DynaPro® Plate Reader™ II User’s Guide

M3101 Rev C Copyright © 2014, Wyatt Technology Corporation

WYATT TECHNOLOGY Corp., makes no warranties, either express or implied, regarding this instrument, computer software package, its merchantability or its fitness for any particular purpose. The software is provided “as is,” without warranty of any kind. Furthermore, Wyatt Technology does not warrant, guarantee, or make any representations regarding the use, or the results of the use, of the software or written materials in terms of correctness, accuracy, reliability, currentness, or otherwise. The exclusion of implied warranties is not permitted by some states, so the above exclusion may not apply to you. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of Wyatt Technology Corporation. ® DynaPro, DYNAMICS, Wyatt Technology, and its logo are registered trademarks of the Wyatt Technology Corporation. ™ Plate Reader and Protein Solutions are trademarks of Wyatt Technology Corporation. A variety of U.S. and foreign patents have been issued and/or are pending on various aspects of the apparatus and methodology implemented by this instrumentation. Table of Contents

Chapter 1: Introduction Overview ...... 6 The Instrument ...... 6 DynaPro Plate Reader II Operations ...... 7 The Software ...... 7 How the Manual Is Organized ...... 8 Contacting Wyatt Technology ...... 9 Where to Go from Here ...... 9 Chapter 2: Safety, Warnings, and Cautions Laser Specifications and Safety Notes ...... 11 Chapter 3: Installation and Setup Unpacking the Instrument ...... 13 Installing DYNAMICS Software ...... 13 System Requirements ...... 13 User Accounts with Restricted Privileges ...... 14 CD Contents ...... 14 Installing the Software ...... 14 Installing the DynaPro Plate Reader II ...... 15 Nitrogen Connection ...... 16 Chapter 4: Components Front Panel View ...... 18 Back Panel View ...... 19 Top Panel View ...... 20 Laser ...... 21 Laser Beam Warning ...... 21 Laser Enable Standby Time ...... 21 Collecting and Viewing Well Images ...... 22 Chapter 5: Using the Display Window Using the Display Tabs ...... 24 Main Tab ...... 26 Selecting Display Settings for the X, Y Axes ...... 26 Adjusting the Display Range ...... 27 Setting Temperature ...... 28 ACF Tab ...... 30 Alarm Tab ...... 32 System Tab ...... 34 Camera Tab ...... 36 Comm Tab ...... 37

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 3 Contents

Chapter 6: Using the Plate Reader Introduction ...... 39 Instrument Warm-up ...... 39 Choosing the Correct Well Plate and Well Plate Insert ...... 39 Well Plate Insert Installation ...... 40 Bar Code Labels ...... 42 Preparing Samples ...... 42 Filtering Samples ...... 42 Filtering Options ...... 43 Centrifuging Samples ...... 43 Loading the Well Plate ...... 44 Loading the sample manually: ...... 44 Loading Well Plates Automatically ...... 45 Loading the Plate Holder ...... 46 Controlling the Plate Reader from DYNAMICS ...... 47 Plate Reader Control Buttons ...... 47 Well Labeling Conventions Used By Dynamics ...... 48 Measuring Automatically ...... 51 Frequently Asked Questions ...... 52 Chapter 7: Hardware Maintenance General Maintenance ...... 56 Replacing the Fuses ...... 57 DynaPro Plate Reader II Firmware Upgrades ...... 57 Appendix A: Sources for Well Plates Preferred Well Plates ...... 58 Compatible Well Plates ...... 59 Appendix B: Technical Specifications Appendix C: Troubleshooting Low Count Rates Appendix D: Connecting to a Network or PC Components ...... 64 Instrument connections: ...... 64 LAN connection: ...... 64 Computer connections: ...... 65 Crossover cable: ...... 65 Ethernet cable: ...... 66 Ethernet to USB adapter: ...... 66 Ethernet switch: ...... 67 Connecting to a LAN ...... 68 Instrument and Computer to LAN: ...... 68 Connecting via USB ...... 69 Connecting via Ethernet when not on a LAN...... 71 Instrument to computer not on LAN using an Ethernet switch: ...... 72 Instrument Network Settings ...... 73 Accessing instruments with DYNAMICS ...... 74 Troubleshooting and diagnostics ...... 74 Verifying instrument connections ...... 74

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 4 1 Introduction

When you can measure what you are speaking about, and express it in numbers, you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meager and unsatisfactory kind: it may be the beginning of knowledge, but you have scarcely, in your thoughts, advanced to the stage of science.

—Lord Kelvin William Thomson Kelvin, the 19th century physicist and mathematician who wrote that paragraph, would have been very comfortable with the DynaPro Plate Reader II and software. The DynaPro Plate Reader II offers an unparalleled level of automation in dynamic light scattering. Designed to meet the demanding needs of labs requiring speed, ease-of-use, and sophisticated software; low sample volumes (as low as 4 μL per well) and high sensitivity are achieved with our proprietary optical and system designs. The DynaPro Plate Reader II system can determine the size (hydrodynamic radius) and size distribution of particles, polymers, and biopolymers from 0.5 nm to 1000 nm. With temperature control of the samples (Temperature Controlled version), the flexibility, versatility, and ease of measuring replicates of the DynaPro Plate Reader II instruments make them exceptional measuring systems. Read on to learn more about the DynaPro Plate Reader II line of dynamic light scattering instruments.

CONTENTS PAGE OVERVIEW ...... 6 THE INSTRUMENT...... 6 THE SOFTWARE ...... 7 HOW THE MANUAL IS ORGANIZED...... 8 CONTACTING WYATT TECHNOLOGY...... 9 WHERE TO GO FROM HERE...... 9

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 5 Chapter 1: Introduction

Overview

The DynaPro® Plate Reader™ User’s Guide describes how to set up and use the DynaPro Plate Reader II. Please see the DYNAMICS User’s Guide for details on data analysis. The following is a brief description of the hardware and software that makes up the DynaPro Plate Reader II.

Figure 1-1: DynaPro Plate Reader II

The Instrument The DynaPro Plate Reader II is a dynamic light scattering instrument that can be used for a broad range of applications requiring both accuracy and high sensitivity. For example, the DynaPro Plate Reader II is ideally suited for studies of nanoparticles, proteins, vesicles, viruses, and colloids. The DynaPro Plate Reader II is straightforward to operate. Knowledge of the underlying physics of molecular sizing is not essential. The block diagram below summarizes the basic operation of the DynaPro Plate Reader II.

Incident laser light PC with DYNAMICS ht lig Sample in red for Data Analysis atte Well Plate ° sc 158

Correlator SPCM

Figure 1-2: DynaPro Plate Reader II Block Diagram

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 6 Chapter 1: Introduction

DynaPro Plate Reader II Operations Samples are pipetted into a well plate, and the well plate is placed into the Plate Reader instrument. After the well plate is placed into the Plate Reader and the door is closed, the sample is illuminated by a semiconductor laser with a wavelength of approximately 830 nm. The light scattered by the sample is collected and guided via a fiber optic cable to an actively quenched, solid state Single Photon Counting Module (SPCM), which contains an Avalanche Photodiode (APD). The photons are then converted to electrical pulses which are sent to a multi-tau hardware correlator. The DynaPro Plate Reader II analyzes the time scale of the scattered light intensity fluctuations by a mathematical process called auto correlation. To perform the very fast data manipulation necessary to obtain results in real time, the DynaPro Plate Reader II uses the latest generation of correlator running multiple tau algorithms. The translational diffusion coefficient (Dt) of the molecules in the sample is determined from the decay of the intensity auto correlation data. The hydrodynamic radius (Rh) of the sample is then derived from Dt using the Stokes-Einstein equation,

k T R = ------b h πη 6 Dt

where: kb is the Boltzmann constant, T is the temperature in degrees K, and η is the solvent viscosity.

The Software DYNAMICS for Windows is data collection and analysis software for the DynaPro molecular sizing system. Tuned specifically for batch and automated characterization of unfractionated molecular samples, DYNAMICS applies sophisticated analysis tools to the dynamic light scattering (DLS) data from the DynaPro Plate Reader II to give information on molecular sizes and sample polydispersity. The DYNAMICS software gives researchers the tools to quickly assess important sample properties without relying on chromatography or other fractionation techniques. Please refer to the DYNAMICS User’s Guide for specific information about the software and details on dynamic light scattering theory and data analysis.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 7 Chapter 1: Introduction

How the Manual Is Organized The chapters and appendices in this manual are organized as follows: Chapter 1, “Introduction” introduces the DynaPro Plate Reader II and this manual, and describes the support options available from Wyatt Technology. Chapter 2, “Safety, Warnings, and Cautions” describes the use of warning symbols in this manual and on the instrument. Chapter 3, “Installation and Setup” takes you through the necessary first steps for unpacking, connecting, and testing the instrument. Chapter 4, “Components” takes you on a guided tour of the instrument. Chapter 5, “Using the Display Window” shows you how to navigate and change settings in the DynaPro Plate Reader II Display Window or from a remote terminal. Chapter 6, “Using the Plate Reader” shows you how to load samples and record data using the DynaPro Plate Reader II. Chapter 7, “Hardware Maintenance” has procedures for keeping the instrument in good working order, and includes a procedure for aligning the optics block. Appendix A, “Sources for Well Plates” provides information about specific well plates that have been characterized by Wyatt Technology. Appendix B, “Technical Specifications” provides information for installation and operation of the DynaPro Plate Reader II. Appendix C, “Troubleshooting Low Count Rates” provides instructions for diagnosing failure modes that result in lower than expected count results. Appendix D, “Connecting to a Network or PC” covers connecting the DynaPro Plate Reader II to either a network through the ethernet, or to a host PC through the ethernet-to-USB converter.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 8 Chapter 1: Introduction

Contacting Wyatt Technology

We solicit and encourage questions and comments about this manual and the DynaPro Plate Reader II instrument. Please contact: Wyatt Technology Corporation 6300 Hollister Ave. Santa Barbara, CA, 93117 Telephone: (805) 681-9009 FAX: (805) 681-0123 E-mail: [email protected] If you have a question about your DynaPro Plate Reader II, first look in this manual or consult the online help that comes with DYNAMICS for Windows. If you cannot find an answer, please contact Wyatt Technology Technical Support at [email protected].

Where to Go from Here

Read Chapter 2, “Safety, Warnings, and Cautions” to ensure the safety of your lab and personnel, then continue to Chapter 3, “Installation and Setup” to check out your shipment and make some necessary initial checks and adjustments.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 9 2 Safety, Warnings, and Cautions

We have used special formatting along with messages to especially alert users to potential hazards involved in this technology, following this standard:

You will see this Caution to indicate that failure to follow this message, Caution! may result in damage to equipment.

Note: You will see this Note to indicate additional information which may be of interest.

For best results and user safety, the following warnings and cautions should always be followed when handling and operating your DynaPro Plate Reader II.

DANGER! DO NOT USE WITH FLAMMABLE SOLVENTS! Using volatile solvents in the Temperature Controlled DynaPro Plate Reader II can create deadly vapors, fire, and explosions.

DANGER! The power supply provided with your DynaPro Plate Reader II is designed to convert high voltage to the level required for operation. Do not open the power supply or defeat any of its safety interlocks. Serious injury or Death may result.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 10 Chapter 2: Safety, Warnings, and Cautions

DANGER! The DynaPro Plate Reader II has no user serviceable parts. For your safety, do not dismantle the Plate Reader. Do not bypass any of the safety systems and interlocks that are in place for your health. If the instrument is not functioning properly, do not apply power.

Caution! The DynaPro Plate Reader II components can reach temperatures of 85 °C, hot enough to cause serious burns. Wear heat resistant gloves and exercise extreme caution when installing or removing well plates.

Laser Specifications and Safety Notes

The lasers used in the DynaPro Plate Reader II are Class IIIb lasers. However the DynaPro Plate Reader II itself is classified as a Class 1 Laser Product according to IEC60825-1:1993+A1+A2 and CFR Title 21 Subchapter J. Note these environmental specifications apply to the laser subsystem and not to the instrument as a whole. This means that under normal operation, no laser radiation should escape from the instrument, and no protective equipment must be worn. However the following warning applies:

Use of controls or adjustment or performance of Caution! procedures other than specified herein may result in hazardous radiation exposure.

Note: All safety labels are in English. If you need safety labels in a language other than English, please contact Wyatt Technology.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 11 3 Installation and Setup

This chapter helps you get the DynaPro Plate Reader II unpacked, tested, and connected. You will also make some first time adjustments.

CONTENTS PAGE Unpacking the Instrument ...... 13 Installing DYNAMICS Software...... 13 Installing the DynaPro Plate Reader II ...... 15 Nitrogen Connection ...... 16

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 12 Chapter 3: Installation and Setup

Unpacking the Instrument

Please read the shipping parts list (packing slip) included with your instrument shipment and check that everything arrived in good condition. 1. Carefully examine the shipping container. If it is damaged or shows signs of mishandling, contact Wyatt Technology immediately. 2. Unpack the instrument. 3. Place the DynaPro Plate Reader II on a level surface and inspect the instrument for damage. If you see any damage, contact Wyatt Technol- ogy immediately. 4. Check that the boxes contain all of the items listed as included with your instrument shipment in addition to the instrument (the packing slip sent with the instrument contains the most up-to-date list).

Installing DYNAMICS Software

DYNAMICS is light scattering instrument control software for molecular research. It is used with the DynaPro Plate Reader II and other Wyatt Technology instruments. DYNAMICS software not only collects and stores data, it assists you with data interpretation. It also frees you to perform other tasks by automating routine and user-defined experiments. You can read more about DYNAMICS in your DYNAMICS User’s Guide.

System Requirements As of the date of publication of this manual (March 2014), the minimum system resources DYNAMICS requires are listed below. For current DYNAMICS system requirements please refer to our website; http://www.wyatt.com/DYNAMICS. • Either 32 or 64-bit editions of Windows 7 or Windows Vista (including Business, Enterprise, or Ultimate Editions), or Windows XP Professional 32-bit Edition • Computer with an Intel Core 2 Duo processor • 2 GHz or better processor speed • 4 GB of RAM or better • At least 1 GB of available disk space, and at least 100 MB of available hard-disk space for application and documentation • CD-ROM Drive (optional for installation) • Ethernet connection to a network or USB 2.0 port for instrument communications • Display with 1280 x 768 resolution or higher • Support for DirectX 9 graphics with 128 MB of graphics memory

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 13 Chapter 3: Installation and Setup

User Accounts with Restricted Privileges If DYNAMICS is to be run from a user account with restricted privileges, it is necessary to install DYNAMICS under the account to be used. If DYNAMICS is installed globally, you must have Windows Power User privileges to run DYNAMICS.

CD Contents Your installation CD includes the following items for using the DynaPro Plate Reader II. • DYNAMICS software files (required for operating the DynaPro Plate Reader II) • PDF file of the DYNAMICS User’s Guide • PDF file of the DynaPro Plate Reader II User’s Guide for your hardware configuration • Additional software utilities

Installing the Software Install the software as follows: 1. Restart your computer to ensure that no other programs are running, and that any previously installed DYNAMICS components are not running. 2. Insert the DYNAMICS CD in your CD drive. On most systems, the DYNAMICS setup procedure will start automatically. If the setup procedure does not start automatically, use Windows Explorer or the Run dialog to run the setup.exe file in the DYNAMICS folder on the CD. 3. Answer the prompts in the setup procedure.

4. To verify installation of DYNAMICS, open the Windows Start menu and look for Programs→ Wyatt Technology→ DYNAMICS 7.1.7 or higher.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 14 Chapter 3: Installation and Setup

Installing the DynaPro Plate Reader II

The installation procedure for the DynaPro Plate Reader II involves some initial tests to see that everything is working properly. To install the DynaPro Plate Reader II, do the following: 1. Place the instrument on a flat, clean surface, standing on its feet and positioned to allow air flow through all ventilation holes—DO NOT block the holes on the top of the instrument; they are functional. To keep the instruments electronics cool, allow a minimum of 10 cm (4 in) of open space on all sides and 30 cm (12 in) of open space above the instrument, and a minimum of 15 cm (6 in) of open space at the back panel. See Chapter 7, “Hardware Maintenance” for more informa- tion about the required environment and how to keep the DynaPro Plate Reader II in peak condition.

Note: Please contact Wyatt Technology if the DynaPro Plate Reader II will be installed inside an area enclosed on three or four sides.

2. Make sure the supplied power plug is correct for the local power outlet. The DynaPro Plate Reader II is equipped with a universal power sup- ply, which operates anywhere in the world. It accepts inlet voltages between 90 Vac to 250 Vac and line frequencies from 50 Hz to 60 Hz. 3. Connect one end of the supplied ethernet cable to the ethernet port on the back of the DynaPro Plate Reader II and the other end to your local area network. Alternatively, you can use the supplied ethernet- to-USB converter and connect to the USB port on the host computer. When the DynaPro Plate Reader II is on the local area network, it may be accessed and controlled from any machine on the network. When using the USB converter, it can be accessed only by the host computer. See Appendix D for more details about implications for network secu- rity from the two different configurations. 4. Switch on the instrument and allow 30 min for the temperature to sta- bilize. The power switch is on the front panel. The DynaPro Plate Reader II is now installed and ready to use.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 15 Chapter 3: Installation and Setup

Nitrogen Connection

A nitrogen line must be attached to prevent condensation within the instrument when the temperature is set below 20 °C. The nitrogen bleed rate with the door closed is approximately 60 cc/min at 60 psi. With the door open, a high-flow purge valve is opened to prevent condensation on the optics and well plate. To preserve the gas supply, nitrogen valves are closed when the instrument temperature is set above 20 °C. Refer to “Nitrogen Connection” on page 16 and “Alarm Tab” on page 32 for more information regarding the nitrogen purge connection. The following adapter and tubing is shipped with the instrument:

Wyatt Part Number 90 Degree Nitrogen Adapter Barb length 5.08 mm (0.200") P6703-02 Tubing Barb 5.08 mm OD (0.200")

White Polyethylene Tubing S6607-04 ID 4.32 mm (0.170 in), OD 6.35 mm (¼ in)

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 16 4 Components

This chapter gives you a guided tour of the DynaPro Plate Reader II components. If you have just installed the DynaPro Plate Reader II, read this chapter to become familiar with the various instrument parts and their functions.

CONTENTS PAGE FRONT PANEL VIEW ...... 18 BACK PANEL VIEW ...... 19 TOP PANEL VIEW ...... 20 LASER ...... 21 COLLECTING AND VIEWING WELL IMAGES ...... 22

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 17 Chapter 4: Components

Front Panel View

The front panel (see Figure 4-1) contains the main power switch (On/Off), the display window, and display controls for operating the instrument and monitoring data. Located behind a door for diagnostic purposes only is a DB9 connector.

Loading Door

Keypad

Diagnostics LCD Display or Power Connector Touch-screen Switch Figure 4-1: DynaPro Plate Reader II Front Panel

LCD Display: The LCD display allows you to monitor, control and configure the DynaPro Plate Reader II. Chapter 5, “Using the Display Window” describes the functions of the tabs available on the LCD display. Keypad: The keypad allows you to control the LCD display. “Using the Display Tabs” on page 24 describes how to use the keypad. DB9 Connector: The DB9 connector behind the front door is for diagnostic purposes only.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 18 Chapter 4: Components

Back Panel View

The back panel contains the AC power module, ethernet connector, nitrogen purge connector, and cooling fan. The main power fuses are located in the AC power module and are described below.

Nitrogen Purge Connector

Fan

Fuse Holder

Power Ethernet Air Filter Plug Figure 4-2: Back Panel

Power Inlet: the power inlet can receive 90-250 V, 50/60 Hz power. Ethernet: the Ethernet connections is used to connect the instrument to your Local Area Network. Nitrogen Purge: Used with the Temperature Controlled version. Refer to “Alarm Tab” on page 32.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 19 Chapter 4: Components

Top Panel View

The DynaPro Plate Reader II is loaded from the door on the top panel. It also has ventilation holes which must be kept clear for proper temperature regulation. The door is controlled from the front panel or from the DYNAMICS software. To identify components inside the door, see Figure 4-4.

Figure 4-3: Loading Door

Camera Guide pins

Optics (under beam)

LED light strips

Figure 4-4: Inside the Thermal Box, with plate insert removed

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 20 Chapter 4: Components

Laser

The 75 mW linearly polarized diode laser provides the light source for the system. The laser system provides very high power density at the 2 illuminated sample by means of a narrow beam diameter (the 1 ⁄ e diameter of the Gaussian beam profile is 40 μm). This small beam diameter also helps reduce the noise contributions of larger particulate contaminants (such as dust).

Laser Beam Warning The laser in the DynaPro Plate Reader II is interlocked so that it is off when the door is open, and is physically not accessible to the user when the door is open. However, it is good laboratory practice with any laser source, irrespective of its power, to AVOID LOOKING INTO THE BEAM.

Laser Enable Standby Time When the laser is enabled, or set to On, either by Dynamics upon connection to the instrument or manually on the Main tab of the LCD, the laser On/Off button on the Main tab of the LCD will stay yellow and flash for 30 s. During this period, measurements cannot be taken. Once the 30 s has passed, the On/Off button on the Main tab will turn solid green, indicating that the laser is fully operational. At this time measurements can be taken. In addition, every time the DynaPro Plate Reader II door is opened, the laser is automatically turned off. Once the door is closed again and the laser is automatically turned back on, the user must allow 30 s of laser warm up before measurements can be taken.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 21 Chapter 4: Components

Collecting and Viewing Well Images

You can take optical pictures of wells as you perform an experiment with the DynaPro Plate Reader II. Later you can view and export these images. Refer to the DYNAMICS User’s Guide.

The wells are illuminated from above, and the camera looks up through the sample. You can view the images to determine if sediment, dust, very large particulates, oil droplets, or bubbles are present. You can also tell if the well has dried out. If the results for a well are questionable, check the image for that well. If there are no problems with the well, the image should look like the previous well image. The following figure shows examples of wells with various types of problems:

The camera is mounted within the temperature controlled sample space (see Figure 4-4), and it experiences the same temperature environment as the samples. Above 50 °C the instrument disables the camera to protect it, and requests for images result in a “camera over temperature” message. The camera is re-enabled when the temperature drops below 49.5 °C.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 22 5 Using the Display Window

This chapter describes how to navigate and change settings in the DynaPro Plate Reader II Display Window.

CONTENTS Using the Display Tabs...... 24 Main Tab...... 26 Selecting Display Settings for the X, Y Axes ...... 26 Adjusting the Display Range...... 27 Setting Temperature...... 28 ACF Tab ...... 30 Alarm Tab ...... 32 System Tab ...... 34 Camera Tab...... 36 Comm Tab...... 37

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 23 Chapter 5: Using the Display Window

Using the Display Tabs

You navigate through the display tabs using the buttons to the right of the Display Window. The commands found on the display tabs can be issued by DYNAMICS software as well.

Note: If a command is given from the front panel while running DYNAMICS, a warning appears in a pop-up window that reads Take control and resubmit command? Choose either OK or Cancel. If OK is chosen, the front panel command will override the command previously set by DYNAMICS. Use caution when issuing commands from the front panel because overriding DYNAMICS allows the possibility of disrupting a DYNAMICS data collection.

Panel tabs

Temperature Lock

Alarm Status Figure 5-1: Main Display Tab Front panel button description Esc selects the current panel tab, then the left or right arrows navigate through the panel tabs or select the number of the panel tab (1 through 6) to select the first field in that tab. Tab cycles through various fields in the current tab. Enter displays the options of the selected field. Use the arrow keys to change an option, then Enter to select. If the field is a check box, Enter toggles the option.

Note: If the instrument is equipped with a touch-screen display, tap on a tab or field to select a tab or field.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 24 Chapter 5: Using the Display Window

Warning and Alarm Lights

Color Meaning Yellow Not ready Green Ready Red Hazard Alarms The DynaPro Plate Reader II will set an alarm when an abnormal operating condition is detected. When there is an alarm, the alarm button on the Main tab (and the Alarm tab) turns red. If you select this button, it will take you to the Alarm tab. The indicator remains lit until the alarm is reset. See “Alarm Tab” on page 32 for further information.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 25 Chapter 5: Using the Display Window

Main Tab

The Main tab contains the most commonly used DynaPro Plate Reader II functions. The display shows graphical representations of two of the data streams collected by the instrument. The Blue data stream is on the left axis and the Red data stream is on the right axis.

Selecting Display Settings for the X, Y Axes You can select the data channel you want displayed in each axis.

Left y-axis selector x-axis selector Right y-axis selector

Figure 5-2: Main Tab Left and Right Y-axis Selectors • Tab to the field and press Enter to display the data channels. • Use the up and down arrow keys to scroll through the parameters. Press Enter to select. The left Y-axis data channel displays in blue; the right Y-axis data channel displays in red. X-axis Selector The X-axis selector sets the time range from 1 min to 2 hours. To change the time, see the Set Time field under “System Tab” on page 34.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 26 Chapter 5: Using the Display Window

Adjusting the Display Range You can adjust the range displayed in the graph in a variety of ways. This also applies to the Set Scale button in the ACF tab. To use the zoom and pan buttons: 1. Tab to the Set Scale button. The zoom/pan buttons are displayed. 2. Press the left arrow to zoom in. 3. Press the right arrow to zoom out. 4. Press the up arrow to pan up. 5. Press the down arrow to pan down.

Pan up

Zoom in Zoom out

Tab to here Tab to here to display to display the zoom/ Pan down the zoom/ pan buttons pan buttons

Figure 5-3: Zoom and pan buttons

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 27 Chapter 5: Using the Display Window

To change the scale numerically: 1. Tab to the Set Scale button. 2. Press Enter. The Set Scale window is dis- played. 3. To toggle positive and nega- tive, tab to the +/- button and press Enter. 4. To change values, tab to the Max field and enter a value. Tab to the Min field and enter a value. Press Enter. Figure 5-4: Setting the Scale Numerically Autoscale Changes the scaling so the display fills the window. Alarm Select the color-coded Alarm button to go to the Alarm tab. See “Alarm Tab” on page 32. Door Opens and closes the door. When the label reads closed, the door is closed. When the label reads open, the door is open. Laser Sets the laser to On or Off. When On, the laser does not produce light until the “Laser Power (%)” field on the System tab is set to a non-zero value. When the laser is enabled, or set to On, either by Dynamics upon connection to the instrument or manually on the Main tab of the LCD, the laser On/Off button on the Main tab of the LCD will stay yellow and flash for 30 s. During this period, measurements cannot be taken. Once the 30 s has passed, the On/Off button on the Main tab will turn solid green, indicating that the laser is fully operational. At this time measurements can be taken. In addition, every time the DynaPro Plate Reader II door is opened, the laser is automatically turned off. Once the door is closed again and the laser is automatically turned back on, the user must allow 30 s of laser warm up before measurements can be taken.

Setting Temperature The Temperature Controlled DynaPro Plate Reader II operates at regulated temperatures between 4 °C and 85 °C. The Main tab includes a temperature control box labeled Set to which is located on the lower left corner of the screen. You set the sample temperature by using the numeric keypad to enter the value.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 28 Chapter 5: Using the Display Window

You set the sample temperature by using the numeric keypad to enter the value. The Temperature Controlled DynaPro Plate Reader II operates at regulated temperatures between 4 °C and 85 °C. The instrument default temperature ramping rates are as follows: • For a temperature change of less than 3 °C from the initial temperature, the default ramping rate is 0.3 °C/min. • For a temperature change of between 3 and 5 °C from the initial temperature, the default ramp rate is 0.5 °C/min. • For a temperature change of between 5 and 20 °C, the default ramp rate is 1 °C/min. • For any temperature change greater than 20 °C, the temperature setpoint is immediately made the final set point, with no ramping. • The recommended maximum ramp rate when performing a thermal ramping experiment is 0.25 °C/min.

Tab here to adjust the sample temperature.

Figure 5-5: Set Sample Temperature

Dry gas (i.e., nitrogen) at a pressure of at least 50 psi is required to set the temperature set point below 20 °C. Note that the nitrogen purge valve is only activated if the door is open and the sample temperature is below 20 °C. When it is activated the nitrogen flows at a higher flow rate.

Note: The vertical green lines shown in Figure 5-5 indicate stage movement.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 29 Chapter 5: Using the Display Window

ACF Tab

The ACF Tab displays the auto correlation function. The data displayed in Red represents the Auto Correlation Function of the last full acquisition. The data displayed in Blue represents the current Auto Correlation Function.

Figure 5-6: Auto Correlation Function tab Prev Prev moves the Plate Position one column to the left. If the plate position is currently set to column 1, the plate position moves to the final column of the row directly above the current row. If the plate is in the first column of the first row then the plate does not move. Next Next moves the plate position one column to the right. If the plate position is currently set to the final column, the plate position moves one row down and to column 1. If the plate is in the final column of the final row then the plate does not move. Row Drop Down Menu Row Drop Down Menu allows the user to select a desired row. Column Field The Column Field allows the user to enter the desired column. Go Go moves the optics block to the row and column chosen in Row Drop Down Menu and Column Field.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 30 Chapter 5: Using the Display Window

Integration Time(s) The Integration Time(s), also referred to as the Acquisition Time(s), is the time over which the collection of data is taken. The percentage bar below it displays where the computer is in that process. Count Rate (Hz) Displays the count rate in units of Hertz. SPCM Status Green indicates that the SPCM is collecting data; red indicates that the SPCM is either powered off or the count rate is zero. Note that very high count rates may cause the indicator to turn red momentarily. Adjusting the Display Range Adjusting the display range is the same as on the Main tab. See “Adjusting the Display Range” on page 27 for more details. The Auto Correlation function can either be adjusted with the Auto Scale or Set Scale.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 31 Chapter 5: Using the Display Window

Alarm Tab

The Alarm tab displays sensor information and lets you adjust alarm settings. An event screen text display is shown which includes the last few alarms and the time at which they occurred. Audio Alarm, when selected, causes the audio alarm to sound when a Red alarm condition is detected.

Event screen text display

Figure 5-7: Alarm tab Event Screen This text display, located on the left side of the Alarm tab, shows a log of plate reader events including alarms. Alarm Indicators Here is a list of the alarms and their meanings: • Door Error is triggered when the stage loses communication with the DYNAMICS software or the on board computer. • N2 pressure is triggered when the temperature is less than 20 °C, but the nitrogen pressure is less than 50 psi. In this case, the alarm activates and resets the system temperature to 25 °C. This prevents condensation from damaging the optics if the nitrogen connection is not made, or if the tank runs empty. • Stage Movement Error is triggered when the stage does not finish a command or reports an error to the on board computer. • Stage Communicating is triggered when the stage loses communication with the DYNAMICS software or the on board computer.

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• Auto Attenuating is triggered when the instrument is auto attenuating. • Camera Active is triggered when the stage loses communication with the DYNAMICS software or the on board computer. • Plate Insert ID Error is triggered when the on board camera cannot read the bar code located on the underside of the well plate insert (more details regarding the insert are found in “Well Plate Insert Installation” on page 40). The camera attempts to read the bar code each time the door is closed. The error may occur if the insert is not in place, or the bar code on the insert is damaged, or the camera is malfunctioning. • SPCM Enabled is triggered when the Single Photon Counting Module is enabled. • Over temperature is triggered in the Temperature Controlled DynaPro Plate Reader II if any of the heaters malfunction and the temperature rises over 110 °C. This alarm turns off all heating and cooling. • Purge Enabled is triggered in the Temperature Controlled DynaPro Plate Reader II if the door is told to open while the instrument is at a temperature below 20 °C, nitrogen will purge the system for 10 s to 12 s prior to the door opening and will continue to purge while the door remains open.

Note: DO NOT leave the door open when the Nitrogen Purge is enabled and the set temperature is below 20 °C. Nitrogen Purge will flow nitrogen into the sample space at a higher flow rate and can quickly deplete external nitrogen tanks.

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System Tab

The System tab contains additional options for some of the selections on the Main tab.

Select here to adjust the sample temperature.

Figure 5-8: System Tab Sample Temp Displays the current temperature of the DynaPro Plate Reader II. Set to The Temperature Controlled DynaPro Plate Reader II includes a Set to command located on the upper left corner of the System tab which allows you to adjust the temperature. Set to is the same function as “Setting Temperature” on page 28 as shown on the Main tab. Well Count You can change the well count to accommodate 96, 384, or 1536-well well plates. Note that the well count setting on the front panel only affects the Move to Well commands issued by the front panel and will not affect those issued by DYNAMICS. Attenuation % This is the level of attenuation seen by the Single Photon Counting Module. Auto Attenuate This function sets the attenuation so that the Single Photon Counting Module sees an optimal count rate. If the count rate falls out of range during a data collection, Auto Attenuate removes the collection, re- attenuates, and then re-takes the collection.

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Select the Configure Auto Attenuation button to adjust Auto Attenuation parameters.

Parameter definition: • Target count rates: Auto Attenuation will adjust laser power and attenuation to within this range for each measurement. • Threshold count rates: If the count rate varies outside this range during a measurement, Auto Attenuation will automatically restart. This can happen, for instance, because a large particle has moved through the measurement volume. Laser Power (%) Sets the percentage of full laser power used by the instrument. Serial Number Serial number displays the instrument’s serial number. Firmware Version Firmware version displays the version of the firmware currently installed. Load Factory Defaults Load Factory Defaults is used to reset the instrument to the settings installed when the instrument was shipped. These settings include various networking parameters and hardware settings such as auto attenuation parameters. Set Time Set Time sets the time. Restart Instrument Restart Instrument turns off the DynaPro Plate Reader II and turns it back on. This is typically used only when installing a firmware update.

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Camera Tab

The Camera tab allows you to visually record aspects of your well samples prior to or after measurements.

Figure 5-9: Camera Tab—Default view no lights on

The camera operates in two modes: The Live mode allows you to monitor the camera image in real-time. Images cannot be taken in this mode. The Acquire Well Image mode allows you to select a specific well and acquire an image of the well sample. You can not acquire an image during a measurement. When in the Live mode, the Lights can be turned on/off from either the top, bottom, or both by selecting the check boxes. The Camera Position lists the well currently being viewed by the camera. The DLS Optics Position lists the well currently being measured by the DLS detector.

When in the Acquire Well Image mode, the Well pull-down menus allow you to move a well location above the camera. The Acquire button instructs the instrument to take an image of the currently selected well.

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Comm Tab

The Comm tab allows you to connect to a computer network.

Figure 5-10: Comm Tab

Configure Ethernet contains two communications options: • Obtain an IP address automatically: Once the instrument is connected to a computer or LAN, the IP address and subnet mask will be assigned automatically. When using this option, it may take several minutes for the IP address to be assigned. During this time, the IP address and subnet mask on the front panel will read 0.0.0.0. Once the IP address and subnet mask have been assigned, both will be automatically updated on the front panel, and should no longer read 0.0.0.0. At this point, it should be possible to connect to the instrument from the computer. • Use the following IP address: If you wish to use a static IP address and subnet mask, please contact your IT department to obtain a valid address and mask. Enter the information into the IP address and subnet mask fields. • Remote Access PIN: Sets the PIN for users wanting to use the Aurora mobile app to monitor the front panel.

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This chapter describes how to load samples and record data using the DynaPro Plate Reader II. This chapter assumes you are familiar with the DYNAMICS User’s Guide and you have DYNAMICS software installed and running on the PC.

CONTENTS PAGE Introduction ...... 39 Instrument Warm-up...... 39 Choosing the Correct Well Plate and Well Plate Insert...... 39 Well Plate Insert Installation...... 40 Bar Code Labels ...... 42 Preparing Samples...... 42 Loading the Well Plate ...... 44 Loading the Plate Holder...... 46 Controlling the Plate Reader from DYNAMICS...... 47 Plate Reader Control Buttons ...... 47 Well Labeling Conventions Used By Dynamics ...... 48 Measuring Automatically ...... 51 Frequently Asked Questions ...... 52

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Introduction

The DynaPro Plate Reader II offers an unparalleled level of automation in dynamic light scattering. Designed to meet the demanding needs of labs requiring speed, ease-of-use, and sophisticated software; low sample volumes (as low as four microliters per well) and high sensitivity are achieved with our proprietary optical and system designs.

Instrument Warm-up

Once powered ON, the DynaPro Plate Reader II requires at least 30 min in order for the instrument to establish temperature stabilization of both the laser and SPCM. Using the device prior to proper stabilization can adversely affect data quality.

Choosing the Correct Well Plate and Well Plate Insert

The DynaPro Plate Reader II requires the use of either a 96-, 384-, or 1536-well well plate with a clear, flat bottom with good optical quality. The DynaPro Plate Reader II is shipped with a collection of Greiner glass- bottom plates (96, 384, 1536). These optically high quality plates yield the best data. In addition, several Corning 384-well plastic bottom plates are shipped, along with the appropriate insert in any of the three sizes requested.

The Plate Reader is compatible with many other well plates. However, because all well plates have slightly different optical depths, it is necessary to match the DynaPro Plate Reader II well plate insert to the well plates you plan to use in your research. The well plate inserts are available in 0.5 mm increments from 7 mm to 11 mm (7.5 mm and 9 mm inserts are shipped with the instrument). Please see Appendix A for a listing of the well plates that have been tested with the DynaPro Plate Reader II, their associated well plate inserts, and requisite minimum volume.

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Well Plate Insert Installation

When changing to a new type of well plate, it may also be necessary to change the well plate insert in order to ensure the sample is located at the correct optical depth. Please see Appendix A for a listing of DynaPro Plate Reader II compatible well plates and their associated well plate inserts.

Insert optical depth “Z”

Guide pins

Figure 6-1: Installed Insert 1. Use a Phillips screwdriver to loosen all four (4) of the captive screws located on the corners of the insert. 2. Remove the insert from the Plate Reader by lifting the insert straight up and out.

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Guide pins

Spring pins

Figure 6-2: Installing Insert 3. Install the replacement insert into the Plate Reader, allowing the guide pins to center with the mating holes located on either side of the insert. It is easiest to slide the insert over the pin to the rear of the instrument first. The insert will click into place over the front most guide pin.

Do not over tighten the screws! Caution! Applying too much torque may strip screw heads.

4. Tighten the captive Phillips screws in all corners of the insert. Do not over tighten the screws.

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Bar Code Labels

Each well plate insert is labeled with a serial number, the insert depth (“Z” number), and a 2-D bar code—which encodes the serial number and depth. The label is affixed to the underside of the insert, where it may be viewed by the instrument camera. Each time the instrument door is closed, the camera reads the bar code and stores that information. The information from the bar code is displayed on the instrument front panel System tab under “Insert:”, and is also stored within a DYNAMICS experiment file and may be accessed within DYNAMICS. If the bar code on the label isn’t read when the door is closed (incorrect insert or unreadable bar code), then the instrument indicates “None” as the “Insert:” value and an alarm will sound.

Preparing Samples

The light scattered from a solution of particles is very sensitive to sample impurities and dust. It is important to develop routine procedures for preparing samples and collecting measurements. The information below describes some options for sample preparation. Before beginning your sample preparation, always work on a clean bench, thoroughly wiped with ethanol. In order to prevent contaminants from being transferred to the well plate, be sure to properly store the plates with either the plastic cover, sealing tape, or in a Ziploc bag when they are not being used. In the case of long experimental run times, larger variations can occur when the well in question is not filled sufficiently or solvent has evaporated. The variations in this case are due to the sample meniscus. Please see Appendix A for the minimum required sample volume for your well plate. If running a very long experiment, we recommend using more sample if possible. Alternately you may consider capping or oiling the wells. There are a few suggested procedures for removing dust or other large particle contaminants from your samples. We recommend filtering, but if this is not possible, centrifuging the samples is also effective.

Filtering Samples In order to remove large particles, we recommend using a filter that is appropriate for the size of your molecule. For example, for the BSA monomer, a 0.02 μm filter would be effective in separating out large particles from the sample itself. It is important to remove large non- sample contaminants, as the upper size limit for dynamic light scattering is 1 μm radius. The presence of contaminant particles of this size or larger may cause inaccuracies with your measurements.

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If you are working with larger particles, such as gold and silver colloids, macromolecular assemblies, and (some) polymers, the scattering from the sample itself is often sufficient to counter any scattering contribution from dust particles (assuming reasonable care is taken during your sample preparation, such as by using filtered solvent, and a new and clean well plate.) As such, number fluctuations arising from dust are less likely to be observed, since the scattering contribution is virtually negligible.

Filtering Options If you are filtering your sample, you have several options: If you have less than 300 μL sample to use for a measurement, or are limited by sample volume, then you can use the Wyatt NanoFilter (part number WNF-01), which has a dead volume less than 5 μL. Precious sample loss is minimized. In addition, the Wyatt NanoFilter has been designed to make sample recovery and refiltration simple. If you are not limited by sample volume and have more than 300 μL of sample, use a traditional syringe filter. Make sure that the filter material used is compatible with your sample. If you are measuring BSA, the hydrodynamic radius of the monomer is 3.5 nm. This means that even oligomers of BSA are well below 20 nm radius. The average Rh may be larger if there are aggregates present in your sample. You may use the BSA standard and syringe filters that came with your instrument shipment, and compare the Rh value to the BSA Certificate of Analysis.

Centrifuging Samples Rather than filtering your samples, you may prefer to centrifuge them. As a consequence of centripetal force, larger dust or contaminant particles migrate to the bottom of the centrifuge tube, thereby eliminating the need to remove the dust particles via standard filtration techniques. Recommended spin rates and times vary with the sample. However, 10 min to 15 min at 1000 g to 5000 g is typical. When removing the sample for loading, remember that only the top portion of the sample is dust-free. Because of the need for volume control, a volumetric pipette works best for transferring the sample from the centrifuge tube to the well plate. Dust can be removed from the pipette tip with a quick blast of filtered compressed air.

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Loading the Well Plate

Note: Please contact Wyatt Technology Technical Support if you are planning to run thermal experiments over a long period of time (2 or more hours) without capping the samples with paraffin or silicon oil.

You can load the samples manually or automatically into the well plates. To eliminate dust contamination, we recommend keeping the well plates covered at all times, with the plastic lid, film, or a plastic Ziploc bag. Always make sure to keep the plate on a clean surface, and if the bottom appears dusty, deliver a quick blast of filtered nitrogen to the underside of the well plate.

Loading the sample manually: With a syringe or pipette (narrow tip), insert the recommended volume of sample into the well by placing the needle or tip on the bottom corner of the well and slowly injecting the liquid. Better results may be obtained by placing the tip in the center of the well, depending upon the size and shape of the well. A single pipettor works well and a multichannel pipettor will rapidly accelerate the loading process for a large number of wells. Manual Loading Tips Some well plates may require as much as 200 μL of sample volume. Please see Appendix A for a listing of the minimum sample volume required for alternate well plates. Bubbles in Sample Bubbles can be highly deleterious with respect to data quality if they are in the path of the laser or the detector. If the bubbles are small and on the side of the well, data quality will probably be unaffected. If bubbles are visible, we recommend removing them to achieve optimal data quality. An effective and quick way of removing bubbles is with a well plate compatible centrifuge. Spin the loaded well plate at 2000 g for 1-2 min to remove all the bubbles from the sample. Dissolved gas will form on the bottom of the well if your sample temperature increases after centrifuging. In this case, the plate should be re-spun. Using Oil to Prevent Evaporation Placing a low volume of oil on top of sample prevents sample evaporation and contaminants. Evaporation is particularly deleterious when using a 1536 well plate because low volumes of sample evaporate rapidly, in as little as half an hour. If sample is evaporating, you may observe that the auto correlation signal is increasing and showing better quality data, because the concentration of the sample is concurrently increasing from evaporation of the buffer. However, at some point the sample level will drop below the measurement region, determined by the laser-detector beam intersection. At this point, useful data will no longer be taken.

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For best results, centrifuge the sample with oil or be careful not to create any bubbles. We recommend adding sample to the wells, centrifuging the plate, adding oil, and then centrifuging again. Oil should never touch the bottom of any well. Though it is less dense than water and floats to the top, oil sticks to the bottom of well plates and alters their optical properties. Two recommended oils are Paraffin and Silicone Oil. The volume of oil is inconsequential as long as it covers the entire sample meniscus. A standard oil volume for a 384 well plate is 20 μL and for a 1536 well plate is 5 μL, depending on the well volume size and sample.

Caution! Check that your sample is not affected by the oil that you use.

An added precaution is to filter the oil, though this is often an unnecessary step. If you decide to filter your oil, use a filter of at least 200 nm and a filter than can accommodate a large volume because the oil is highly viscous.

Loading Well Plates Automatically • We recommend that you perform several practice runs using solvents, such as water, to make sure completely bubble-free liquid is loaded into the wells. • We also recommend that you utilize a logical layout while loading your samples. A logical sample loading plan can assist with data analysis of large quantities of data.

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Loading the Plate Holder

You load the well plate into the plate holder as follows: 1. Gain access to the sample chamber by opening the Plate Reader door from the Main tab on the front panel LCD. When the door is opened, an interlock is activated that causes the plate holder to automatically move to the load/eject position.

Note: Prior to loading the well plate into the plate holder, visually examine the bottom of the plate to make sure it is clean and free of obvious scratches and contamination that may affect the scattered light. We have found that blowing off the bottom of the plate with a quick blast of filtered nitrogen is the best method for cleaning the plate without leaving anything behind from the cleaning process. If data quality is still affected, try cleaning the bottom of the plate by gently wiping with lens paper wetted with ethanol.

2. Once the plate holder has reached the Load/Eject position (and has stopped moving), carefully place the well plate onto the plate holder. Make sure the well plate is sitting flat and square and that well A1 is located in the upper left-hand corner as indicated by the “A1” mark engraved on the well plate insert. (Note that the corner labeled Figure 6-3: Installed Well Plate A1 is located at the front left in the instrument.) Firmly slide the well plate into the upper left corner of the plate holder as this will ensure the correct position of the plate. The solenoid-actuated latch arm will hold the plate securely in the upper left corner position once the door is closed. The solenoid- actuated latch arm should not be relied upon to move the plate into the proper upper left corner position. The main function of the latch arm is to keep the plate from sliding out of the upper left corner position. 3. Close the Plate Reader door from the Main tab on the front panel. When the door is closed the optics translation stage is free to move. The solenoid-actuated latch arm will hold the plate in the upper left corner of the insert. Once the door is closed and the laser is automati- cally turned back on, the user must allow 30 s of laser warm up before measurements can be taken.

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Controlling the Plate Reader from DYNAMICS 1. From the Dynamics menu bar, select File→ New to open a new exper- iment or File→ Open to open an existing experiment. 2. First, establish a connection between DYNAMICS and the DynaPro Plate Reader II, by selecting the “Connection To Hardware” icon. For manual control, launch the Plate Reader Control by selecting the Plate Reader Control icon from the experiment window tool bar. If you frequently use the DynaPro Plate Reader II, you may use File→ Open Preset or use the Event Scheduler to control the Plate Reader (see the DYNAMICS User’s Guide for more information).

Connect the Plate Reader Launch the Plate Reader to DYNAMICS Control Panel.

Figure 6-4: Launching the Plate Reader Control

The Plate Reader Control provides complete manual control of the Plate Reader. Use the buttons in the Plate Reader Control to specify actions for the Plate Reader.

Plate Reader Control Buttons Close Door: closes the plate reader door. Open Door: opens the plate reader door and moves the plate holder to the Load/Eject position. Current: Displays the actual position of the plate loader. Move To: Select the Row and Column of the well to be measured, then press Move. Prev Well and Next Well: These commands move the position of the plate one well before or after the current well.

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Well Labeling Conventions Used By Dynamics The following diagrams describe the well labeling system used by DYNAMICS for 96-, 384-, and 1536-well well plates. The wells on the 96-well and 384-well plates are labeled using a standard grid labeling system with letters indicating rows and numbers indicating columns. Refer to Table 6-1: 96-well Well Plate and Table 6-2: 384-well Well Plate. The wells on the 1536 plate are labeled using sub-divided blocks of 16 wells each. Table 6-3: 1536-well Well Plate, represents only a portion of the well plate, an actual 1536 plate has 12 major columns and major rows A through H. Table 6-4: 1536-well Well block detail shows four well blocks with the wells individually labeled. The colored blocks in Table 6-3 correspond to the colored blocks in Table 6-4.

Table 6-1: 96-well Well Plate

123456789101112 A A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 C C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 D D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 F F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 G G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 H H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 48 Chapter 6: Using the Plate Reader 123456789101112131415161718192021222324 I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 I13 I14 I15 I16 I17 I18 I19 I20 I21 I22 I23 I24 J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24 A1 A2B1 A3 B2 A4 B3 A5 B4E1 A6 B5 E2 A7 B6 E3 A8 B7 E4 A9 B8 A10 E5 B9 A11 E6 A12 B10 A13 B11 E7 A14 B12 A15 B13 E8K1 A16 B14 E9 A17 B15 K2 A18 B16 E10 A19 B17 K3 E11 A20 B18 E12 K4 A21 B19 E13 A22 B20 E14 K5 A23 B21 E15 A24 B22 K6 E16 B23 E17 K7 B24 P1 E18 E19 K8 P2 E20 E21 K9 P3 E22 K10 P4 E23 K11 E24 K12 P5 K13 P6 K14 K15 P7 K16 K17 P8 K18 P9 K19 K20 P10 K21 P11 K22 P12 K23 P13 K24 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 C1 C2D1 C3 D2 C4 D3 C5 D4 C6 D5 C7 D6H1 C8 D7 H2 C9 D8 H3 C10 D9 H4 C11 C12 D10 H5 C13 D11 C14 D12 H6 C15 D13 C16 H7 D14 C17 D15 H8 C18 D16 C19N1 D17 H9 C20 D18 N2 H10 C21 D19 C22 H11 D20 N3 H12 C23 D21 H13 C24 D22 N4 H14 D23 N5 H15 D24 H16 N6 H17 H18 N7 H19 H20 N8 H21 N9 H22 H23 N10 H24 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 N23 N24 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13O1 G14 G15 O2 G16 G17 O3 G18 O4 G19 G20 O5 G21 G22 O6 G23 G24 O7 O8 O9 O10 O11 O12 O13 O14 O15 O16 O17 O18 O19 O20 O21 O22 O23 O24 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 I J F L E P B C D H K N A G O M Table 6-2: 384-well Well Plate Plate Well 6-2: 384-well Table

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Table 6-3: 1536-well Well Plate

123456

123412341234123412341234 a b A c d a b B c d a b C c d a b D c d a b E c d

Table 6-4: 1536-well Well block detail

12

12341234 a A1a1 A1a2 A1a3 A1a4 A2a1 A2a2 A2a3 A2a4 b A1b1 A1b2 A1b3 A1b4 A2b1 A2b2 A2b3 A2b4 A c A1c1 A1c2 A1c3 A1c4 A2c1 A2c2 A2c3 A2c4 d A1d1 A1d2 A1d3 A1d4 A2d1 A2d2 A2d3 A2d4 a B1a1 B1a2 B1a3 B1a4 B2a1 B2a2 B2a3 B2a4 b B1b1 B1b2 B1b3 B1b3 B2b1 B2b2 B2b3 B2b4 B c B1c1 B1c2 B1c3 B1c4 B2c1 B2c2 B2c3 B2c4 d B1d1 B1d2 B1d3 B1d4 B2d1 B2d2 B2d3 B2d4

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Measuring Automatically

The DynaPro Plate Reader II also provides automatic software control over the selection and measurement of each well of the plate. Automatic control is provided through the Event Scheduler. See the DYNAMICS User’s Guide for complete information on using the Event Scheduler. The Event Scheduler provides several software commands that can be joined together into a script. See the DYNAMICS User’s Guide for a complete description of the commands and sample scripts. Commands include: • Move to well • Move to next well • Label measurement as well # • Save data as • Do/Loop for repetitive actions • Move to column • Increase a row • Collect acquisitions • Collect acquisitions and an image • Set temperature (Temperature Controlled version only, refer to “Setting Temperature” on page 28.) • Set laser power The Event Scheduler operates the DynaPro Plate Reader II in a hands- free mode, enabling you to perform other activities. The set of software commands can be scripted to measure partial or complete rows or plates. We recommend that you organize your samples in such a way as to maximize the efficiency of the scheduler. For example, in a serial fashion along a row.

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Frequently Asked Questions

This section answers the following questions: • I have used cuvette based DynaPro instruments before. How do I know I will get the same answers with the DynaPro Plate Reader II? • I am concerned about variations in the plate itself. What effect do the plates have on the results? • What sample volume and concentration are required for measurement? • I do not plan on filling up all the wells on the plate. Do I have to throw out the plate if I use only a few of the wells? • Can well plate covers, packing tape, or silicone oil be used to prevent evaporation from the wells? • What do I do about lower than expected count rates? I have used cuvette based DynaPro instruments before. How do I know I will get the same answers with the DynaPro Plate Reader II? The DynaPro Plate Reader II design has been thoroughly and rigorously tested to ensure the accuracy, reproducibility, and reliability of measurements. Various proteins, polymers, and other particles have been measured using the DynaPro Plate Reader II under a variety of conditions. The DynaPro Plate Reader II provides the same high quality data you expect from any cuvette based DynaPro instrument, while also providing you with the following important capabilities: • experimental redundancy; measuring duplicate conditions for high accuracy • greater efficiency due to hands-free operation Can well plate covers, packing tape, or silicone oil be used to prevent evaporation from the wells? For particularly long (many well) experiments, especially at elevated temperatures, capping the wells can be a useful means of helping preserve the quality of data through the entire experimental run. Filling the wells with more than the specified minimum volume (i.e. 1.5 to 2.5 times the minimum volume, although without overfilling the well) is recommended for elevated temperature experiments, to account for evaporation. Opaque sealing tapes will interfere with data quality. Clear sealing tapes might help. There are several recommended manufacturers of suitable microplate sealing tapes: • Optically clear sealing tape is available in uncut lengths (i.e. reel-type dispensing) from Hampton Research. The product name is “Crystal Clear Sealing Tape.” • Pre-cut sealing tapes are also available. Wyatt recommends using the manufacturer's applicator to apply the tapes over the microplate.

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Table 6-5: Microplate Sealing Tape

3M: 9795 Nunc: 235307 Nunc: 236269 Thermo Scientific: 15036 For most experiments, sealing tapes will mitigate evaporation of aque- ous solutions from 20 °C to 85 °C. However, due to the variability in materials, plate surfaces, the manual application of sealing tapes, and the formation of condensate on the tapes, Wyatt cannot guarantee the degree to which evaporation is mitigated, nor can Wyatt guarantee or specify the optical performance of the Plate Reader when these tapes are applied to the top of a microplate. Solvent might evaporate and condense on the tape, potentially adversely affecting data quality. Paraffin or silicone oil capping has been found to be more effective at preventing solvent evaporation. Note, however, that the presence of silicone oil within the wells can slightly reduce the amplitude of the auto correlation function, thereby reducing device signal-to-noise. I am concerned about variations in the plate itself. What effect do the plates have on the results? Our rigorous qualification testing of the DynaPro Plate Reader II design determined that despite the microscopic variations in the optical characteristics of each well of each plate, the DynaPro Plate Reader II successfully and accurately performs Dynamic Light Scattering measurements. In other words, unless the plate is defective or contaminated, the plate has a negligible effect on the quality of the data. Well-to-well variations within the well plate affect the amplitude of the auto correlation function, effectively changing the signal-to-noise ratio (S:N) of the measurement. Changes in measurement S:N do not affect the calculated molecular size or polydispersity. However S:N variations do affect the experimental precision. (Note that the DynaPro Plate Reader II is inherently slightly less sensitive than the DynaPro NanoStar. Thus, slightly higher concentrations are required for measurement.) We recommend that you build redundancy into your experiments (by repeating experiments in duplicate or triplicate rows, columns, or wells). This helps mitigate not only the effects of potentially defective wells, but also the effects of improperly loaded samples. What sample volume and concentration are required for measurement? Sample volume The minimum sample volume required for measurement varies from plate to plate due to the designs and shapes of these plates. The typical minimum sample volume required for measurement using the Corning 384 well plate (manufacturer’s part number 3540) is approximately 20 μL. Please see Appendix A for minimum sample volumes required for other Plate Reader compatible well plates.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 53 Chapter 6: Using the Plate Reader

Concentration As with all DynaPro Dynamic Light Scattering instruments, the minimum concentration required for measurement is related to the molecular weight or size of the particle. The DynaPro Plate Reader II minimum concentration is specified using 14 kDa Lysozyme at 0.125 mg/ml in an aqueous solution with 50 μl of sample in a Greiner 384 glass bottom well plate (manufacturer’s part number 781 892). Larger particles can be measured under relatively more dilute conditions. The maximum measurable concentration varies by technique, sample, and instrument-related limitations. In order to perform a valid dynamic light scattering experiment, we recommend the maximum concentration be below the multiple scattering limit (haze-free or non-turbid samples). The sample must be properly buffered to prevent interparticle charge effects, particularly at high concentrations. Instrument-related limitations, due to detector saturation, are generally mitigated through the automated software control of laser power and attenuation of detector signal. I do not plan on filling up all the wells on the plate. Do I have to throw out the plate if I use only a few of the wells? Use of the plate can be continued as long as it contains wells that have not been used. We recommend covering the plate in between sample measurements (using plate lids, sealing tape, or parafilm, for example) and placing it in a sealable bag. We do not recommend cleaning used plates; any residue or contaminant will affect the light scattering measurement.

What do I do about lower than expected count rates? Please see Appendix C for some tips on how to troubleshoot lower than expected count rates.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 54 7 Hardware Maintenance

The DynaPro Plate Reader II requires little maintenance. This chapter gives guidelines for keeping the instrument clean and in good working order.

Note: Only trained personnel are authorized to perform work inside of the instrument. These authorized individuals are the individuals or group responsible for the safe use and maintenance of the equipment. Authorized personnel must have been trained by a Wyatt representative. Training may be achieved at Light Scattering University®, as part of on-site instruction during installation or on-site visit, or other type of instruction provided by a Wyatt representative. Please contact Wyatt Technology Technical Support at [email protected] for any questions.

CONTENTS PAGE General Maintenance...... 56 Replacing the Fuses ...... 57 DynaPro Plate Reader II Firmware Upgrades...... 57

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 55 Chapter 7: Hardware Maintenance

General Maintenance

For general maintenance, we suggest you do the following: • Keep the instrument on a flat, clean surface, standing on its feet, and positioned to allow air flow through all ventilation holes. To keep the instruments electronics cool, allow a minimum of 10 cm (4 in) of open space on all sides and 30 cm (12 in) of open space above the instrument, and a minimum of 15 cm (6 in) of open space at the back panel.

Note: Please contact Wyatt Technology if the DynaPro Plate Reader II will be installed inside an area enclosed on three or four sides.

• Keep the case clean. Periodically wipe down the outside case of the instrument with a clean, moist cloth to keep it free from dust or surface stains. • Allow the instrument to warm up for 30 min before taking measurements. • Remove plates when finished with measurements. When not in use, do not store or leave well plates in the Plate Reader. • Keep the Plate Reader loading door closed to help keep the internal compartment clean. • Periodically remove and clean the foam filter (part number P6010- 0922) for the instrument’s air intake fan. The air intake fan is located on the rear panel of the instrument on the right (see Figure 7-1). a. Lift the filter bracket off of the rear panel of the instrument and remove the air filter from the filter bracket. b. Wash the air filter with mild soap and water. c. When dry, place the air filter back into the filter bracket. d. Attach the filter bracket with filter back onto the rear panel of the instrument. We recommend that any time a problem arises, you should turn off the instrument immediately to avoid any possible damage. Refer to this manual or contact Wyatt Technology to identify and resolve the problem. Some errors can cause damage to the internal components if the user allows the instrument to be powered-up for extended periods of time before the problem is remedied. If you find a problem that is not described in this manual, or if the suggestions given here do not appear to work, please contact Wyatt Technology at [email protected].

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 56 Chapter 7: Hardware Maintenance

Replacing the Fuses

Filter Bracket

Fuse Holder

Figure 7-1: Back panel 1. Disconnect the power cord. 2. Open the cover of the AC Power module using a small blade screwdriver or similar tool. 3. Replace the burned out fuse with a 5 A, 250 V slow blow fuse. The fuse block contains two fuses. Both of them must be installed for Figure 7-2: Fuseholder and Fuses the instrument to operate cor- rectly. 4. Replace the cover of the AC Power module and reconnect the power cord.

DynaPro Plate Reader II Firmware Upgrades

For peak performance and stability of your DynaPro Plate Reader II, check regularly for the most recent firmware. The firmware upgrades are available on the Wyatt web page. Go to www.wyatt.com/Firmware and click on the link to the DynaPro Plate Reader II firmware

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 57 A Sources for Well Plates

The following tables list well plates and the associated insert required by each to ensure a compatible optical depth when inserted into the DynaPro Plate Reader II. Also refer to the minimum sample volume for each well.

Preferred Well Plates

Table A-1 lists preferred well plates which have been tested and shown to yield the highest quality data. Table A-1: Wyatt Technology Preferred Well Plates

Manufacturer Wyatt Part Well Plate Name Plate Material

(Part Number) Number (°C) Insert Range Number of Wells of to Be Used Volume (µL) Volume Temperature Plate Reader Minimum Sample Aurora 1536 Lobase Brooks Life Science P8806-1536 Black cyclic olefin -10 to Assay Plates, nontreated, Systems (1090- polymer well walls, 1536 7.5 6 120 square well 00110) clear polymer base 1536W SensoPlate, ST, Greiner Bio-One, P8803-1536 Black plastic well Flat Bottom, BLK/Glass, Inc. (783 892) walls, Glass well 1536 7.5 5 4 to 37 Lid Glass Bottom, Sterile bottom Low Volume, 384 Well, Corning, Inc. (3540) P8802-384 Black plastic well Black with Clear Flat walls, polystyrene 384 9 20 0 to 60 Bottom, Polystyrene, Not well bottom Treated, Microplate 384 SensoPlate, ST, Flat Greiner Bio-One, P8803-384 Black plastic well Bottom, BLK/Glass, Lid Inc. (781 892) walls, Glass well 384 9 50 4 to 37 Glass Bottom, Sterile bottom Aurora 384 LV/EB, 100% Brooks Life Science P8806-38401 Black COP well Cyclo Olefin Polymer Systems (1012- walls, clear film -10 to 384 9 30 (COP), 200 micron Clear 00110) bottom 120 Film Bottom

Corning® 96 Well Half Corning, Inc. (3880) P8802-09602 Black polystyrene Area Black with Clear Flat well walls, clear Bottom Polystyrene Not polystyrene base 96 9 100 0 to 60 Treated Microplate, 25 per Bag, without Lid, Non sterile 96 SensoPlate, ST, Flat Greiner Bio-One, P8803-096 Black plastic well Bottom, BLK/Glass Lid, Inc. (655 892) walls, Glass well 96 9.0 100 4 to 37 Glass Bottom, Sterile bottom

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 58 Appendix A: Sources for Well Plates

Compatible Well Plates

Table A-2 lists well plates which have been tested and shown to be compatible with your plate reader, although the data quality may be lower than with the well plates listed above in Table A-1.

Table A-2: Other Compatible Well Plates

MFG Part Well Plate Name Manufacturer Plate Material Number Range (°C) Range to Be Used Volume (µL) Volume Temperature Number of Wells of Number Minimum Sample Minimum Plate Reader Insert Greiner Bio-One Plate Greiner Bio-One, 783 096 Black polystyrene well 1536WELL Ps Blk CS60 Inc. walls, clear polystyrene base, 1536 7.5 4 0 to 60 medium binding hydrophobic 1536 Well Cycloolefin Greiner Bio-One, 792 870-906 Clear cycloolefin Storage Plates with Inc. copolymer well walls, -10 to 1536 8.5 4 Optimized Geometry for low clear cycloolefin base 120 Evaporation, clear Aurora 384 LV™/EB™, 100% Brooks Life 1011-00110 Black cyclo olefin -10 to COP, 100 micron clear film Science Systems polymer well walls, 384 8.5 30 120 bottom clear polymer base 384 LV™/EB™, 100% Cyclo Brooks Life 1012-00110 Proprietary Black COP -10 to Olefin Polymer (COP), 200 Science Systems polymer well walls, 384 9 25 120 Micron Clear Film Bottom clear film bottom Corning® 384 Well Black Corning, Inc. 3711 Black plastic well non-treated, black, lid: No, walls, Polystyrene well 384 9 30 0 to 40 non-sterile; γ-irradiated bottom 384 Well Polystyrene Greiner Bio-One, 781 096 Black polymer well -20 to Microplates, black Inc. walls, clear polymer 384 9.5 60 60 base 384 Well Small Volume™ Greiner Bio-One, 788 092 Black polystyrene well LoBase Polystyrene Inc. walls, clear -20 to 384 8 20 Microplates, black polystyrene base, 60 hydrophilic 384 Well Small Volume™ Greiner Bio-One, 788 096 Black polystyrene well LoBase Polystyrene Inc. walls, clear -20 to Microplates, black polystyrene base, 384 8 20 60 'medium binding' hydrophobic 384 well SZ, LoBase, COC Greiner Bio-One, 788 876 Black cycloolefin Chimney style, Black with Inc. copolymer well walls, -20 to 384 8.5 15 µClear, nontreated clear cycloolefin base, 60 hydrophobic 384 Well Sensoplate™, SZ Greiner Bio-One, 788 892 Black polystyrene well LoBase, black with glass, Inc. walls, clear glass base 384 8 10 4 to 37 sterile, with lid

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 59 Appendix A: Sources for Well Plates

Table A-2: Other Compatible Well Plates (Continued)

MFG Part Well Plate Name Manufacturer Plate Material Number Range (°C) to Be Used Volume (µL) Temperature Number of Wells Number Minimum Sample Plate Reader Insert 384 Well Small Volume™ Greiner Bio-One, 788 896 Black polystyrene well 384 8.5 10 4 to 37 LoBase SensoPlate™, black Inc. walls, glass base Standard and Low-Volume Brooks Life MGB101- Black polymer well 384 8.5 60 0 to 40 MatriPlate™ Microplates Science Systems 1-2-LG-L walls, clear glass base ScreenMates 384 Well Black/ Matrix 4323 Black plastic well Clear Bottom, Polystyrene Technologies walls, Polystyrene well -20 to 384 9 50 Microplate, Flat Bottom, Corporation bottom 60 Medium Binding 384 Well Optical Bottom Nalge NUNC 242764 Black plastic well Plates Polystyrene/Polymer International walls, Polymer well 384 8.5 5 4 to 38 Base bottom Polystyrene; Clear; 70µL/ Thermo Electron, 95040000 Clear polystyrene well well; Non sterile, no lid Labsystems walls, polystyrene well 384 8 30 0 to 60 bottom Quartz Plate Wyatt Technology P8801-384 Frosted quartz well n.s. to walls, quartz well 384 8 50 900 bottom 96 Well Flat Bottom Plate Corning, Inc. 3631 Black Polystyrene well Polystyrene Untreated walls, polystyrene well 96 9.5 100 0 to 40 bottom 96 Well UV Plate Corning, Inc. 3635 Clear acrylic well walls, Acrylic well 96 9 165 0 to 40 bottom Corning® 96 Well Half Area Corning, Inc. 3679 Clear acrylic well UV Plate walls, acrylic well 96 9 80 0 to 40 bottom ® Greiner Bio One, 675801 Clear COC well walls, 96 Well Half Area UV Star -80 to Inc. clear COC film base 96 9 60 Clear/Clear® Microplate, no 40 lid 96 Well Chimney style, black, Greiner Bio One, 655087 Black polystyrene -20 to 96 9.5 150 µClear bottom, sterile Inc. wells, clear bottom 60 96 Well Optical Bottom Nalge NUNC 265301 Black plastic well 20 to Plates, Polystyrene/Polymer International walls, Polymer well 96 8.5 165 40 Base bottom Standard and Low-Volume Brooks Life MGB096-1-2- Black polymer well 96 10 300 0 to 40 MatriPlate™ Microplates Science Systems LG-L walls, clear glass base Quartz Plate Wyatt Technology P8801-096 Frosted quartz well n.s. to walls, quartz well 96 8 100 900 bottom

Disclaimer: The well plate information provided above is true and correct to the best knowledge of Wyatt Technology Corporation. Please communicate with individual well plate manufacturers to confirm the above information.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 60 B Technical Specifications

Measurement Specifications: Supported well plate formats: 96-, 384-, and 1536-well well plates Minimum concentration: 0.125 mg/mL Lysozymea Maximum concentration: Practically none. The onboard attenuator and laser control auto- matically vary the light level, resulting in a dynamic range of con- centration measurement of more than 3 million. Minimum volume: 4 μLb Temperature range: 4 °C to 85 °C with an ambient temperature of 24 °C.c Dry gas pressure 50 psi to 80 psi (0.34 MPa to 0.55 MPa) required for operation below 20 °C. At 60 psi, instrument uses 60 cc/min dry gas. (a standard tank lasts about 1 month). Physical Specifications: Dimensions: 60 cm (L) x 36 cm (W) x 25 cm (H); 23.5 in x 14 in x 10 in Power: 350 W, 90 V to 250 V @ 50/60 Hz, universal power input Shipping weight: 26 kg (instrument weight 18.5 kg) Laser Specifications: Type: Polarization maintaining fiber coupled laser Wavelength: 830 nm Optical power: 75 mW

a. Minimum concentration specified with 50 μL of sample in a Greiner Bio-One 384 well SensoPlate (Greiner Bio-One) with 100 s acquisition. b. Minimum volume specified with 2 mg/mL Lysozyme in a Greiner Bio-One 1536 well SensoPlate (Greiner Bio-One) with 25 s acquisition. c. Absolute accuracy of +/- 0.5 °C from 4 °C to 50 °C, and +/- 1.0 °C from 50 ° C to 85 °C.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 61 C Troubleshooting Low Count Rates

There are many possible low count rate failure modes. CASE A: If the count rate equals zero (0.0) then in all likelihood the failure is associated with the detector or correlator. Examples of failure that result in a 0.0 count rate include defective detector, defective correlator, or defective cable between detector and correlator. All such cases require a return of the instrument to Wyatt for troubleshooting and repair. CASE B: If the count rate is very low for 2.0 mg/mL BSA, (that is to say above zero but maybe only 1000 counts per second, which is even lower than is expected for clean water)—the data will be invalid. Potential causes are: 1. The laser is set to “OFF.” (Using the DynaPro Plate Reader II front panel, navigate to the Main tab, press the tab key on the keypad sev- eral times until the “Laser” button is highlighted in the bottom right corner. Press the green enter key once and the “Laser” value should toggle from “OFF” to “ON”). 2. The laser power is set too low. (Once DYNAMICS is connected, use the experiment window to navigate to Parameters→Instrument. Sev- eral properties will be available for variation; double click the value box next to the “laser power (%)” property and enter 100). 3. The attenuation level is set too high and auto attenuation has been disabled. (Once DYNAMICS is connected, click the instrument control panel icon, in the instrument control panel window, click on the “Auto- Attenuation” check box to place a check in the box if it isn't already there.) 4. The laser or another component is misaligned or malfunctioning. We recommend that you first work sequentially through all possibilities above. In case of #4, the instrument must be returned to Wyatt for service. Please contact Wyatt Technology Technical Support at [email protected] for assistance.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 62 D Connecting to a Network or PC

These instructions contain an overview for connecting your DynaPro Plate Reader II to a computer for data collection. The instructions are divided into seven sections:

CONTENTS PAGE Components...... 64 Connecting to a LAN ...... 68 Connecting via USB ...... 69 Connecting via Ethernet when not on a LAN...... 71 Instrument Network Settings ...... 73 Accessing instruments with DYNAMICS...... 74 Troubleshooting and diagnostics...... 74 Please read over the Components section to gain an understanding of the components to be used. Then read over either “Connecting to a LAN” on page 68, “Connecting via USB” on page 69, or “Connecting via Ethernet when not on a LAN.” on page 71 depending on your configuration. Finally, read over “Instrument Network Settings” on page 73 for instrument settings. Please read “Accessing instruments with DYNAMICS” on page 74 for instructions on accessing instruments via the DYNAMICS software. Finally, if you experienced problems connecting to your instrument, please read “Troubleshooting and diagnostics” on page 74 for diagnostics and trouble-shooting.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 63 Chapter :

Components

Instrument connections: Figure D-1 is a detail of the instrument back panel. The Ethernet port, designated with an arrow, is to be used for all connections in these instructions. Please see “Connecting via USB” on page 69 for instructions on establishing a USB connection.

Figure D-1: Detail of the back panel of the DynaPro Plate Reader II. The arrow designates the Ethernet port.

LAN connection: Figure D-2 shows a typical wall socket connection to a Local Area Network (LAN). If you are going to connect the instrument to a LAN, you will need access to this type of socket.

Figure D-2: Wall socket LAN connection indicated by an arrow.

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Computer connections: Computer connections are made via the Ethernet or USB port. Figure D-3 shows these ports on a standard laptop computer. “Connecting to a LAN” on page 68 and “Connecting via Ethernet when not on a LAN.” on page 71 describe instrument connections made via the Ethernet port. “Connecting via USB” on page 69 describes connections made via the USB port.

Ethernet Port USB Ports Figure D-3: Ethernet and USB ports on the computer.

Crossover cable:

Note: Many newer computers will automatically detect the Ethernet cable and adjust to the requirements of the interface, whether it is a switch or another computer. This allows using a standard Ethernet cable for the connection between two computers.

A crossover cable can be used to make a direct connection from the instrument to an Ethernet port on a computer or to an Ethernet to USB adapter. Please note that the crossover cable shipped with Wyatt Technology instruments is yellow to distinguish it from a standard Ethernet cable. Please be careful to only use the yellow crossover cable where indicated.

Figure D-4: The Ethernet crossover cable shipped by Wyatt Technology is yellow.

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Ethernet cable: A standard Ethernet cable is sometimes referred to as a patch cable, or a straight-through cable to distinguish it from the crossover cable. Ethernet cables provided by Wyatt Technology are black, blue, white, or gray, but never yellow (yellow is reserved for the crossover cable). For these instructions, the Ethernet cable will always be black.

Figure D-5: Standard Ethernet cable. For these instructions, the standard cable is always black.

Ethernet to USB adapter: This device can be used to connect an Ethernet cable to a USB port on the computer. Using this adapter, it is possible to have the computer connected to a LAN via the computer’s Ethernet port, and the instruments connected to the computer via USB. The Ethernet to USB adapter supplied by Wyatt Technology will look similar to this. The first time you connect an Ethernet to USB adapter to your computer, you may be prompted to install USB drivers for the device. To do so, follow the onscreen instructions.

Ethernet USB Connector Port

Figure D-6: Standard Ethernet to USB adapter. The Ethernet cable is plugged into the Ethernet port (shown with the arrow) and the USB connector is plugged into a USB port on the computer.

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Ethernet switch: Ethernet switches are used to connect several Ethernet cables to one resource, such as the LAN socket in Figure D-2. The Ethernet switch supplied by Wyatt Technology will look similar to the eight-port switch shown below. Please note that Although Ethernet cables can be connected to the switch in any order or position, best practice is to use the Uplink port to connect to a LAN while leaving port 8 empty. Also, the switch has an external AC adapter (not shown) to provide power to the switch.

Figure D-7: Eight-port Ethernet switch.

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Connecting to a LAN

If an instrument is connected to a LAN, it can be accessed by any computer plugged into the same LAN. Plug the instrument into a LAN wall socket using a standard Ethernet cable. The computer that is to communicate with the instrument must be on the same LAN.

Figure D-8: Connection for one instrument to LAN.

Instrument and Computer to LAN: If there is only one LAN wall socket available for both the instrument and computer, it is necessary to use an Ethernet switch to connect both the computer and instrument to the LAN. In this configuration, the computer can access the LAN and the instrument, and the instrument can be accessed from any other computer on the LAN.

Figure D-9: One instrument and a computer can both be connected to the LAN using an Ethernet switch.

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Connecting via USB

If it is not possible or desired to have the instruments connected to a LAN, it is possible to connect to the instruments via USB. In this way, the instruments can be isolated from the LAN, even while the computer maintains its own Ethernet connection with the LAN.

Instrument to USB via a crossover cable:

Note: Many newer computers will automatically detect the Ethernet cable and adjust to the requirements of the interface, whether it is a switch or another computer. This allows using a standard Ethernet cable for the connection between two computers.

Connect the yellow crossover cable from the instrument to the Ethernet to USB adapter. Plug the Ethernet to USB adapter into an available USB port on the computer. You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer. To install the drivers, follow the Windows instructions. Please note that the network communication setting in the Communications tab of the instrument display is Ethernet for this configuration, and not USB Virtual Ethernet.

Figure D-10: One instrument to USB via yellow crossover cable.

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Instrument to USB using an Ethernet switch: Connect the instrument to the Ethernet switch using a standard Ethernet cable. Then connect the Ethernet switch to the Ethernet to USB adapter using a standard Ethernet cable. Plug the Ethernet to USB adapter into an available USB port on the computer. You may be prompted to install drivers for the Ethernet to USB adapter the first time it is plugged into the computer. To install the drivers, follow the Windows instructions.

Figure D-11: Connecting one instrument to USB using an Ethernet switch.

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Connecting via Ethernet when not on a LAN.

If the computer is not on the LAN, it is possible to use the Ethernet port directly to connect to the instruments.

Instrument to computer not on LAN using crossover cable:

Note: Many newer computers will automatically detect the Ethernet cable and adjust to the requirements of the interface, whether it is a switch or another computer. This allows using a standard Ethernet cable for the connection between two computers.

Connect the yellow crossover cable from the instrument directly to the Ethernet port on the computer.

Figure D-12: Connecting one instrument directly to a computer that is not on the LAN using the yellow crossover cable.

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Instrument to computer not on LAN using an Ethernet switch: Connect the instrument to the Ethernet switch using a standard Ethernet cable. Then connect the switch to the computer Ethernet port using a standard Ethernet cable.

Figure D-13: Connecting one instrument to the computer using an Ethernet switch.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 72 Chapter :

Instrument Network Settings

Figure D-14 shows the standard settings on the instrument front panel that will work with all of the above connection schemes. As shown in Figure D-14, there is a choice of Obtain an IP address automatically or Use the following IP address: for the ethernet configuration. If you select Obtain an IP address automatically, your LAN or computer must implement Dynamic Host Configuration Protocol (DHCP). Once the instrument is connected, the IP address and subnet mask will be assigned automatically. This will even work with the USB connections described in “Connecting via USB” on page 69. When using this option, it might take several minutes for the IP address to be assigned. During this time, the IP address and subnet mask on the front panel will read 0.0.0.0. Once the IP address and subnet mask have been assigned, both will be automatically updated on the front panel, and should no longer read 0.0.0.0. At this point, it should be possible to connect to the instrument from the computer. If you want to use a static IP address and subnet mask, please contact your IT department to obtain a valid address and mask.

Figure D-14: Standard settings on instrument front panel for instrument connectivity.

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Accessing instruments with DYNAMICS

To access an instrument connected via a LAN, USB, or via Ethernet when not on a LAN using DYNAMICS, please refer to the DYNAMICS User’s Guide.

Troubleshooting and diagnostics

Prior to troubleshooting any issues, ensure that the DynaPro Plate Reader II firmware is updated to the current version. See “DynaPro Plate Reader II Firmware Upgrades” on page 57 for instructions on how to do this. If you are experiencing instrument connectivity problems, please go over these steps. If you still cannot connect to your instrument after going over this section, please contact Wyatt Technology for assistance or visit www.wyatt.com for the latest troubleshooting guides.

Verifying instrument connections Please verify that the instrument is communicating with the computer. Open a Windows cmd prompt, as shown in Figure D-15. At the command line, type “ping” plus the IP address of the instrument as shown on the instrument front panel (see Figure D-14). If the instrument is connected properly, the result should be similar to that shown in Figure D-15.

Figure D-15: Using ping to verify the instrument connection.

If the instrument is not connected properly, the result should be similar to that shown in Figure D-16.

Figure D-16: Failure to connect to instrument using ping.

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 74 Index

Numbers D 1536-well 48 DB9 connection 18 384-well 48 DHCP 37, 73 96-well 48 diagnostic connection 18 diagnostics A www.wyatt.com 74 ACF tab 30 display alarm ACF tab 30 auto attenuating 33 alarm tab 32 N2 pressure 32, 33 autoscale 28 overheat 33 button description 24 purge enabled 33 Comm tab 37 SPCM enabled 33 main 24 stage communicating 32 navigating 24 tab 32 setting the scale 28 turning off 25 system tab 34 APD 7 system tab temperature controlled 33, auto correlation 7 34 avalanche photodiode 7 display range B adjusting 27 back panel 19 display. See LCD display Block Diagram 6 dynamic light scattering theory 7 Dynamics 6, 7 C installing 13, 14 CD Contents 14 software description 7 cleaning 15 DynaPro plate reader Comm tab 37 adjusting the display range 27 DCHP 37 connecting to 63 Compatible well plate 58 connecting via USB 69 concentration display window 23 maximum 61 LAN connection 64 minimum 61 load factory defaults 35 connecting network settings 73 to LAN 64 overview 6 to network 63 restart instrument 35 to PC 63 warning lights and alarms 25 via ethernet 71 zoom/pan buttons 27 via USB 69 connectors E back panel 19 Eject Plate 47 crossover cable 65 electronic fuse 19 Current environment plate position 47 location 15 equipment list 13

DynaPro Plate Reader II User’s Guide (M3101 Rev C) 75 Index ethernet cable 66 minimum sample volume 58 ethernet connection Move To 47 one instrument 72 N ethernet crossover cable 65 ethernet port 64 network settings 73 ethernet switch 67 Next Well 47 event history 32 nitrogen line installation 16 Event Scheduler 51 nitrogen purge 16 F O firmware upgrades 57 On/Off switch 18 Frequently Asked Questions operation below 20 °C. 61 plate reader 52 P front panel 18 packing list 13 H parts list 13 hazards, definitions of 10 plate hydrodynamic radius 7 loading 46 Plate Reader 38 I choosing correct inserts 39 installation choosing correct plates 39 DynaPro plate reader 15 loading samples 44 Installing Dynamics 14 Plate Reader Control instrument connections 64 buttons 47 verifying 74 launching 47 instrument firmware version 35 plates instrument serial number 35 choosing correct 39 recommended 39 K recommended inserts 40 keypad 18 Power 61 L power switch 18 LAN connection Prev Well 47 one instrument and computer 68 R laser Recommended well plate 59 description 21 restart instrument 35 On/Off 28 Restricted Privileges 14 warning 21 Laser Specifications 61 S LCD display 18 Samples load factory defaults 35 loading 44 Load Plate 47 loading tips 44, 45 Loading measuring automatically 51 plate 46 preparing 42 samples 44 volume and concentration 53 tips 44, 45 shipping list 13 location 15 software logging in to wyatt.com 57 Dynamics 6, 7, 13, 63, 74 low count rate installation 13 troubleshooting 62 SPCM 7 alarm 33 M specifications 61 maintenance static IP address 37, 73 general 56 Stokes-Einstein equation 7 manufacturer supported well plate formats 61 standard well plate 58 System Requirements 13 Measuring automatically 51 76 DynaPro Plate Reader II User’s Guide (M3101 Rev C) Index system tab 34 warning set time 35 laser 21 temperature controlled 33, 34 lights and alarms 25 well labeling system T 96-, 384-, 1536-well 48 Technical Specifications 61 well plate 39 temperature controlled 33, 34 choosing correct 39 touch-screen display 18 compatible 58 troubleshooting insert 39 www.wyatt.com 74 loading 46 troubleshooting low count rates 62 manufacturer 59, 60 U Wyatt Technology Corporation unpacking the instrument 13 corporate headquarters 9 upgrades 57 wyatt.com 57 USB adapter 66 X USB connection X-axis one instrument 70 selector 26 via crossover cable 69 Y V Y-axis data channel 26 ventilation 56 Z W zoom/pan buttons 27 warm up time 16, 39, 56

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