PharosFX™ Molecular Imager® System Hardware Instruction Manual for Catalog Numbers 170-9450 PharosFX System (532 nm) 170-9460 PharosFX Plus System (532 nm) 170-7890 170-7892 External Laser (488 nm) External Laser (488 and 635 nm) This instrument is for Laboratory Use Only Copyright 2005 Bio-Rad Laboratories Inc.
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.
Table of Contents Section 1 General Information ......................................................................1 1.1 1.2 About this Manual ......................................................................................1 Safety Information......................................................................................1 1.2.1 General Cautions ...........................................................................1 1.2.2 General Warnings ..............................................
Section 5 Care and Maintenance ................................................................30 5.1 5.2 Scanner Maintenance ..............................................................................30 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 .
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.
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.
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.
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.
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.
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.
Table 2.1.
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).
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.
Single Pixel Intensity 2-D view 3-D vie w Fig. 2.8. Two and three-dimensional representations of a digitized image. 2.4 Overview of the Imaging Process 2.4.1 Steps in Fluorescence Imaging The procedure for imaging fluorescent samples has four steps. Step 1 involves thoroughly cleaning the sample tray with ethanol, using a clean soft cloth or lint free paper towel. Step 2 involves placement of the sample onto the tray.
Upon completion of the four-step process, the storage phosphor screen can be erased and the cycle repeated with a new sample. Fig.2.9. Phosphor Imaging work flow Section 3 System Installation 3.1 Operating Requirements 3.1.1 System Location The PharosFX scanners and all peripherals should be located in an area that is free of excessive dust or moisture, strong magnetic fields or ionizing radiation.
External Laser Module The External Laser Module has been designed for flexible placement. It can be located either to the left or right of the scanner, or placed directly below the scanner in an under-bench position. The maximum distance between the external laser and the scanner is 2 meters, as determined by the optical fiber that connects the two instruments. Once the External Laser has been installed and aligned it is not recommended to move it around to prevent any possible optical misalignment.
3.1.3 Host Computer Recommendations The scanner is capable of producing large image files of high resolution; these can be up to 120 megabytes in size. To easily manipulate such large files, a powerful computer is required. The host computer MUST meet or exceed the specifications as detailed below. Table 3.
Unpacking the Laser Scanner and Tray Note: Explicit packing/unpacking instructions are located in a clear document envelope attached on the outside of the shipping container. Refer to these instructions at all times prior to unpacking or packing. Unpack the laser scanner by following the steps below: 1. Cut the metal strap on the instrument packaging 2. Slide the cardboard cover off vertically 3. Remove the front and rear packaging 4. Grip the bottom of the scanner on both sides and place on the bench top.
1. 2. 3. 4. 5. Fig. 3.1. Steps for unlocking the scan head. Unpacking the Imaging Screens K-type imaging screens are shipped in a sealed cardboard box. The phosphor matrix is protected by a sheet of paper that must be removed prior to erasure and use. Retain all packaging for screen storage. 3.2.3 Electrical and Communication Connections Power Insert the power cord into the power entry module on the rear panel of the scanner.
USB2 Connection The laser scanner must be connected to the host computer via a USB2 interface (Figure 3.2). The USB2 link ports are located on the right-hand side of the rear of the scanner unit. The appropriate USB2 cable is included with the scanner. Fig. 3.2. View of USB2 connection 3.2.4 Quantity One Software Installation Please refer to your software instruction manual for comprehensive software installation procedures. Section 4 Operating the PharosFX System 4.
Fig. 4.1. PharosFX LCD and control panel. An LCD "Contrast" button is located on the control panel (Figure 4.1). This button can be used to adjust the brightness of the LCD display. The contrast function cycles so that holding the button down will cause the display to get lighter and then darker again. The "On Line" button on the control panel should be depressed only in the unlikely event that the acquisition software cannot halt scanner operation. 4.2 Fluorescence Detection 4.2.
Table 4.1 Sample Preparation and Accessories Accessory Sample Tray 170-7811 Use for • Agarose Gels • Acrylamide Gels • Blots/Membranes • Colorimetric Gel Documentation • Unmounted Storage Phosphor Screens Preparation Notes • No gels thicker than 8 mm. • Gels should be wet. • Blots/Membranes should be moist. • Use Sample Holders (170-7813) to keep sample from moving during scan. • When performing Colorimetric Gel Documentation work, utilize the transilluminating screen supplied.
5. Slowly insert the tray with the sample all the way into the scanner, until the hold latch engages and you hear a click. 6. Close the front door of the scanner. 7. The sample can now be scanned using the acquisition software. 8. To remove the tray, simply wait until scanning is complete, open the scanner door and pull the tray out. Note: For wet samples, use a clean, lint-free absorbent paper towel to remove excess liquid from around the sample.
4.2.4 Inserting Additional Excitation and Emission Filters PharosFX and PharosFX Plus are supplied pre-configured with emission filters that support most common fluorescence applications and as such, users should not routinely have to access the filter areas. If users wish to install additional filters and perform custom fluorescence experiments this can be easily done as the emission filter wheels can be accessed through the port in the scanner's front panel (Figure 4.3). Fig. 4.3.
3. Depress and release the filter wheel advance switch until the desired emission filter location is presented. Filter cubes are identified by a wavelength and catalog number label, which should be clearly visible. 4. If a filter cube is to be removed from the slot do so by gently pulling the cube towards you 5. To install a new filter, hold the cube with the mounting slots on the left hand side.
The standard emission filter configurations are as follows: Table 4.
Fig. 4.7. PharosFX External Laser Module connected to the scanner 4.3.1 External Laser Safety Information The External Laser Module (Figure 4.7) is certified according to 21 CFR 1040 of the CDRH, as a Class I laser device. The laser contained within the External Laser Module produces 10 milliwatts at 488 nm. This laser can also be upgraded to include a 635 nm laser producing up to 10 milliwatts at 635 nm. The cover of the system is designed to protect the user and should not be removed.
4.4 Phosphor Imaging Procedures 4.4.1 How to Prepare K-Type Imaging Screens Fig. 4.8. K-type imaging screens. All K-type imaging screens (K, K-HD, K/Tritium) are not erased prior to shipment from the factory and should be erased for 20 minutes prior to first use. Subsequent erasures should only take 10 minutes. Note: Optimal image quality and sensitivity can only be achieved with a thoroughly erased screen.
To erase the imaging screens, place the imaging screen against the front panel of the Eraser-K with the white phosphor side facing the white diffuser plate of the unit. Set the timer to the desired setting or to erase continuously set the timer to the HOLD position. The erasure process typically requires only 10 minutes, assuming the previous sample has not charged the phosphor in the screen above 20,000 pixel density units. An erasing time guideline is shown in Table 4.3.
Fig. 4.10. Imaging Cassette. A protective sheet should be placed between the sample and the screen to minimize the chance of radioactive contamination (Figure 4.11). Fig. 11. Use of protective sheet in Imaging Cassette. Wet Radioactive Samples Precautions must be taken to prevent wet samples from contaminating the imaging screen. Wet thin samples should be completely enclosed in a heat-sealable bag and moist samples must be covered with plastic wrap before being exposed to the imaging screen.
Plastic wraps will attenuate weak beta radiation signals according to the following table. Table 4.4 Attenuation of radiation by various plastic wraps. Wrap Type 2 mm Seal-A-Meal Bag 0.5 mm Saran Wrap 32 P 16% 6% 14 C 82% 50% 3 H >99.9% 99.7% Application Wet samples Moist samples Note: When enclosing a sample in plastic wrap or a heat-sealable bag, make sure that there are no large air bubbles or surface wrinkles on the side of the sample that will come into contact with the screen.
4. Place the Imaging Screen-K directly on top the sample with the phosphor (white) side facing down onto the sample. It is recommended that the sample is at least 1 cm from all edges of the screen to prevent possible edge effects. 5. Make a note of the sample's upper-left and lower-right grid coordinates for later use in selecting the scan area. 6. Close the cassette and press until the release button catches and a snap sound is heard. 4.4.
Fig. 4.13. Steps for inserting a phosphor screen into the scanner. Scanning the Radioisotopic Samples 1. Open the scan window in the acquisition software. Under the File menu, choose PharosFX Plus. In the acquisition window choose Select and use the layered menu to identify your application. (i.e. for radioisotopes using the Imaging Screen-K, select Radioisotopes/ K-Screen). 2. Choose the scan area that you would like to image. 3. Select the desired resolution setting.
Section 5 Care and Maintenance 5.1 Scanner Maintenance With regular use the PharosFX scanner should provide years of trouble-free operation, without the need for operator maintenance. If you suspect that the PharosFX system requires servicing, please contact your local Bio-Rad office. It is recommended that the casing of the scanner be periodically inspected to verify that no panels are loose or distorted. ! Caution: Do not remove the cover from the PharosFX system, as this voids the warranty.
! Caution: Do not use powdered detergents to clean the screen as undissolved particles may damage the screens coating. Cleaning Protocol for Imaging Screen-K and Imaging Screen-HD 1. Handle the screen only by the edges. Avoid touching the screen coating with anything sharp, such as fingernails. The screen is coated with a thin layer of plastic and scratching this protective layer could damage the screen, making that area unusable. 2.
2. Turn off the eraser. 3. Wait five minutes for the bulbs to cool. 4. Unplug the power cable. 5. Open the four hex screws on the front of the eraser, using the hex tool stored on the rear panel of the unit. 6. Remove the white plastic filter. 7. Remove the foam on both sides and replace the defective bulb. 8. Reassemble by reversing steps 4 to 7. Step 6. Step 7a Step 7b Fig. 5. Screen Eraser bulb replacement steps. Caution: Use only 15 W bulbs.
Section 6 Troubleshooting 6.1 Factors Affecting Image Quality Fluorescent samples Resolution Any movement of the sample during scanning can cause a blurred image. Be sure that excess liquid around gel samples is removed and that the imager is level. Sample holders are supplied with the glass sample tray. These have suction feet that allow attachment to the glass and will hold samples in place. Sample height will affect the resolution of the image.
Sensitivity Optimal sensitivity can only be achieved with a thoroughly erased screen. Close contact of the sample with the screen will also impact the imaging sensitivity. Ensure that the sample is pressed close to the screen surface. 6.
Appendix 1 PharosFX System Specifications System Technical Specifications Specification Linear dynamic range Pixel resolution Image resolution Pixel density Signal decay Scanning area 1:65535 50, 100, 200 or 800 µm, selectable 2 line pairs/mm or 250 µm 16-bit (0–65,535) 32 P 50% retention in 24 hr 35 x 43 cm Main Component Specifications Laser Scanner Specification Dimensions Construction Excitation source Weight Electrical Maximum power Input voltage range Fuses Operating Requirements Storage Require
Appendix 2 PharosFX Warranty Information The FX system and all peripheral items are warranted for a period of one year against defects in materials and workmanship. If any defects should occur during this period, Bio-Rad Laboratories will either replace or repair the defective parts free of charge. For the exact terms of warranty, please see the Instrument Warranty Card shipped with the instrument. Defects caused by the following actions are specifically excluded: 1. Improper system operation or abuse. 2.
Appendix 3 Ordering Information Accessories Excitation Sources Catalog # Description 170-7890 External Laser, 488 nm, includes 170-9459 filter 170-7893 635 nm External Laser Upgrade for 170-7890, includes 170-7865 filter 170-7892 External Lasers, 488 nm and 635 nm, includes 170-9459 and 170-7865 filters Filters 170-9459 Filter 530nm BP, for ECL Plus, AttoPhos, SYBR Green, Alexa Fluor 488, FITC, Cy2, and Pro-Q Emerald 170-7863 Filter 555LP, applications similar to 170-7866 and 170-7896 filters 17
Catalog # Description Software 170-9600 The Discovery Series™ Quantity One 1-D Analysis Software 170-9620 PDQuest 2-D Gel Analysis Software Basic License 170-9630 PDQuest 2-D Gel Analysis Software Advanced License Miscellaneous 931-0071 3 meter USB cable 161-0722 Bio-Rad Cleaning Concentrate 165-3414 Gel Clip 38
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