autoclone equation ID numbe displays in the well. 15 To pogam an entie ow at a time. Id numbe fom the pogam equation box. Numbe next to the ow of wells you want to sot into. 5-19

112 Autoclone SORTING OPTION SORTING 16 To pogam an entie column at a time. Id numbe fom the pogam equation box. Lette unde the column of wells you want to sot into. 17 To pogam an entie plate at a time. Id numbe fom the pogam equation box. Fill Plate button. WARNING Risk of biohazadous contamination. Keep sample stage aea and sot collection aea doos closed duing sample acquisition. Aeosols may be ceated which may contain biohazadous mateials. 18 Ensue H.V. ON button is lit and Activate. 5-20

113 Autoclone SORTING OPTION CALIBRATION CALIBRATION When Calibate is selected in the Autoclone Contol menu, the system pefoms automatic calibation of the Autoclone soting option am movement. Calibate the Autoclone soting option wheneve the cytomete softwae is eloaded o eplaced, o the CYTO.HEX file is tansfeed. Fine tune the calibation wheneve the steam is not centeed in the plate wells. Makes Lx Cw Hg AUTOCLONE SCREEN 96 WELLS 12 ROW 8 COL CURRENT SELECTED Autoclone Cove Off Inc ROW Dec ROW ROW COL Inc COL Dec COL MOVE 13 SELECTED LOCATION Autoclone Waste On Home Load Retact Sceen Adjust Sceen Sot Sceen Options Sceen MAIN Sceen Befoe you Fine Tune the calibation you must: low cell oizontal Flow cell vetical Beam vetica Make sue the system is optically aligned. Refe to the Special Pocedues and Toubleshooting manual. H.V. ON Beam hoizontal 5-21

114 Autoclone SORTING OPTION CALIBRATION Use Sot Test to get tight sot steams. Refe to Chapte 7, SORTING, in the Opeato s Guide. Toggle Right Sot Enable OFF. Tun the STREAM knob until the left sot steam is flowing staight down. Toggle Deflection High Voltage off and on to check the steam position. With high voltage on, the left sot steam should look the same as the sheath steam does when high voltage is off. Sot steam Slide adapte Slide/ plate holde Leave Sot Test on. Fine Tune Calibation 1 Slide a 96-well plate onto the plate holde. 5-22

115 Autoclone SORTING OPTION CALIBRATION 5 2 At the cytomete MAIN menu select Autoclone Options and Contol. 3 Select Inc ROW and Inc COL until 12-D is displayed to the ight of SELECTED. 4 Select MOVE TO SELECTED LOCATION. 5 Toggle Autoclone Waste Off. 6 Select Inc ROW and Inc COL until 1-D is displayed to the ight of SELECTED. 7 Select MOVE TO SELECTED LOCATION. 5-23