BIO RAD PharosFX 170-9450, PharosFX 170-9460, PharosFX 170-7890, PharosFX 170-7892 Hardware Instruction Manual

0 (0)

Hard ware Instruction Man ual

for Catalog Numbers

170-9450 PharosFX System (532 nm) 170-9460 PharosFX Plus System (532 nm) 170-7890 External Laser (488 nm)

170-7892 External Laser (488 and 635 nm)

For Technical Service, Call Your Local Bio-Rad Office or, in the US, Call 1-800-4BIORAD (1-800-424-6723)

This instrument is for Laboratory Use Only

PharosFX

™

Molecular Imager®System

Welcome

Dear Customer, On behalf of Bio-Rad Laboratories, we would like to thank you for investing in the Molecular

Imager PharosFX Product Family. This combination of systems allows you the highest level of flexibility for the best in isotopic and fluorescence imaging and we are sure that it will provide you with many years of high quality imaging.

One of the best ways to familiarize yourself with the capabilities of your new PharosFX system is to read this manual. In it, you will learn how to set up the system and operate all hardware components. It is also recommended that you read the accompanying software manual, to familiarize yourself with general acquisition functions and data analysis. After reading this manual, please keep it close to your PharosFX system so that it can be conveniently referred to.

Your PharosFX system is protected by a comprehensive instrument warranty agreement. Please read this warranty (Appendix 3) thoroughly, so that you fully understand the coverage it provides and are aware of your rights and responsibilities. One of the responsibilities of system ownership is regular maintenance. Following the maintenance instructions provided with this manual will help to keep your system and peripherals functioning optimally and will protect your investment. Please also keep in mind that Bio-Rad offers a range of comprehensive service agreements that can be tailored to meet your specific needs.

Bio-Rad Laboratories is dedicated to your total satisfaction and would be pleased to answer any questions or concerns that you may have.

How to Contact Bio-Rad Laboratories

In the United States you can reach Bio-Rad Laboratories at the following numbers: For general information Toll free: 1-800- 4BIORAD

1-800-424-6723 Fax: 1-510-741-5802 email: lsg.techserv.us@bio-rad.com

For service or technical assistance Toll free: 1-800-424-6723 Fax: 1-800-741-5802

Outside the United States contact your local Bio-Rad Laboratories office. For information concerning Bio-Rad Laboratories and its products, visit our Worldwide Web

site at http://www.bio-rad.com.

Table of Contents

Section 1 General Information......................................................................1

1.1 About this Manual......................................................................................1

1.2 Safe t y I nf o r m at i o n......................................................................................1

1.2.1 General Cautions...........................................................................1

1.2.2 General Warnings..........................................................................2

1.2.3 Power Safety Information...............................................................2

1.2.4 Laser Safety Information ................................................................3

1.2.5 Screen Eraser Safety Information...................................................4

Section 2 Introduction...................................................................................4

2.1 System Capabilities ...................................................................................4

2.2 System Description....................................................................................4

2.3 Theo r y o f O p e r at i o n...................................................................................8

2.3.1 Fluorescence Detection Mechanism...............................................8

2.3.2 Storage Phosphor Detection Mechanism .......................................9

2.3.3 Data Processing and Analysis........................................................9

2.4 Overview of the Imaging Process.............................................................10

2.4.1 Steps in Fluorescence Imaging ....................................................10

2.4.2 Steps in Storage Phosphor Imaging.............................................10

Section 3 System Installation......................................................................11

3.1 Operating Requirements..........................................................................11

3.1.1 System Location...........................................................................11

3.1.2 AC Power Requirements..............................................................12

3.1.3 Host Computer Recommendations...............................................13

3.2 System Setup..........................................................................................13

3.2.1 Shipping Check............................................................................13

3.2.2 Unpacking....................................................................................13

3.2.3 Electrical and Communication Connections..................................15

3.2.4 Software Installation.....................................................................16

Section 4 System Operation ......................................................................16

4.1 Starting the Scanner................................................................................16

4.2 Fluorescence Operating Procedures........................................................17

4.2.1 Sample Preparation .....................................................................17

4.2.2 Inserting a Fluorescent Sample into the Scanner .........................18

4.2.3 Scanning Fluorescent Samples....................................................19

4.2.4 Inserting Emission Filters .............................................................20

4.3 Fluorescence Imaging Using the External Laser Module..........................22

4.3.1 External Laser Safety Information ................................................23

4.3.2 Fiber Optic Cable Alignment.........................................................23

4.4 Phosphor Imaging Operating Procedures................................................24

4.4.1 How to Prepare K-Type Imaging Screens ....................................24

4.4.2 How to Erase Imaging Screens....................................................24

4.4.3 How to Prepare Samples .............................................................25

4.4.4 How to Use the Exposure Cassette..............................................27

4.4.5 How to Scan the Imaging Screen.................................................28

Section 5 Care and Maintenance................................................................30

5.1 Scanner Maintenance..............................................................................30

5.2 Care for PharosFX Plus Accessories.......................................................30

5.2.1 General Care of Imaging Screens................................................30

5.2.2 Radioactive Contamination Check................................................30

5.2.3 Cleaning Imaging Screens ...........................................................30

5.2.4 Storage of Imaging Screens.........................................................31

5.3 Exposure Cassette and Platform Maintenance ........................................31

5.4 Screen Eraser Maintenance.....................................................................31

5.4.1 Changing Bulbs............................................................................31

Section 6 Troubleshooting..........................................................................33

6.1 Factors Affecting Image Quality...............................................................33

6.2 Problem Solving Guide............................................................................34

Appendix 1System Specifications ................................................................35

Appendix 2Warranty Information..................................................................36

Appendix 3Ordering Information...................................................................37

Section 1 General Information

1.1 About this Manual

This manual provides instructions for installing, operating and maintaining the PharosFX system. This manual uses certain conventions to facilitate understanding of the text material and to assist operators in using the PharosFX system.

Notes, Cautions and Warnings

Notes, cautions and warnings are used to highlight certain operating procedures and recommendations.

A note indicates a special procedure, an exception to normal operation or something else of specific interest to the reader. Notes are preceded by the word “Note” in italics.

A caution precedes an operational step that could damage the instrument or destroy data unless the operator takes certain precautions. Cautions are located in the main text, are preceded by a Caution: statement and are accompanied by a “Caution Symbol” in the left margin.

A warning precedes an operating procedure that could cause injury to the operator if not followed correctly. Warnings are located in the main text, are preceded by a Warning: statement and are accompanied by a "Warning Symbol" in the left margin.

1.2 Safety Information

Your safety and the safety of others is very important to us. To help you make informed decisions about safety, we have provided comprehensive operating procedures and safety information in this manual and on labels affixed to instrumentation. This information will alert you to any potential hazards.

1.2.1 General Cautions Caution: Always install the scan head locking screw before moving the PharosFX and

avoid subjecting the PharosFX system to vibration. (See section 3.2.2) Caution: After transport, always remove the scan head locking screw before supplying

power to the PharosFX scanner. (See section 3.2.2) Caution: Other than emission filter wheel access port, do not remove instrument covers.

There are no user-serviceable parts inside. Refer all servicing to qualified Bio-Rad personnel or their agents. If you experience technical difficulties with the instrument, contact Bio-Rad to schedule a service appointment.

Caution: The instrument should not be modified or altered in any way other than moving or changing emission filters. Alteration of this instrument voids the manufacturer's warranty and may create a potential safety hazard for the user.

Caution: Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than that for which it is intended or by the modification of this instrument when not performed by qualified Bio-Rad personnel or an authorized agent.

1.2.2 General Warnings Warning: There are hazardous voltages inside the PharosFX scanner. Do not attempt to

defeat the door interlock or remove the instrument's cover. These are designed to prevent user injury.

1.2.3 Power Safety Information

The PharosFX system is designed and certified to meet both I.E.C. 61010 safety standards and Center for Devices, Radiological Health (CDRH) laser safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This safety certification does not extend to uncertified equipment or accessories, even when connected to the PharosFX system.

This instrument and its accessories should not be altered or modified in any way. Alteration of the PharosFX or its accessories will void the manufacturer's warranty, void the I.E.C. 61010 and CDRH certification, and create a potential safety hazard for the user.

Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not performed by Bio-Rad or an authorized agent.

Figure 1.1 shows two serial number certification labels. These are found at the rear of the PharosFX system and the rear of the External Laser. This label provides manufacturing data about the instrument, its voltage settings and CDRH compliance information.

Fig. 1.1.a. Instrument serial number information on the rear of the PharosFX Scanner.

Note: For easy customer access, serial number information for the PharosFX scanner is

also located on the right hand side of the overlay located behind the scanner door.

Fig. 1.1.b. Instrument serial number information on the rear of the External Laser.

1.2.4 Laser Safety Information

This instrument and its accessories are certified according to 21 CFR 1040 of the CDRH, as a Class I laser device and IEC / EN 60825-1+A1+A2 as a class 1 laser device. (Figure 1.1.a).

The laser contained within the PharosFX and PharossFX Plus scanning unit is configured with a laser that generates energy up to 15 milliwatts at 532 nm. The cover of each scanner has redundant interlocks and is designed to protect the user.

The optional External Laser Module contains an Argon-Ion laser that produces laser energy of up to 10 milliwatts at 488 nm. This External Laser Module can also be outfitted with an additional laser diode that produces laser energy of up to 10 milliwatts at 635 nm. The cover of this system is designed to protect the user at all times.

Warning: Do not remove the cover for any reason or defeat the interlock. Attempting to operate the unit with the cover removed may damage the instrument and expose the operator to energy from the laser.

Warning: Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous laser energy exposure.

Caution: The top cover should be removed by trained service personnel only. Do not attempt to operate the product with the cover removed. The PharosFX system should be serviced only by Bio-Rad or its trained representatives.

Laser warning labels (Figure 1.2) are located externally on the top cover and rear of the instrument and internally on the top surface of the mounting plate, at the rear right-hand corner and the top of the laser module.

Fig. 1.2. Laser warning label.

1.2.5 Screen Eraser Safety Information Warning: The Screen Eraser must be plugged into a grounded electrical outlet.

Section 2 Introduction

2.1 PharosFX System Capabilities

The PharosFX Product Family provides options for imaging and analyzing the following sample types:

Fluorescence (for PharosFX, and PharosFX-Plus Models)

The PharosFX detects almost any visibly excited fluorescent dye. The internal and optional external lasers allow optimal excitation of single-color or multi-color fluorescent samples. Computer controlled filter wheels with 6 available emission filter positions allow detection of many multi-color fluorescent signals. The system is also capable of performing sequential detection of multiple fluorescent dyes on the same gel or blot.

Colorimetric Detection

A Transilluminating Screen, made of white plastic, is provided with the sample tray for PharosFX and PharosFX Plus for documentation of gels stained with colorimetric stains, such as Coomassie and Silver Stain.

Radioisotope Emissions (for PharosFX Plus Only)

Detects a broad range of isotopes, including

P, 33P, 35S, 14C, and 3H. The PharosFX uses storage phosphor screen technology that is at least ten times more sensitive to isotopic emission than x-ray film. The system is compatible with most available phosphor imaging screens based on the BaFBr:Eu storage phosphor chemistries

2.2 System Description

Fig. 2.1. A Typical PharosFX scanner and peripherals.

The PharosFX (Plus) imaging system consists of:

• Laser scanner

• Sample Tray set, including: glass sample tray, transilluminating screen, and sample holders

• Pre-installed filters for fluorescence detection: 640BP, 605BP, blank filter holder for custom filters. PharosFX Plus has an additional 390BP filter for phosphor screens

• External Laser Module is optional

(1) Laser Scanner

The PharosFX and PharosFX Plus have an internal laser that emits light at 532 nm only. All PharosFX units can scan at resolutions of 50, 100, 200, and 800 microns and have a linear dynamic range that extends over 4.8 orders of magnitude (1:65536). In contrast, x-ray film has a linear dynamic range that is limited to only 1.5 orders. The scanner also contains fully automated emission filter wheels with 6 filters. These combined features permit the PharosFX to image almost any fluorescent dye and to scan multi-color fluorescence applications, in addition to storage phosphor imaging with the PharosFX Plus.

(2) Sample Tray

The glass Sample Tray is used as a scanning platform for fluorescent gels or blots and K-type phosphor screens. Its spill resistant design exhibits very low background fluorescence and high chemical resistance.

Additional Multi-Sample trays can be purchased to accommodate thick agarose gels, gels within glass plates (1707819 Multi-Sample Tray II), metal backed storage phosphor screens,and microtiter plates (1707812 Multi-Sample Tray).

Fig. 2.2. a) Multi-Sample Tray, b) Multi-Sample Tray II

(3) Optional External Laser Module

The optional external laser module provides one or more additional excitation source(s). The current configurations for lasers are:

a) 488 nm Only b) 488 nm and 635 nm

Fig. 2.3. External Laser Module

(4) Storage Phosphor Imaging Screens

Bio-Rad offers a range of storage phosphor screens to match different user requirements. Table 2.1 summarizes the key features of each screen. The screens are composed of a barium fluorobromide matrix doped with europium (BaFBr:Eu); they can be used with traditional autoradiography cassettes without a darkroom, are easy to handle and are used solely for the detection of isotopic emissions.

PharosFX Plus offers a variety of phosphor storage applications in addition to all the functionality of the PharosFX. All phosphor screens are reusable and unharmed by repeated exposure to radioactivity. Screens are sensitive to β particles and X-rays. All screens are flexible and easy to handle. Exposure takes place in standard X-ray cassettes. All phosphor screens require erasure prior to re-exposure, and their lifetime is extended when they are cared for properly.

Imaging Screen-K

This is a general-purpose screen designed for all common radioisotopic emitters, such as

P, 33P, 35S, and 14C. These screens are available in 35 x 43 cm and 20 x 25 cm formats.

Screens are guaranteed for 1 year.

Imaging Screen-K/Tritium

This is a special imaging screen available for imaging

H. These screens require special care and handling and are reusable only if cared for properly. Screens are 20 x 25 cm and are covered by a 6-month warranty.

Table 2.1. Imaging Screens Specifications and Recommended Applications

(5) Sample Exposure Cassettes

The sample exposure cassettes ensures that a close contact is made between the sample and imaging screen. The cassettes contain a grid marked exposure area, to which the sample is mounted. This allows the sample to be firmly pressed against the imaging screen to generate a high quality image.

Fig. 2.4. Sample exposure cassette and phosphor screen

(6) Screen Eraser

The screen eraser removes any residual signal or background from the imaging screen. The complete erasure process "zeros" or "blanks" the screen to a basal level, which is critical for maximizing sensitivity, linear response, quantitative accuracy and image quality. The Screen Eraser-K is used with the K-type screens and any other commercially available phosphor screens which are based on the BaFBr:Eu chemistry (Fig. 2.5).

Screen

Name

maging

creen

maging

creen

/Tritium

Application Key Features Sizes

32P,33

•

BaFBr:Eu formulation

•

Easy-to-use format

•

Compatible with standard X-ray cassettes

•

More durable

•

BaFBr:Eu formulation

•

Sensitive to weak signal

•

Easy-to-use format Compatible with standard X-ray cassettes

(cm)

35 x 43

mounted

20 x 25

mounted

20 x 25 170-7845

Catalog Number

170-7841 170-7842

170-7843 170-7844

Fig. 2.5. Screen Eraser.

(7) Control and Analysis Software

Quantity One software is included with the PharosFX and PharosFX Plus systems. Quantity One permits user-friendly, application driven control of the scanning system and accurate 1-D analysis of the captured image data.

2.3 Theory of Operation

2.3.1 Fluorescence Detection Mechanism

The fluorescence process occurs when a molecule absorbs light of a certain wavelength and excites electrons to a transient higher energy state (Figure 2.6, step 1). When the electrons return to ground state, energy is released in the form of photon emission at a wavelength which is longer than the illumination source (step 2). For example, ethidium bromide (EtBr) absorbs light of 532 nm and emits light at 595 nm. Using optical filters, the emission wavelength can be separated from the excitation wavelength and detected using a photomultiplier tube.

Fig. 2.6. The fluorescence detection mechanism.

2.3.2 Storage Phosphorescence Detection Mechanism

When a radioactive emission strikes the phosphor screen, phosphor oxidation occurs and a high-energy site is formed (Figure 2.3, step 1). When such an activated site is subsequently illuminated with certain wavelengths of visible light (step 2), the reduction reaction occurs. Trapped energy is released as photons that are in turn captured by a photomultiplier tube (step 3).

Fig. 2.7. The storage phosphor detection mechanism.

Note: Storage phosphor screens are reusable after erasure.

2.3.3 Data Processing and Analysis

Phosphorescence and fluorescence signals are captured as a 16-bit digital file. This file can then be analyzed and manipulated by the appropriate image analysis software for visualization and quantitation.

Traditionally the image is displayed in a two-dimensional format, where the darkness of each pixel is proportional to the signal intensity at that sample location (Figure 2.8, left). For the purpose of image analysis however, it is helpful and more accurate to think of the data as a three-dimensional structure, where the signal intensity at each pixel becomes the height or z-axis dimension (Figure 2.8, right). Sample spots or bands can also be visualized as peaks in a profile analysis along the length of a gel lane or perceived as topographic volumes when quantitating the total signal from a specific band or spot.

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