Reference. COULTER EPICS ALTRA Flow Cytometer COULTER EPICS ALTRA HyPerSort System. PN CA (August 2010)

Transcription

1 COULTER EPICS ALTRA Flow Cytomete COULTER EPICS ALTRA HyPeSot System Refeence (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 Issue, 7/98 Softwae vesion 1.0 Issue B, 11/98 Changes made wee elated to the HyPeSot option. Changed Pages: iii to xii, 1-2, 1-3, 3-1 to 3-3, 3-5, 3-6, 3-10, 3-11, 3-18, 4-1, 4-3, 4-6, 4-7, 4-10, Glossay-1, Glossay-2, Index-1 to Index-9, Tademaks, and back cove. Issue C, 2/00 Complete evision. Issue CA, 8/10 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, xiii HOW TO USE YOUR COULTER EPICS ALTRA FLOW CYTOMETER MANUALS, xiii ABOUT THE REFERENCE MANUAL, xiii CONVENTIONS, xiv 1 USE AND FUNCTION, INTENDED USE, SYSTEM FUNCTION, SYSTEM DESCRIPTION, 1-1 Main Assemblies, SENSOR MODULE, 1-2 Sample Intoduction System, 1-3 HPSS Contol Panel, 1-3 Sot Collection Aea, 1-3 Detecto and Sensing Compatments, 1-3 Gaphics Monito, 1-4 Touch Sceen Monito, PNEUMATICS CABINET, ELECTRONIC PEDESTALS, OPTICAL TABLE, FLOWCENTRE MULTIMEDIA WORKSTATION, 1-5 Disk Dives, 1-6 Keyboad, 1-6 Mouse, 1-6 Colo Monito and Gaphics Cad, REAGENTS AND QUALITY CONTROL (QC) MATERIALS, 1-6 Sheath Fluid, 1-6 Cleaning Agent, 1-6 QC Mateials, MATERIAL SAFETY DATA SHEETS (MSDS), INSTALLATION, GENERAL, 2-1 -v

6 CONTENTS 2.2 SPECIAL REQUIREMENTS, 2-1 Access Limitation, 2-1 Decontamination, 2-1 Accessibility, 2-1 Heat Dissipation And Ventilation, 2-2 Ambient Tempeatue and Humidity, 2-2 Electical Powe, 2-2 EMC/EMI Compliance, CALIBRATION OF Autoclone SORTING OPTION AFTER INSTALLATION, SYSTEM CONNECTIONS, OPERATION PRINCIPLES, SYSTEM OVERVIEW, SAMPLE PRESENTATION, 3-1 Hydodynamic Focusing, 3-1 Flow Cell, 3-1 Sheath Velocity, 3-2 Sample Coe, 3-2 Diamete, 3-2 Velocity, 3-2 Sheath Pessue, 3-3 Sample Delivey Rate, LASER BEAMS, 3-3 Lase Pinciples, 3-3 Beam Shaping, 3-4 Beam Shaping Lenses, 3-5 Beam-Shape Selection Chat, 3-6 Beam-Shaping Assemblies, 3-7 Diffaction Limited Spot Size, 3-8 Beam-Steeing Optics, SENSING AREA, 3-9 Scatte, 3-9 Fluoescence, 3-9 Light Collection, 3-10 Video Camea, 3-10 Fowad Scatte Detecto, 3-11 SotSense Flow Cell Tip, 3-11 Fluoescence Lenses, 3-12 Optical Filtes, vi

7 CONTENTS 3.5 SIGNALS, 3-14 Detectos, 3-14 Fowad Scatte Detecto, 3-15 Photomultiplie Tubes (PMTs), 3-15 Signal Pocessing, 3-16 Integation, 3-18 Amplification, 3-18 Time of Flight, 3-18 Fluoescence Compensation, HISTOGRAMS, 3-19 Disciminatos, 3-20 Peak-Sense-and-Hold (PSH), 3-20 Analog-to-Digital Convesion (ADC), 3-20 Data Listing, 3-20 Amophous Gates, 3-21 One-Paamete Histogams, 3-21 Two-Paamete Histogams, LISTMODE DATA, SORTING, STATISTICS, 3-22 Linea Region Statistics, 3-22 Log Region Statistics, vii

8 CONTENTS 4 SPECIFICATIONS/CHARACTERISTICS, SYSTEM SPECIFICATIONS, 4-1 Dimensions, 4-1 Weight, 4-1 Ambient Tempeatue and Humidity, 4-1 Detectos, 4-1 Optical Filtes, 4-1 Standad ALTRA Flow Cytomete Filtes, 4-1 With Red HeNe Lase, 4-2 With HeCd Lase, 4-2 Lases, 4-2 Excitation Optics, 4-2 Analog-to-Digital Convete (ADC), 4-2 Cytomete Displays, 4-2 Sample Concentation, 4-2 Sample Size Range, 4-3 Pneumatics/Fluidics, 4-3 Sample Volume, 4-3 Sample Containe, 4-3 Flow Cells, 4-3 SotSense Enhanced Quatz, 4-3 High Velocity Quatz, 4-3 BioSense Enhanced Quatz, 4-3 Sense-in-Ai (Jet-in-Ai), 4-3 Sheath Fluid, 4-3 Micopocessos, 4-3 FlowCente Multimedia Wokstation, 4-3 Cytomete Contolle, 4-4 Memoy, 4-4 FlowCente Multimedia Wokstation, 4-4 Data Stoage, 4-4 Gaphics Input, 4-4 Data Acquisition, 4-4 Sample Identification, 4-4 Histogam Resolution, 4-5 Statistics, 4-5 Single-paamete histogams, 4-5 Dual-paamete histogams, 4-5 All histogams, 4-5 Regions, 4-5 Signal Pocessing, 4-6 Soting, 4-6 Powe Requiements, 4-7 Installation Categoy, 4-7 Pollution Degee, 4-7 File Fomats, 4-7 -viii

9 CONTENTS 4.2 PERFORMANCE CHARACTERISTICS, 4-7 Analysis Thoughput, 4-7 Fluoescence Detection Range, 4-8 Fluoescence Sensitivity, 4-8 Light Scatte Sensitivity, SYSTEM OPTIONS, 4-8 Ai-Cooled Lases, 4-8 Cyonics - Agon, 4-8 Red HeNe, 4-8 Geen HeNe, 4-8 Omnichome - HeCd 74 kit, 4-8 Wate-Cooled Lases, 4-8 Coheent - Innova Entepise II Seies, 4-8 Coheent - Innova 300 Seies (Model 305), 4-9 Coheent - Innova 70 Spectum, 4-9 Autoclone Soting Option System, 4-9 Gated Amplifie, 4-9 Lage Paticle Soting Option, 4-10 Fowad Fluoescence Detecto Option, 4-10 Second Monito, 4-10 Flow Cells, 4-10 Beam Shapes, 4-10 Pintes, 4-10 Zip Dive, 4-10 Recodable CD-ROM, 4-10 jaz Dive, 4-11 Modem, CE COMPLIANCE, PRECAUTIONS/HAZARDS, PRECISION, 5-1 Flow Rate, 5-1 Sample Dispesal, LIGHT INTERFERENCE, SHEATH AND SAMPLE SUSPENDING LIQUIDS, 5-1 Sheath Fluid, 5-1 Soting, 5-2 Steile Soting, CLEANLINESS, 5-2 Spills, 5-2 Biohazad Ai Filte, 5-2 Geneal Cleanliness, 5-2 -ix

10 CONTENTS 5.5 DATA STORAGE, 5-2 Data Backups, 5-2 Diskette and CD-ROM Cae, BIOLOGICAL HAZARDS, LASER SAFETY, 5-3 Safety Pecautions, 5-3 Radiation Hazads, 5-4 A OPTICAL FILTERS, A-1 A.1 INTRODUCTION, A-1 Tansmittance Specta, A-1 A.2 FILTERING MECHANISMS, A-1 Intefeence Filtes, A-1 Absobance Filtes, A-2 A.3 FILTER HANDLING, A-2 A.4 FILTER NOMENCLATURE, A-2 Long-Pass (LP), A-3 Shot-Pass (SP), A-3 Blocking (BK), A-3 Band-Pass (BP), A-3 Neutal Density (ND), A-3 A.5 FILTER TRANSMITTANCE SPECTRA, A-4 GLOSSARY, GLOSSARY-1 INDEX, INDEX-1 TRADEMARKS -x

11 CONTENTS ILLUSTRATIONS 1.1 ALTRA HPSS Flow Cytomete, Senso Module, Lase Aangement, Powe and Cable Connections, Standad Flow Cell and Body, HPSS Flow Cell and Body, Coss Cylindical Beam-Shaping, Beam Shapes, Beam Steeing Assembly, Optical System, Fowad Scatte Detecto, SotSense Flow Cell Tip, Filte Slots, Filte Configuation, Example, Photomultiplie Tube (PMT), Signal Pocessing, Integal and Peak Signals, Fluoescence Ovelap, Analog-to-Digital Convesion, Agon Lase, Manufactue's Waning Labels, Red Helium-Neon Lase, Manufactue s Waning Labels, Optional Geen Helium-Neon Lase, Manufactue s Waning Label, Optional COHERENT INNOVA ENTERPRISE II Lase, Manufactue s Waning Labels, Optional COHERENT INNOVA 70 SPECTRUM Lase, Manufactue s Waning Labels, Optional Helium Cadmium Seies 74 Lase, Lase Waning Labels, Optional COHERENT INNOVA 300 Seies Lase Waning Labels, Lase Waning Labels at Sensing Aea, Fowad Fluoescence Detecto Option Sensing Aea, Lase Waning Labels, Detecto and Sensing Compatments Lase Intelock Switches, Detecto and Sensing Compatments Cove, Lase Waning Labels, Cetification Label, Rea Cove and Left Cove Lase Waning Labels, Instument Oute Cove Lase Waning Label, 5-13 A.1 Tansmittance Specta, A-1 A.2 488BP Tansmittance Spectum, A-5 A.3 525BP Tansmittance Spectum, A-5 A.4 575BP Tansmittance Spectum, A-5 A.5 488DL Tansmittance Spectum, A-5 A.6 550DL Tansmittance Spectum, A-6 A.7 600DL Tansmittance Spectum, A-6 A.8 640LP Tansmittance Spectum, A-6 A.9 488BK Tansmittance Spectum, A-6 A DL Tansmittance Spectum, A-7 A SP Tansmittance Spectum, A-7 A BP Tansmittance Spectum, A-7 A LP Tansmittance Spectum, A-7 A DL Tansmittance Spectum, A-8 A BP Tansmittance Spectum, A-8 -xi

12 CONTENTS TABLES 4.1 Sot Modes, 4-7 A.1 Filtes, A-3 -xii

13 INTRODUCTION This intoductoy section contains the following topics: How to use you COULTER EPICS ALTRA flow cytomete manuals About the Refeence 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 REFERENCE MANUAL You ALTRA flow cytomete Refeence manual is a souce of infomation on what the system does. This infomation is oganized as follows: s s Chapte 1, Use and Function Contains the intended use of the instument, the eagents used and a shot desciption of the majo components and options. Chapte 2, Installation Contains the instument equiements and instuctions how to install the instument. -xiii

14 INTRODUCTION CONVENTIONS s s s s s Chapte 3, Opeation Pinciples Contains a desciption of flow cytomety, the nomal sample flow though the instument, how light collection and signal pocessing ae accomplished and how paametes ae deived. Chapte 4, Specifications/Chaacteistics Details the instument and pefomance specifications and the pefomance chaacteistics and lists the available options. Chapte 5, Pecautions and Hazads Contains safety infomation including lase safety and label location. Appendix A Povides efeence mateial on optical filtes. Index Use the index to easily locate specific infomation in this manual. 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). 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. -xiv

15 1USE AND FUNCTION INTENDED USE The COULTER EPICS ALTRA flow cytomete and ALTRA HyPeSot System (HPSS) (Figue 1.1) ae geneal use laboatoy instuments. They ae intended, by design, to be vesatile scientific instuments capable of a divese ange of applications using a wide vaiety of eagents and eagent systems. Execise cae wheneve the instument is configued fo use with a paticula eagent o settings have been made to collect specific infomation. Whee thee ae no published potocols, o the use of a eagent system with the ALTRA flow cytomete is being established, the entie system must be validated to assue that the system configuation is coect and the data geneated is consistent with the expectations of the application. Most pocedues and infomation in the poduct manuals apply to all ALTRA flow cytometes. Infomation that applies only to ALTRA HyPeSot System (HPSS) flow cytometes contains HPSS in the pocedue s name. 1.2 SYSTEM FUNCTION The system measues fluoescence and lase light scatteed fom cells o othe micoscopic paticles passing though a lase beam. Fowad light scatte coelates with paticle size. Side (90 ) light scatte coelates with the ganulaity o intenal complexity of a cell. Fluoescence intensity gives the affinity of the sample fo cetain dyes, o the inheent fluoescence of the sample paticles. Samples fo the system must be single cell o paticle suspensions. The system measues up to 15,000 cells pe second. Fo light scatte measuements, cell diamete can ange fom 0.5 to 40 µm. Fo smalle paticles, thee is a Fowad Fluoescence Detecto option. See Heading 4.3, SYSTEM OPTIONS fo a desciption of this option. Fo fluoescence measuements, the sample can be macomolecula o as lage as 40 µm in diamete. Fo lage paticles, thee is a Lage Paticle Soting Option. See Heading 4.3, SYSTEM OPTIONS fo a desciption of this option. The system coelates and stoes seveal measuements of cell chaacteistics, and gaphically displays the distibution of those chaacteistics within the sample. The system can sepaate two cell o paticle subsets based on the measued chaacteistics. This is called cell soting. 1.3 SYSTEM DESCRIPTION Most functions on the system ae unde the contol of a compute. You must pepae the sample fo analysis and instuct the compute how to pefom the desied test. The compute then acquies data and analyzes it as instucted by the test potocol. The system does not equie knowledge of compute pogamming to ceate a potocol. Opeation is by easy-to-undestand menus. You select a pepogammed potocol o define you own by selecting menu options at the FlowCente Multimedia Wokstation. 1-1

16 USE AND FUNCTION SENSOR MODULE Main Assemblies The system compises the following assemblies and subassemblies: FlowCente Multimedia Wokstation CPU Colo monito Keyboad Mouse Cytomete Powe/lase switches Pneumatics cabinet Sheath/waste compatment Electonic pedestals Senso module: Sample intoduction system, Sot collection aea, HPSS contol panel (optional), Sample/fluidics stage contol panel, Detecto and sensing compatment, Touch sceen, Gaphics monito Figue 1.1 ALTRA HPSS Flow Cytomete Cytomete Touch sceen Gaphics monito (Scope sceen) Powe/Lase switches Sample/Fluidics stage contol panel Detecto and sensing compatment Sample aea FlowCente multimedia wokstation C HyPeSot System contol panel (Optional) Electonic pedestals Sot collection aea Sheath/Waste compatment Pneumatics cabinet 1.4 SENSOR MODULE The senso module (Figue 1.2) contains the: Sample intoduction system HPSS contol panel (optional) Sot collection aea. Detecto and Sensing compatments Gaphics (Scope sceen) and Touch sceen monitos 1-2

17 USE AND FUNCTION SENSOR MODULE 1 Figue 1.2 Senso Module HPSS contol panel (Optional) Detecto and sensing compatment Touch sceen monito Gaphics monito (Scope sceen) Sot collection aea Sample intoduction system C Sample Intoduction System The sample stage takes samples fo analysis in 12- x 75-mm, 12- x 76-mm, and 17- x 100-mm test tubes. Sample pickup is by positive pessue. Sample flow is guided into the sheath steam at the flow chambe. Sheath fluid comes fom a containe in the sheath/waste compatment. The sheath is pessuized fom an intenal compesso; the HPSS uses an extenal 100-psi compesso. A bank of contols to the left of the sot collection aea povides manual contol of sheath and sample flow. Fo HPSS Cytometes, additional contols ae available on the HPSS contol panel. HPSS Contol Panel Sheath and Sample pessue anges ae selected in this aea. The ange value displayed hee is added to the value selected on the Contol Values sceen to detemine the nominal opeating pessue. The actual Sheath, Sample, and Diffeential pessues can be selected fo display hee. Indicatos fo the sheath and waste tank levels and sample doo status ae also on this contol panel. Sot Collection Aea The sot collection aea contains the deflection plates fo soting, the waste dain, slide holdes and test tube holdes. The Autoclone soting option is also accessed though this aea. Detecto and Sensing Compatments The detecto compatment is next to the sensing compatment. The compatment contains: Five fluoescence detectos; detectos ae photomultiplie tubes (PMTs). The side-scatte detecto; it can also be used as a fluoescence detecto. 1-3

18 USE AND FUNCTION PNEUMATICS CABINET 12 filte slots; fo insetion of filtes and dichoic mios that measue diffeent colos of fluoescence. The collection lenses and filtes. Imaging Optics assembly, optional. The sensing compatment contains: SotSense flow cell/body Beam-shaping lens assembly Fowad-scatte detecto Collection lenses fo fluoescence and side scatte Viewing optics fo the video camea. The SotSense flow cell/body intoduces the sample into the sheath steam and makes sample cells flow in single file. This flow passes though the focused lase beam. The beam-shaping assembly focuses the lase light to illuminate the sample. Afte illumination, the sample can be soted o collected in a waste dain located in the sot collection aea. Gaphics Monito The gaphics monito (scope sceen) has a 720 x 348 esolution fo high-esolution gaphics. The sceen acts like an oscilloscope to display eithe two signal pulses o fou scattegams. The sceen is also used to display the camea view fo system maintenance and soting. Touch Sceen Monito The touch sceen monito is 80 chaactes by 25 lines. The touch sceen is used to display and change instument settings. You can also display and change many settings at the FlowCente Multimedia Wokstation. To use the touch sceen, just point at a menu option. An aay of infaed beams cisscoss the touch sceen. Detectos aound the edge of the sceen locate whee you finge inteupts the beams, activating the option you selected. 1.5 PNEUMATICS CABINET The pneumatics cabinet is on the lowe ight, below the cytomete. It contains: The Solenoid valve manifold Sheath, inse and waste containes Pessue egulatos. The compesso is extenal. 1.6 ELECTRONIC PEDESTALS Two electonic pedestals suppot the optical table and senso module. These contain all soting and data acquisition electonics, powe supplies and the cytomete compute. 1-4

19 USE AND FUNCTION OPTICAL TABLE OPTICAL TABLE Duing nomal opeation you ae neve exposed to lase adiation. Fo you potection, intelocks block the lase beam when coves ae emoved that might expose the lase. You can potect the lase fom unauthoized use by a keyswitch. The lases ae mounted on a section of industy-standad optical table (with M6 theaded holes on 25-mm centes) fo inceased stability and vesatility. Seven diffeent lases ae available fo the ALTRA flow cytomete. See Heading 4.3, SYSTEM OPTIONS fo a desciption of the lases. Figue 1.3 shows a typical ai-cooled lase system. An ai-cooled agon lase that opeates fom a common 110 Vac single-phase powe outlet. An ai-cooled 10-mW HeNe lase that opeates fom a common 110 Vac single-phase powe outlet. Figue 1.3 Lase Aangement HeNe (Lase 2) Agon (Lase 1) Lase beams Top view C 1.8 FLOWCENTRE MULTIMEDIA WORKSTATION The FlowCente Multimedia Wokstation has the following featues: Micosoft Windows 98 and MS-DOS opeating systems ALTRA flow cytomete softwae EXPO softwae (optional) Data acquisition Data display Data stoage Data analysis Data management 1-5

20 USE AND FUNCTION REAGENTS AND QUALITY CONTROL (QC) MATERIALS Potocol stoage and definition Cytomete contol Cell soting setup. Disk Dives The disk dive section contains: 13-GB EIDE had disk 3.5-in MB dive 650-MB CD-ROM. Keyboad Use the IBM PC/AT compatible keyboad fo system input and contol. Mouse Use the mouse fo gaphics input and menu selection. Colo Monito and Gaphics Cad The FlowCente Multimedia Wokstation has a colo monito (15 in., 18 in., o 21 in.) and an SVGA-compatible gaphics cad with built-in micophone and speakes. 1.9 REAGENTS AND QUALITY CONTROL (QC) MATERIALS Sheath Fluid Use IsoFlow sheath fluid o a physiological saline solution filteed to 0.2 µm fo the sheath fluid. The sheath fluid should be nonfluoescent, paticle fee, tanspaent to the lase light, and compatible with the chaacteistics of the sample that is being measued (such as size o viability). The sheath fluid must not cause cells to swell o shink. The efactive index of the sheath fluid must diffe fom that of the sample paticles fo good light scatte measuements. Cleaning Agent Use a cleaning agent duing daily shutdown to flush the sample tubing and pevent potein buildup. Beckman Coulte ecommends that you use COULTER CLENZ cleaning agent. QC Mateials Which QC mateials you use depends on what chaacteistics you ae measuing. Some of the cell and fluoescent QC mateials available fom Beckman Coulte ae: Flow-Check optical alignment fluoosphees EPICS IMMUNO-BRITE fluoescent lineaity fluoosphees Flow-Set fluoosphees fo fluoescence and light scatte standadization CYTO-COMP Reagents and CYTO-COMP Cells to set fluoescence compensation COULTER IMMUNO-TROL Cells o CYTO-TROL Contol Cells to assess monoclonal antibody eactivity and to monito instument pefomance. 1-6

21 USE AND FUNCTION MATERIAL SAFETY DATA SHEETS (MSDS) MATERIAL SAFETY DATA SHEETS (MSDS) To obtain an MSDS fo Beckman Coulte eagents used on the ALTRA flow cytomete: 1. In the USA, eithe call Beckman Coulte Custome Opeations ( ) o wite to: Coulte Copoation A Beckman Coulte Company Attn: MSDS Requests P.O. Box Miami, FL Outside the USA, contact you local Beckman Coulte Repesentative. 1-7

22 USE AND FUNCTION MATERIAL SAFETY DATA SHEETS (MSDS) 1-8

23 2INSTALLATION GENERAL Each ALTRA flow cytomete system is tested befoe shipping. Intenational symbols and special handling instuctions ae pinted on the shipping caton to infom the caie of the pecautions and cae applicable to electonic instuments. CAUTION Risk of instument damage. Impopely uncating the system by anyone othe than a Beckman Coulte epesentative may damage the instument. Do NOT uncate the system. You Beckman Coulte Repesentative is esponsible fo uncating, installing and initially setting it up. When you eceive you system, caefully inspect all catons. If you see signs of mishandling o damage, file a claim with the caie immediately. If sepaately insued, file the claim with you insuance company. 2.2 SPECIAL REQUIREMENTS Access Limitation Because the system contains a lase, isolate it fom nonlase instuments. Classify the aea as a contolled zone, and limit admittance to authoized pesons. Keep a copy of ANSI standad Z136.1, SAFE USE OF LASERS, nea the instument fo efeence. Copies ae available fom: Ameican National Standads Institute (ANSI) 1430 Boadway New Yok, NY Decontamination Be caeful when woking with pathogenic mateials. Means must be available to decontaminate the instument, ventilating ai and waste liquid. Refe to the following fo futhe guidance on decontamination. Biohazads Safety Guide, National Institute of Health. Classifications of Etiologic Agents on the Basis of Hazads, Centes fo Disease Contol, U.S. Public Health Sevice. See the Shutdown Chapte in the ALTRA flow cytomete Opeato's Guide fo futhe infomation on decontamination. Accessibility Fo opening sevice access doos, allow at least 46 cm (18 in.) behind and 46 cm (18 in.) on the ight side of the instument; and allow 76.2 cm (30 in.) above the instument. Allow at least 25 cm (10 in.) behind the FlowCente Multimedia Wokstation fo cable cleaance. 2-1

24 INSTALLATION SPECIAL REQUIREMENTS Heat Dissipation 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) and 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. Ambient Tempeatue and Humidity Keep oom tempeatue between 18 and 29 C (64 and 85 F). Keep the humidity between 5 and 80%, without condensation. Electical Powe You system has one of these two powe configuations: A 100/115 Vac, 20 A (16 A continuous), 50/60 Hz dedicated line to powe the cytomete, FlowCente Multimedia Wokstation and HeNe lase. Plus, a 100/115 Vac, 20 A, 50/60 Hz dedicated line to powe the ai-cooled agon lase. A 220/240 Vac, 10 A, 50/60 Hz dedicated line to powe the cytomete, FlowCente Multimedia Wokstation and HeNe lase. Plus, a 220/240 Vac, 10 A, 50/60 Hz dedicated line to powe the ai-cooled agon lase. In both configuations, the compute FlowCente Multimedia Wokstation and monito must use the powe outlets on the cytomete. The safety gound path must be capable of the full cuent of the cicuit (confimed thid-wie eath gound). The pinte equies anothe sepaate powe outlet and must be odeed accoding to you county's paticula powe supplies. EMC/EMI Compliance The ALTRA flow cytomete is tested to comply with applicable standads and caies a CE mak to signify compliance with these standads. 2-2

25 INSTALLATION CALIBRATION OF Autoclone SORTING OPTION AFTER INSTALLATION CALIBRATION OF Autoclone SORTING OPTION AFTER INSTALLATION If you system has the Autoclone soting option, you must pefom this abbeviated calibation pocedue afte installation o einstallation of the ALTRA softwae: 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. DANGER 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. 2-3

26 INSTALLATION CALIBRATION OF Autoclone SORTING OPTION AFTER INSTALLATION 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 2-4

27 INSTALLATION CALIBRATION OF Autoclone SORTING OPTION AFTER INSTALLATION 2 8 Follow the instuctions on the Retact sceen and pess: OK 9 Replace the Autoclone cove. 2-5

28 LASER RADIATION WHE N OPEN AND INTERLOCK DEFEATED AVOID EYE OR SKIN EXPOSURE TO DIRECT O R SCATTERED RADIATION UT LT INSTALLATION SYSTEM CONNECTIONS 2.4 SYSTEM CONNECTIONS The only connections extenal to the unit ae the two powe cods fom the ea pedestal and a powe connecto fo an extenal compesso and pessue/vacuum inputs. The FlowCente Multimedia Wokstation and cytomete cable connections ae shown in Figue 2.1. Figue 2.1 Powe and Cable Connections Monito Sound cable to monito Mouse PEDESTAL PWR CAMERA SERVICE VIDEO OUT SERIAL COMM DATA LISTER OUT A Compute Keyboad B UT LT PEDESTAL PWR SERVICE CAMERA VIDEO OUT SERIAL COMM DATA LISTER OUT A To second monito (optional) To netwok LANtastic (optional) To pinte (optional) To modem connection (optional) C To colo pinte (optional) B 2-6

29 3OPERATION PRINCIPLES SYSTEM OVERVIEW The system measues fluoescence and light scatte of cells on a cell-by-cell basis. The coelated measuements ae pesented in histogams. With the pope eagents and sample pepaation, you can analyze these histogams fo a pofile of the sample chaacteistics. You can sot two classes of paticles on the basis of these chaacteistics. Flow cytometic measuement and soting can be divided into the following pocesses. Fo accuate measuement, the flow cytomete: Pesents the sample, one cell at a time, to the sensos and lase beams. Regulates and shapes the lase beams. Collects and sepaates the fluoescence and scatteed lase light the cell emits when passing though the lase beam. Convets the light to electic pulses with voltages popotional to the intensity of the light. The electic pulses must be amplified and pocessed. Convets the peak voltage of the pocessed pulse to a popotional channel numbe fo a histogam o listmode data. Conditions the steam fo soting and makes sot decisions on the basis of the measuements. 3.2 SAMPLE PRESENTATION To analyze each cell of a sample suspension flowing though a lase beam, the cells must be sepaated fom each othe and the lase beam must be small enough that two cells cannot be in the beam at the same time. Fo this eason the lase beams ae focused to a vey naow coss section. The steam of sample paticles flows though the lase beams at a ight angle to the beams. To sepaate the cells, this steam is also focused using hydodynamic focusing. Hydodynamic Focusing Sheath fluid flows though a consticting nozzle (flow cell). Sample suspension is injected into the middle of the sheath though a small tube (sample insetion od). The flow of sample suspension and sheath fluid though the flow cell is lamina and the fluids do not mix. The sample steam (sample coe) is suounded by the sheath steam as the fluids flow though the flow cell. Flow Cell The flow cell is mounted on the optical table. At the top is the sample insetion tube. A pot on the flow cell supplies sheath fluid. The uppe pat of the flow cell is conical, naowing to a squae, 250-µm quatz channel (76 µm high-velocity quatz o 150 µm with the HPSS option) at the sensing aea. A lens is mounted on the flow cell fo light collection; see Figue 3.1. See Figue 3.2 fo the HPSS flow cell and body. Afte passing though the sensing aea, the fluids exit though a 76-µm o 100-µm oifice (70-µm oifice with the 70-µm 3-1

30 OPERATION PRINCIPLES SAMPLE PRESENTATION HyPeSotSense flow cell, o diectly fom the 76-µm channel of the 76-mm HyPeSotSense flow cell, o 76-µm oifice with the high-velocity quatz fo the HPSS) Figue 3.1 Standad Flow Cell and Body Peak tubing. Figue 3.2 HPSS Flow Cell and Body Peak tubing Peak adapte Peak adapte Sample insetion od Feule Sample insetion od Feule Sheath Vacuum Sheath Vacuum 0-ing 0-ing B Lens Sheath steam B Lens Sheath steam Sheath Velocity Sheath velocity is influenced by seveal factos including sheath pessue and size of flow cell tip oifice. Fo standad sample analysis/soting, the velocity in the 250-µm channel is 2 m/second. Fo the HPSS, the velocity in the sensing aea is 10 to 28 m/second. Sample Coe As the flow cell naows, the steam diametes decease and the steam velocities incease. Resolution and sensitivity patly depend on the velocity and diamete of the sample coe. Diamete Changing the diamete of the sample coe changes the esolution. Because the intensity of lase light is less at the edges of the beam and geate at the cente, all cells should follow the same path though the cente of the lase beam fo the best sensitivity and esolution. The numbe of possible paths of a cell though the lase beam is limited by the diamete of the sample coe. The best esolution is achieved with a small coe diamete. Velocity Changing the velocity changes the sensitivity. The longe a cell stays in the lase beam the moe lase light it eceives. With slowe velocities the cell stays in the lase beam longe; scatteed lase light and fluoescence fom the cell incease. The best sensitivity is achieved with a low velocity. 3-2

31 OPERATION PRINCIPLES LASER BEAMS 3 Sheath Pessue Sheath fluid is contained in a 5.0-L tank. Pessue is deliveed to the bottle fom an intenal compesso (extenal pessue fo HPSS). The pessue foces sheath fluid up a pobe at the bottom of the sheath tank and though tubing to the flow cell. Sheath pessue contols the velocity and diamete of the sample coe. The sheath pessue default setting is 12.0 psi (fo HPSS, a maximum of 100 psi). The velocity of sheath fluid though the flow cell depends on the pessue diffeence acoss the flow cell. The exit side is at atmospheic pessue so the velocity of the sheath fluid though the flow cell depends on the sheath pessue. The velocity of the sample coe within the sheath fluid is slightly highe than the sheath velocity. The sheath pessue setting along with the fequency setting influence the sot efficiency and ecovey, and the stability and sensitivity of the soting esults. High sheath pessue and fequency settings esult in the best soting efficiency and ecovey but the sensitivity and stability ae educed. Low sheath pessue and fequency settings esult in the best sensitivity and stability but the sot efficiency and ecovey ae educed. Use the fequencies and sheath pessue settings ecommended fo the flow cell you ae using as listed in Chapte 7, SORTING, in the Opeato s Guide and then adjust these settings accoding to the needs of you laboatoy. Sample Delivey Rate Pessue applied to the sample tube contols sample delivey. Inceasing the pessue inceases the sample delivey ate. The diffeence between the sheath and sample pessues contol the diamete of the sample coe. Keeping the sample pessue constant, you can incease the sheath pessue to incease the velocity and decease the sample coe diamete and sample delivey ate. This helps the esolution of the measuement but deceases sensitivity and analysis ate. If you keep inceasing the sheath pessue, the sample flow could evese, and sheath flows back to the sample tube. Keeping the sheath pessue constant, you can incease the sample pessue to incease the sample coe diamete and sample delivey ate. This inceases the analysis ate at the expense of esolution. Sensitivity and esolution also depend on the shape and intensity of the lase beam, as explained in Heading 3.3, LASER BEAMS. 3.3 LASER BEAMS To illuminate the cells passing though the sensing aea and measue thei fluoescence and light scatte, a unique light souce is needed. The diection, wavelength, and intensity of the light must be as constant as possible. Only a lase poduces light of this quality. Lase Pinciples Lase stands fo Light Amplification by Stimulated Emission of Radiation. When an atom absobs enegy, it is pushed into a highe enegy state; its electons ae put in highe obitals. To go to a moe enegetically favoed state, electons go to lowe obitals. The enegy diffeence between lowe and highe obitals is emitted as a photon of light. This is called 3-3

32 OPERATION PRINCIPLES LASER BEAMS spontaneous emission. Since thee ae only a few diffeent obital tansitions fo an atom of a paticula substance, it only emits a few diffeent wavelengths of light. The atoms in the lases ae excited to highe enegy states to poduce spontaneous emission. Mios at eithe end of the lase eflect the light fom spontaneous emission back and foth though the lasing substance. When a photon of light passes nea an excited atom, the atom emits a photon of light of the exact same wavelength, polaization, phase, and diection as the incident photon. This is called stimulated emission. As the light bounces back and foth in the lase, a lase beam foms between the mios. To get a lase beam of a single wavelength, a pism is inseted in the light path befoe the ea mio. The pism selects the wavelength eflected. To get a lase beam out of the lase, the font mio only eflects back 98% of the light to maintain stimulated emission. The emaining 2% is the lase beam out. A cuent though the lase contols the numbe of excited atoms and thus contols the intensity of the lase beam. A senso at the font of the lase samples the beam and adjusts the cuent up o down to maintain a constant intensity. Beam Shaping The beams fom the ai-cooled lases ae about 700 µm in diamete (about 1300 µm fo the HeCd lase). The beams ae focused to incease the intensity and to keep fom illuminating moe than one cell at a time. The smalle the initial beam size, the hade it becomes to focus the beam to a small spot. Bette esolution is achieved if all the cells eceive the same amount of light as they pass though the lase beam, so the intensity of light acoss the diamete of the sample steam is made as unifom as possible. The system customizes the beam using coss-cylindical lenses (Figue 3.3). 3-4

33 OPERATION PRINCIPLES LASER BEAMS 3 Figue 3.3 Coss Cylindical Beam-Shaping FLOW CELL CROSS SECTION AFTER HORIZONTAL LENS CROSS SECTION AFTER VERTICAL LENS CROSS SECTION BEFORE LENSES HORIZONTAL LENS LASER BEAM DIRECTION LASER BEAM VERTICAL LENS Afte the lenses, the beams ae elliptical in shape. The beams ae wide in compaison to thei height to minimize the vaiation in intensity acoss the cente of the beams. Even if cells do not follow the same exact path though a beam, they eceive the same amount of light. Beam Shaping Lenses The font lens element (neaest the flow cell) is hoizontal and detemines the height of the lase beam (paallel to the flow). The vetical ea lens element detemines the width of the lase beam (othogonal to the flow). See Figue 3.4. Each beam shape is designed to be eithe confocal o defocused. In a confocal assembly, the lenses ae fixed so that they have coincident focal points. In a defocused assembly, the spacing between the lenses is delibeately educed. By offsetting the focal point of the ea lens, the beam width at the flow cell is inceased. Beam width diectly affects sensitivity and esolution. Beam height detemines peak signal pulse width. Fo doublet discimination, cell size must be at least half the beam height. 3-5

34 OPERATION PRINCIPLES LASER BEAMS Figue 3.4 Beam Shapes FRONT LENS LENGTH REAR LENS Y Z X LENS HOLDER 15 X 60 CONFOCAL 15 X 80 CONFOCAL 15 X ACTUAL SIZE 15 X X X 80 CONFOCAL B 10 X The cylindical lenses ae fabicated fom UV gade fused silica having an extended woking spectum fom 200 to >800 nm. All optical sufaces have had dielectic antieflection coatings to minimize scatteing and distotion of the lase beams. Beam-Shape Selection Chat You can use the following chat to select the most appopiate beam shape fo you needs. 3-6

35 OPERATION PRINCIPLES LASER BEAMS 3 Lase Type You Pefe to Wok With Comments Small Ai-Cooled Lage Wate-cooled Highest sensitivity Low sample flow ates High sensitivity and esolution High sample data ates Highest esolution High sample flow ates Closest match to XL optics Doublet discimination 5-μm diamete cells High sensitivity Sense-in-quatz flow cell Resolution can be lost due to inceasing CVs as sample flow ate inceases. Spot size is elliptical, but still somewhat intense. Thee is a balance between sensitivity and esolution because of this. Best choice fo high thoughput. Sensitivity is somewhat deceased. Good fo doublet discimination. Easie to align than 8 x 80. Moe difficult to align. Test fo you application. Doublet discimination fo 5-μm diamete cells. Good choice fo applications involving combinations with small lases. Vey high sensitivity. High esolution, high sample thoughput, moe difficult to align. Test fo you application. Recommended fo HPSS. Beam-Shaping Assemblies The chat below summaizes the key featues of the available ange of beam shapes. Beam Shape 15 x 60 confocal 15 x 80 confocal 15 x 80 Δ x 80 Δ 15 8x80 Δ x 80 Δ 6 Sense-in-ai flow cell Results should be simila to a jet-in-ai system. 40 x 80 confocal Focal Lengths mm (Font/Rea) Focal Point Offset mm D Fo Use with Lase Type Fo Use with Flow Cell Type Spot Size μm (Height/Width) Good fo 5 μm Cells Status 15/60 Confocal Small Sense-in-Quatz 12/51 No Standad 15/80 Confocal Small Sense-in-Quatz 16/85 No Optional 15/80 15 Small Sense-in-Quatz 12/151 No Standad 10/80 15 Small Sense-in-Quatz 8/151 Yes Optional 8/80 15 Small Sense-in-Quatz 6.5/151 Yes Standad 15/80 6 Lage Sense-in-Quatz 6/112 Yes Standad 40/80 Confocal Lage Jet-in-Ai 17/34 No Standad Spot sizes ae based on a wavelength of 488 of nm and a small o lage lase as shown. Appoximate spot dimensions fo othe lase wavelengths and specific lases ae pesented on the next page. Use of the beam expande inceases o deceases the beam spot dimensions by a facto of 3, depending on its oientation. 3-7

36 OPERATION PRINCIPLES LASER BEAMS Diffaction Limited Spot Size The chat below shows appoximate woking beam spot dimensions obtained with vaious combinations of lase and focusing lens. Font Lens Focal Length mm Rea Lens Focal Length, mm (and defocus distance mm) Lase To use the chat: Identify the line of the chat fo the lase and wavelength you ae inteested in. Read-off the beam height and width fom the appopiate columns fo the focal length of the lenses in the beam shape you ae using. Fo example: Wavelength nm Beam Diam. mm Beam Height µm 80 (5.8) Beam Width μm Lase: Coheent 305, Wavelength 488 nm, Beam shape 8 x 80 mm (defocused 5.8 mm) gives: Beam height of 3.4 µm and beam width of 112 µm. Beam-Steeing Optics Beam-steeing optics ae used when the ALTRA flow cytomete is configued with multiple lases. They allow beams to be combined o pecisely sepaated at the flow cell. Dichoic mios, designed to pass and/o eflect specific wavebands, fom the optical component of the steeing assemblies. A single beam steeing assembly (Figue 3.5) is included with the ALTRA flow cytomete when it is configued with the Melles Giot HeNe lase and the Cyonics agon lase. The 80 (15.1) Ai-Cooled Lases Cyonics Agon Melles Giot Red HeNe Melles Giot Geen HeNe Omnichome HeCd Wate-Cooled Lases Coheent Innova Seies Coheent Innova Spectum Coheent Entepise II Seies

37 OPERATION PRINCIPLES SENSING AREA nm beam of the HeNe is eflected by the dichoic towad the flow cell while the 488-nm beam passes diectly though it. By adjusting the beam steeing assembly, you can make the ed HeNe lase coincident with, o spatially sepaated fom, the blue Agon beam at the flow cell. Figue 3.5 Beam Steeing Assembly Vetical otation knob Hoizontal otation knob Height setscew Fine adjustment knob Fine adjustment setscew Rail setscew A 3.4 SENSING AREA Afte beam shaping, the lase beams pass though the sensing aea of the flow cell. The sensing aea is a long hollow chambe, squae in coss section, with quatz sides tanspaent to the lase light. Cells pass though the middle of the lase beams in this chambe. As the cells pass though the lase beams, they scatte the lase light and emit fluoescent light fom fluoescent dyes attached to the cell. Scatte The amount of lase light scatteed at naow angles to the axis of the lase beam is oughly popotional to the size of the cell. The lase light scatteed at ight angles elates to the ganulaity o complexity and is affected by suface iegulaities. Fluoescence The amount of fluoescence fom the cell allows measuement of some aspect of the cell depending on the dyes and eagents used. Fo example, monoclonal antibodies conjugated to a dye show the amount of a paticula antigen on the cell. The dye popidium iodide attaches to the DNA in a cell and shows the amount of DNA. Fluoescent dyes absob light at one wavelength and convet the high enegy light (shote wavelength) into lowe enegy light (longe wavelength). Each dye has unique excitation and 3-9

38 OPERATION PRINCIPLES SENSING AREA emission specta; that is, a ange of light wavelengths that when absobed, poduces a ange of emitted light (fluoescence). Using two o thee dyes that have nealy the same excitation specta, but diffeent emission specta, you can use one lase to excite them to measue seveal chaacteistics of the cell at the same time (multicolo fluoescence). Using both lases on the system, you expand the ange of dyes you can use because you now have two anges of excitation specta. Light Collection To measue the light, the system must collect the light, sepaate the colos of inteest, and pesent the light to the appopiate sensos. Thee light collection systems view the sensing aea: the video camea, the fluoescence lenses, and the fowad scatte senso (see Figue 3.6). Figue 3.6 Optical System Lase 1 Filte block Lase 2 Dichoic lase mio PMT 4 PMT 3 PMT 2 PMT 1 Fluoescence lenses Beam shaping lenses Flow cell Imaging optics (optional) LED LAMP Camea pism PMT 5 Camea Fowad light senso A Video Camea The video camea lets you see the sensing aea fo system maintenance and fo setting up and monitoing cell soting. The camea view appeas on the gaphics monito (scope sceen) of the cytomete. The video camea views the flow-cell tip and the exiting sheath steam. A filte in the video camea blocks most of the lase light. A light souce in the fluoescence detecto assembly pojects an image on the flow cell. The elliptical lase spot and the image fom the light souce can be seen on the gaphics monito (scope sceen). The lase steam intesection is a fluoescent spot in the middle of the flow-cell tip. Making adjustments to align the two spots lets you adjust the elative positions of the flow-cell tip, lase beam, and lens systems with cetainty. 3-10

39 OPERATION PRINCIPLES SENSING AREA 3 Fowad Scatte Detecto Lase light scatteed between 1.4 and 19 fom the axis of the lase beam is collected by the fowad scatte lens and passed to photovoltaic cells. See Figue 3.7. The fowad scatte light is so bight that a neutal density filte must be between the lens and the photovoltaic cells to educe the intensity. A ba on the FALS MASK in font of the detecto blocks the lase beam itself. Each FALS MASK is stamped with the application it is designed fo. Figue 3.7 Fowad Scatte Detecto LENS FALS MASK CAMERA PRISM OBSCURATION BAR A SotSense Flow Cell Tip Thee ae fou majo components to the SotSense Flow Cell tip, Figue 3.8: A theaded housing fo attachment to the flow-cell body A quatz flow chambe with a 250-µm squae flow channel A 100-µm diamete jetting oifice A magnifying lens. The magnifying lens is mounted diectly to the flow cell, eliminating the ai gap and dispensing with the need fo an optical gel. This lens foms the pimay element of the fluoescence collection optics and is optically matched to the main assembly. 3-11

40 OPERATION PRINCIPLES SENSING AREA Figue 3.8 SotSense Flow Cell Tip Sample intoduction od Sotsense flow cell tip (Cut away view) Housing Magnifying lens Flow chambe Jetting oifice C Fluoescence Lenses All lenses ae pecision gound and polished. Exposed optical sufaces have antieflective coatings. Togethe, these maximize signal collection by educing abeations and light scatte losses. The assembly is colo-coected to enhance pefomance ove the entie ange of inteest, fom UV though the full visible spectum ( nm). An optimized spatial filte is integal to the assembly. It inceases the signal-to-noise atio by blocking light that has not come fom the intesection point of the lase and the sample. Light passing to the detectos is collimated fo pecise angle of incidence at beam splittes (45 ) and bandpass filtes (90 ). This pevents boadening o shifting of the analyzed waveband. A pecision focusing mechanism is built into the assembly. It is conveniently positioned and equies only fingetip foce to opeate. The mechanism has zeo backlash and is self-locking. Optical Filtes Optical filtes sepaate the light fom the cell by wavelength. The pinciples of optical filteing ae explained in Appendix A, OPTICAL FILTERS. The filtes ae chaacteized by thei tansmission specta, a gaph of the pecentage of light tansmitted vs. wavelength (also supplied in Appendix A). Filtes can be placed in the filte slots shown in Figue 3.9. You can set up a numbe of diffeent filte configuations to measue diffeent combinations of dyes. Figue 3.10 shows an example of how one filte configuation splits and diects light fom diffeent fluoescent dyes to paticula PMTs. The signals poduced fom each senso ae also indicated in Figue