VICTOR NIVO MULTIMODE PLATE READERIntroduction 6 VICTOR Nivo – User Manual 1.1 Measurement technologies The VICTOR Nivo Multimode Plate Reader can be used to quantitatively measure

VICTOR NIVO™ MULTIMODE PLATE READER

User Manual

MULTI MODE DETECTION

Language Revision Publication Date

EN C 2017-10-12

Notices

The information contained in this document is subject to change without notice.

Except as specifically set forth in its terms and conditions of sale, PerkinElmer makes no warranty of any kind with regard to this document, including, but not limited to, the implied warranties of merchantability and fitness for a particular purpose.

PerkinElmer shall not be liable for errors contained herein for incidental consequential damages in connection with furnishing, performance or use of this material.

Copyright Information This document contains proprietary information that is protected by copyright.

All rights are reserved. No part of this publication may be reproduced in any form whatsoever or translated into any language without prior written permission of PerkinElmer, Inc.

Copyright © 2017 PerkinElmer, Inc.

Trademarks

PerkinElmer is a registered trademark of PerkinElmer, Inc. VICTOR Nivo is a trademark of PerkinElmer, Inc.

Contact address

World Headquarters

PerkinElmer, Inc 940 Winter Street, Waltham, Massachusetts 02451, USA.

(800) 551-2121

Service & Support Contact PerkinElmer

Support: http://www.perkinelmer.com/corporate/contactus

Service Contact Dazdaq (MyAssays)

Knowledge Base: http://www.dazdaq.com Email: [email protected]

Contents

VICTOR™ Nivo – User Manual 3

1 Introduction .................................................................................................................................. 5 1.1 Measurement technologies ............................................................................................................ 6 1.2 Safety ............................................................................................................................................. 6

1.2.1 Safety symbols ................................................................................................................... 6 1.2.2 Usage of the VICTOR Nivo ................................................................................................ 7 1.2.3 Safety instructions and warnings ....................................................................................... 7

1.3 Contents of delivery...................................................................................................................... 11

2 Reader installation ..................................................................................................................... 12 2.1 Installation .................................................................................................................................... 13 2.2 Hardware requirements ................................................................................................................ 13 2.3 Software requirements ................................................................................................................. 13 2.4 Reader positioning ....................................................................................................................... 14 2.5 Power and communication connections....................................................................................... 14 2.6 Turning the reader on and off ....................................................................................................... 15 2.7 Computer configuration and connecting the reader ..................................................................... 15

2.7.1 Connect directly with an Ethernet cable .......................................................................... 15 2.7.2 Connect with Wi-Fi Router ............................................................................................... 19 2.7.3 Making reader IP address a trusted site .......................................................................... 20

3 Initializing the reader ................................................................................................................. 23

4 Measurement protocols ............................................................................................................. 26 4.1 Protocols on the VICTOR Nivo .................................................................................................... 27 4.2 Creating a new protocol ............................................................................................................... 27

4.2.1 Plate Map tab ................................................................................................................... 27 4.2.2 Parameters tab ................................................................................................................ 28

4.2.2.1 Non-kinetic measurements .............................................................................. 28 4.2.2.2 Kinetic measurements ..................................................................................... 33 4.2.2.3 Selected operations list .................................................................................... 36 4.2.2.4 Measurement settings ...................................................................................... 37

4.3 Protocol handling .......................................................................................................................... 49 4.3.1 Copying a protocol ........................................................................................................... 49 4.3.2 Editing a protocol ............................................................................................................. 49 4.3.3 Deleting a protocol ........................................................................................................... 49 4.3.4 Exporting a protocol ......................................................................................................... 50 4.3.5 Importing a protocol ......................................................................................................... 51

5 Measuring with the VICTOR Nivo ............................................................................................. 53 5.1 Plate load / unload........................................................................................................................ 54 5.2 Active / non-active protocols ........................................................................................................ 55 5.3 Starting a measurement ............................................................................................................... 56

5.3.1 Adding a comment to measurement data ........................................................................ 57 5.4 Showing the results ...................................................................................................................... 57 5.5 Exporting the results..................................................................................................................... 58 5.6 Log out ......................................................................................................................................... 59

6 Temperature Control .................................................................................................................. 61 6.1 Temperature Control Overview .................................................................................................... 62 6.2 Temperature control from the main menu .................................................................................... 62 6.3 Warming within the protocol ......................................................................................................... 62

7 Shaker ......................................................................................................................................... 65 7.1 Shaker Operation overview .......................................................................................................... 66 7.2 Shaking from the main menu ....................................................................................................... 66 7.3 Shaking in the protocol ................................................................................................................. 67

8 Handling optical components ................................................................................................... 69 8.1 Optical components and user level .............................................................................................. 70 8.2 Dichroic mirrors ............................................................................................................................ 70

8.2.1 Changing a mirror slide .................................................................................................... 70 8.2.2 Creating a new mirror ...................................................................................................... 71

Contents

4 VICTOR™ Nivo – User Manual

8.2.3 Editing a mirror slide ........................................................................................................ 72 8.3 Optical filters ................................................................................................................................. 72

8.3.1 Loading a filter ................................................................................................................. 73 8.3.2 Unloading a filter .............................................................................................................. 74 8.3.3 Creating a new filter ......................................................................................................... 75

9 Dispenser and gas connectors ................................................................................................. 77 9.1 Dispenser option .......................................................................................................................... 78 9.2 Prime ............................................................................................................................................ 79 9.3 Wash ............................................................................................................................................ 81 9.4 Empty syringe ............................................................................................................................... 82 9.5 Gas connectors ............................................................................................................................ 83

10 Settings ....................................................................................................................................... 85 10.1 User Levels .................................................................................................................................. 86 10.2 Plate Wizard ................................................................................................................................. 87 10.3 Lock instrument for transport ....................................................................................................... 88

11 Maintenance ................................................................................................................................ 89 11.1 Dust removing .............................................................................................................................. 90 11.2 Spillage cleaning .......................................................................................................................... 90 11.3 Cleaning optical components ....................................................................................................... 90 11.4 Changing the air filter ................................................................................................................... 91 11.5 Dispenser maintenance ............................................................................................................... 92

12 Reader specifications ................................................................................................................ 93

Introduction


1 Introduction

Introduction


1.1 Measurement technologies The VICTOR Nivo™ Multimode Plate Reader can be used to quantitatively measure light emitting or absorbing substances both from top and bottom side of the plate, making it suitable for several different assay types including kinetic, cellular, enzymatic and autoimmune assays.

The VICTOR Nivo Multimode Plate Reader can offer five different measurement technologies: • Absorbance (ABS) • Fluorescence Intensity (FI) • Fluorescence Polarization (FP) • Time-Resolved Fluorescence (TRF) • Luminescence (LUM)

1.2 Safety

1.2.1 Safety symbols

Notice Failure to observe may result in invalid measurement results or damage of the instrument

Danger! Indicates a hazardous situation which, if not avoided, can result in death or irreversible injury.

Warning! Indicates a hazardous situation which, if not avoided, can result in severe but normally reversible injury.

Caution! Indicates a hazardous situation which, if not avoided, can result in pain or minor injury

Biohazard

Electrical voltage

Hot surface

Flammable material

Ultraviolet (UV) radiation

Corrosive material

Toxic material

Introduction


1.2.2 Usage of the VICTOR Nivo

Caution! Use only as intended.

• The VICTOR Nivo system, including the original accessories may only be used in conjunction with the methods described in the provided manuals. PerkinElmer does not assume any liability for any other applications or procedures, including use of individual subassemblies or components for other purposes.

• The manufacturer does not assume any liability for any other kind of application, including individual subassemblies and the addition of individual components. This exclusion of liability also applies to all service or repair work which was not carried out by authorized PerkinElmer service personnel.

For research use only. Not for use in diagnostic procedures.

The VICTOR Nivo is an automated Multimode Plate Reader for use in biological-medical and pre-clinical research only. The VICTOR Nivo is particularly suited for cellular and biochemical research applications.

1.2.3 Safety instructions and warnings

The VICTOR Nivo Multimode Plate Reader must be installed and used according to the instructions given by the manufacturer. The service must be done by properly trained personnel authorized by the manufacturer. The violation of these instructions may result in warranty invalidation or malfunction of the equipment.

The VICTOR Nivo Multimode Plate Reader may only be operated in a laboratory environment and by qualified laboratory staff who have been specially trained and are familiar with the contents of this manual. The user must implement common laboratory safety standards for protective clothing and handling and disposing of potentially biohazardous, corrosive and toxic materials. This device is designed for analyzing biological substances at biosafety level 1 and 2 (BSL-1 or BSL-2).

The user must not handle the external power supply or cord if it is damaged. The user must ensure that the power supply cord can be easily unplugged, if electric safety is compromised, for example due to a spill. The power cord may only be connected to the properly installed protective grounding socket.

Introduction


After warming the plate reader, care should be taken when removing the plate from the reader: the tray and plate may be too hot to handle barehanded. The heating elements inside the reader may also be hot. There is a hot surface warning label behind the plate loading door (see picture below).

Warning! Ethanol of other cleaning solvents may cause fire or injury if used in great amounts. Keep the concentration of dangerous solvents as low as possible and ensure the area is well ventilated.

The user must consult the manufacturer prior to using cleaning or decontamination agents, if there is any doubt about their suitability or compatibility.

Warning! Do not handle large amounts of liquids near or above the device and ensure the microtiter plate is correctly placed in the plate holder. If liquids should be spilled into the instrument accidentally, switch it off immediately and clean and decontaminate it, if needed. Clean any spillage leaking out of the instrument with adequate safety precautions, if the liquid is biohazardous, corrosive or toxic. Ensure the spillage has not accumulated inside the instrument and the safe operation of the device before taking it back into use. Consult the manufacturer or call the service in case of any doubt about safe operation or cleaning procedure after the spill.

Introduction


This device is designed for analyzing biological substances at biosafety level 1 and 2 (BSL-1 or BSL-2).

• Do not use more hazardous substances due to limited possibilities of decontamination.

• Observe the safety instructions for the substances used. • Observe the instructions for decontamination and cleaning. • Always make sure to comply with national and international

laboratory safety instructions. • Always wear your personnel safety equipment.

There is a biohazard warning label at the back of the plate loading door (visible when open, see figure below).

As supplied, reader does not provide any particular protection against the hazards evolving from aggressive chemicals, flammable or explosive fluids, biological substances or radioactive substances. Therefore, it is not allowed to use the reader with the above substances.

Warning! The user must inspect that there are no materials spilled outside the wells or preferably use a plate seal to prevent cross-contamination. When using the dispenser, extra care needs to be taken that the correct plate is inserted to the reader. Also, the bottles containing liquids to be dispensed should be placed so that they do not topple.

The user is responsible for ensuring that the material is not heated to the ignition or flash point of the material. Even though the maximum temperature of the measurement chamber is 65oC, the heating plate may heat up to 92oC. The user is responsible for ensuring that possible vapors from toxic or corrosive materials are ventilated before reaching dangerous concentrations.

Introduction


Danger! Carbon Dioxide Gas

In high concentration, carbon dioxide is harmful to health. Depending on the concentration it can lead to headache, tiredness, and dizziness, in higher concentrations even to unconsciousness, spasms, and death. Carbon dioxide is hardly perceptible.

If using the VICTOR Nivo reader with external gas control option, always follow the instructions and safety precautions provided with the gas control option. Do not install the VICTOR Nivo with gas control option in very small rooms or without sufficient room ventilation.

The instrument contains an UV radiation source, which is encapsulated in routine operation. If the cover of the instrument is removed for service or maintenance, the service engineer must unplug the power supply cord to ensure no UV radiation is emitted.

The service engineer must use protective clothing and ensure that the instrument is cleaned and decontaminated prior to opening the covers, if it has been introduced to biohazardous, corrosive or toxic materials.

The instrument must be cleaned and decontaminated prior to transport or disposal, if it has been introduced to biohazardous, corrosive or toxic materials.

Introduction


1.3 Contents of delivery The shipping box contains the following items (if any of the items is missing, please contact the manufacturer): • Packing list • VICTOR Nivo Multimode Plate Reader • External power supply and power cord • Ethernet cable • USB/Ethernet adapter • Wi-Fi router • USB stick containing

• PDF manuals (Installation Guide, Quick Start Guide, User Manual, Site Readiness document)

• VICTOR Nivo filter list • Installation Guide • Quick Start Guide Optional: • Dispenser • Gas control option • Tablet or laptop • Additional filter/dichroics • Test plate

Reader installation


2 Reader installation

Reader installation


2.1 Installation It is important that the reader installation is done according to the instructions given by the manufacturer in the Installation Guide provided with VICTOR Nivo.

2.2 Hardware requirements For using the reader the following hardware requirements will apply:

• The reader can be operated with any desktop PC, laptop or tablet. • For best user interface experience a screen resolution of 1600 x 900 pixels is

recommended. • 4 GB or more internal memory is recommended. • Any printer that is compatible with your computer can be used.

2.2.1 Control device power options

• Please disable any sleep mode of the control device. Data are stored temporarily in the browser cache during the measurements. If the control device switches into sleep mode, data will be lost before it can be stored automatically in the internal instrument data storage at the end of the measurement.

• During longer (kinetic) measurements, it is recommended to operate the control device using the power supply and not the battery.

How to disable the sleep mode (described for Windows® 10):

1. Open Control Panel > All Control Panel Items > Power Options.

2. Click Change plan settings to change the settings of the selected power plan.

Reader installation


3. Change option Put the computer to sleep to Never (for both “On battery” and “Plugged in”, if using a laptop).

2.3 Software requirements The reader can be operated using any device having either Microsoft Windows, Mac OS, iOS or Android installed.

The VICTOR Nivo comes with a pre-installed user interface software that is used to operate the reader and export the results. The user interface is accessed through a web browser. The following web browsers are recommended: • Google Chrome • Microsoft Edge

2.4 Reader positioning Place the reader on a flat and even table surface free of dust, liquids or acidic vapors. Avoid places subjected to direct sunlight and vibrations. Ensure that the power supply cord can be easily unplugged when needed.

2.5 Power and communication connections Ensure that the local main power matches the power requirements of the external instrument power. The power requirements are printed on the power adaptor. Attach the power cord and the Ethernet cable to the reader according to the picture below. The USB connector is used for service purposes only.

Ethernet

Power cord

USB connector for Service

Reader installation


2.6 Turning the reader on and off

• To turn the reader on, briefly press the power button. • To turn the reader off, press and hold down the power button for at least 3 seconds

until the light of the button goes out.

2.7 Computer configuration and connecting the reader The reader has a built-in web server for communication via a standard internet browser. The software for controlling the reader is located in this built-in web server and can be accessed via the browser. Communication between the server and the browser is via standard TCP/IP protocol. The pre-set IP address is:

• 192.168.0.2

There are two alternative ways to connect the PC and the reader:

• Direct communication with an Ethernet cable between control PC and the reader. Best suited if no other network connections are used

• Indirect communication with the included Wi-Fi router. Best suited for use with another network and for multi-user environments with many control PCs.

2.7.1 Connect directly with an Ethernet cable Connect the reader to the computer using the Ethernet cable. Configure the IP address of the computer LAN network to correspond to the nine first numbers of the reader IP address. Please note that these instructions are shown for a Windows 10 operating system.

• Control Panel - > Network and Internet

Open the Network and Sharing Centre. Select Change adapter settings.

Double click Ethernet2. Note that the name of the Ethernet connection can be different. Unplug the Ethernet cable from the reader and re-connect it to see which connection is the right one.

Reader installation


A pop up will appear on the screen.

Click Properties.

Reader installation


Select Internet Protocol Version 4 and click Properties. Select Use the following IP address. Enter IP address 192.168.0.100 and subnet mask 255.255.255.0

Reader installation


The first nine digits in the IP address are used to describe the network and the last three the actual device. To avoid IP conflicts, the LAN network to which the reader is connected must have the same first 9 numbers as the reader, but different last three digits.

Press OK and you are ready to start the reader.

Open your web browser to connect to the VICTOR Nivo. • Type http://192.168.0.2 into the address bar and press the Enter key.

The user interface appears and the instrument is initialized automatically.

• Make this page your default starting page.

Reader installation


2.7.2 Connect with Wi-Fi Router Connect the TP-LINK wireless router (LAN/WAN socket) to the Ethernet socket on the rear of the VICTOR Nivo using the provided LAN cable. Connect the wireless router (Power socket) to the USB power supply using the TP-LINK external USB power cable. Plug the USB power supply into a wall socket. Switch on the VICTOR Nivo using the power switch. Switch on your control device with wireless network capability (e.g. a laptop). • If you are using the optional laptop or tablet from PerkinElmer and start it for the first

time, the Windows 10 first start routine will appear. Configure user name, password, language settings etc.

Connect your control device with the instrument: • Make sure your wireless adapter is turned on. • Connect to the wireless network (see default network name on the router’s service

card, labeled with Wi-Fi Name (SSID)).

• Enter the password (see default password, labeled with Wi-Fi Password/PIN). To change the SSID network name and password of your wireless network, please refer to the router´s manual. Open your web browser to connect to the VICTOR Nivo. • Type http://192.168.0.2 into the address bar and press the Enter key.

The user interface appears and the instrument is initialized automatically.

• Make this page your default starting page.

7 8 9 1 +

Reader installation


2.7.3 Making reader IP address a trusted site To improve the connectivity of the reader it is recommended to add your reader’s IP address to the trusted sites. For this go to:

• Control Panel -> Network and Internet -> Internet Options

A view similar as below will appear on the screen.

Select the Security tab and a view similar as below will appear on the screen.

Reader installation


Select Trusted sites and click the Sites button. A view like below will appear on the screen.

If the box Require server verification (https:) for all sites in this zone is checked, remove the check mark.

Type http://192.168.0.2 into the field Add this website to the zone. By clicking Add you include the reader’s IP address to the trusted sites.

Initializing the reader


3 Initializing the reader



To start the reader, press the power button so that the light turns on. Open the web browser and type the reader’s IP address in the address bar and press the Enter key:

• http://192.168.0.2

The reader initialization will start and ask for a user name.

Please note: This user name is not mandatory, so the field can be left empty. The user name is used to separate the results and protocols for certain users. Standard users can only delete protocols and results created by themselves. The log in does not represent a complete user management, since the password is not mandatory and there is no database for different users. Detailed description of user names and user levels can be found in chapter 10 (Settings).



When the initialization is finished, the PROTOCOLS page will appear on the screen (see picture below). The reader control screen is arranged in the following areas:

1. Status bar: Current reader temperature, temperature control status and connection status

2. Navigation area: Access to different control pages for controlling the reader (referred to with capital letters in this manual)

3. Reader and software control buttons

4. Information bar: Shows which protocol is selected and the current user that is logged in

5. List of available protocols

6. Protocol control buttons

1 2 3 4

5 6

Measurement protocols


4 Measurement protocols



4.1 Protocols on the VICTOR Nivo The control of the reader is based on measurement protocols. A measurement protocol defines the plate format, plate map, measurement type, measurement technologies and measurement parameters. There are two types of protocols; locked default protocols and open custom protocols. Default protocols are marked with a closed red lock indicating that it is not allowed to edit or delete these protocols. As soon as a protocol is produced by a user it appears as a custom protocol and is marked with an open green lock indicating that it is allowed to make changes to these protocols.

Tip: When developing your own first protocols with the VICTOR Nivo, start with a copy of a closely matching default protocol. It will help you better understand the different parameters and workflows used.

Picture below shows the PROTOCOLS page.

4.2 Creating a new protocol Creating a new protocol starts by pressing the Create New button on the PROTOCOLS page. The protocol creation is split into plate map definition and parameters selection. . The user can define the plate format (96 well plate, 384 well plate …), plate map (which wells are measured), measurement type (endpoint, scan, kinetic …), measurement operations (FI, LUM, ABS, …) and measurement parameters (optical filters, measurement time …).

4.2.1 Plate Map tab On the Plate Map tab the user has to define a name for the protocol and can add a comment, if needed. The main purpose of the Plate Map page is to define the plate format and the wells to be measured. All plate formats from 1 – 1536 well plates are supported and most common plate types are included in the plate list. As a first step the plate format has to be selected, followed by choosing a plate type from a list of plates having that format.

The user can select the desired wells by keeping the left mouse button pressed while painting the area. The user can select multiple areas by keeping the control button down while selecting separate areas (see picture below).



4.2.2 Parameters tab On the Parameters tab the user can select the operation(s) that are performed in the protocol and organize them as desired. The user can also define the measurement parameters for each operation (see picture below). More detailed instructions are given in the chapter 4.2.2.4.

4.2.2.1 Non-kinetic measurements

The measurements with the reader can be either kinetic or non-kinetic. Kinetic operations are repeated multiple times. Non-kinetic operations are performed only once.

Measurement operation types (non-kinetic)

There are three different measurement types that can be used with almost all measurement technologies when a non-kinetic operation is selected. These measurement types are “Endpoint”, “Scan” and “Z-Focus scan”.



Endpoint

The measurement type “Endpoint” is used for assays where a chemical reaction has happened before and is either finished or stable at the time where the experiment is run. Usually, the measurement is done once at the end. This is the most commonly used type.

Scan

The measurement type “Scan” is used in situations where an XY-scanning of the well area is needed. The user can define the dimensions of the scanning area and how many measurement positions per well are used.

Z-Focus scan

The measurement type “Z-Focus scan” is selected in situations where the user wants to scan the well in vertical dimension. This feature is most commonly used to find the maximum signal of the well.

Measurement operations (non-kinetic)

Measurement operations define the measurement technology or technologies used in a protocol. An operation can also include other functions than a measurement – for example shaking. The list of available non-kinetic operations is as follows (see picture below). Please note that the list will vary based on the purchased reader model and options installed. • Fluorescence intensity (FI) • Fluorescence polarization (FP) • Time-resolved fluorescence (TRF) • Luminescence (LUM) • Absorbance with spectrometer (ABS) • Absorbance with optical filters (Filter ABS) • Dispenser • Temperature • Shaker • Timing



The Parameters page is divided into three sections. • Left area: Lists of available technologies/operations • Center area: Selected operations and processing sequence in the protocol • Right area: Details and parameters of currently selected operation

FI (Fluorescence intensity)

Fluorescence intensity is one of the most common measurement technologies. Here a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths. The emission light is generated almost simultaneously with the excitation.

The amount of emission light is measured very precisely using a high sensitivity photo multiplier tube (PMT). The result is presented as RFU values (relative fluorescence units).

FP (Fluorescence polarization)

Fluorescence polarization is a measurement technology where a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths, as in FI experiments (see above). The emission light is generated almost simultaneously with the excitation. The difference to fluorescence intensity is that in fluorescence polarization experiments the polarization of the light is taken into account. The excitation light is linearly polarized and exciting the sample in the so-called s-direction. The emitted light is measured twice, one time in s-direction, another time in p- The results of the S- and P-polarizations are presented as RFU values, but the final FP result is presented as Millipolarization (mP) value. The formula for calculating the mP value is as follows:

PSPSmP

+−

=

In the formula S represents the RFU value in S-polarization measurement and P represents the RFU value in P-polarization measurement.

In real experiments, optical elements can also influence the polarization of emission light. In order to take that into account, we recommend to do a re-calculation of the mP value. This step will also enable to calibrate the mP-results to the literature values referenced in an experiment. Therefore, a calibration factor G is introduced in the above formula, giving:



𝑚𝑚𝑚𝑚 = 𝑆𝑆 − 𝐺𝐺𝑚𝑚𝑆𝑆 + 𝐺𝐺𝑚𝑚

With G usually 0.5<G<1.5.

The G-factor has to be calculated experimentally based on a reference sample. For example, FITC has a polarization of 27mP in distilled water which can be used to evaluate the G-factor in a measurement.

TRF (Time-resolved fluorescence)

Time-resolved fluorescence is a measurement technology where a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths. The difference to fluorescence intensity is that in TRF the lifetime of the emission signal is quite long (µsec, vs. psec in FI). The long lifetime of the emission signal makes it possible to use a delay time between sample excitation and emission light collection. During this delay time almost all unwanted auto fluorescence emerging from e.g. buffer, plate, etc. fades away and the real emission signal is very pure. This feature improves the sensitivity of TRF measurements greatly.

Due to the long emission lifetime and the resulting lower intensity compared to FI the TRF signal is measured with a photomultiplier tube using an extremely sensitive photon counting technology. The TRF results are presented as Counts, which represent single photons.

LUM (Luminescence)

Luminescence is a measurement technology where a sample emits light based on a chemical reaction without any excitation. The long signal collection time makes it possible to use an extremely sensitive photon counting technology in luminescence. The luminescence results are presented as Counts.

ABS (Spectrometer-based absorbance)

Absorbance is a very common measurement technology where the amount of light absorbed by the sample is measured. In spectrometer-based absorbance it is possible to record the full spectrum between 230 nm – 1000 nm in a single measurement. The amount of light absorbed by the sample can then be presented for any wavelength. The absorbance values are presented as OD values, which corresponds to the optical density of the sample. The formula for calculating the OD value is as follows:

0

logIIOD −=

In the formula I represents the light intensity after the light has passed through the sample and I0 represents the light intensity without any sample.

Filter ABS (Filter-based absorbance)

Absorbance is a very common measurement technology where the amount of light absorbed by the sample is measured. In filter-based absorbance an optical filter is used to select only the needed wavelength for measurement. The amount of light absorbed by the sample is then presented as OD value, which corresponds to the optical density of the sample. The formula for calculating the OD value is as follows:

0

logIIOD −=




Dispensing

Dispensing operations can be used if the dispenser option is included in the reader. The dispenser allows to dispense liquids directly into the sample wells using one or two dispensing pumps.

Temperature

The Warming On operation is used for situations where an inbuilt temperature control has to be started while the measurement protocol is running. When the Warming On operation starts, the temperature control starts to warm the measurement chamber to the target temperature. When the target temperature is reached the protocol will continue.

The Warming Off operation is selected for situations where an inbuilt temperature control is switched off while the measurement protocol is running. When the Warming Off operations starts, it simply switches the temperature control off and continues the protocol.

Shaker

A Shaking operation is chosen for situations where a plate shaking feature is needed while the measurement protocol is running. When the Shaking operation starts, the plate holder shakes the plate for a defined period of time and continues the protocol.

Timing

Delay operations are used in situations where a certain delay time is needed while the measurement protocol is running. When the Delay operation starts, the reader waits for a defined period of time and continues the protocol. The user can define, if the delay step is performed inside or outside the reader, indicated by the sub-operations “Delay” and “Delay outside”, respectively.



4.2.2.2 Kinetic measurements

The measurements with the reader can be either non-kinetic or kinetic. Non-kinetic operations are performed only once, but kinetic operations are repeated multiple times.

Measurement operation types (kinetic)

Kinetic operations are divided into Plate Kinetics and Well Kinetics. Measurement operations are added afterwards to one or the other. Therefore, the first step during the protocol setup of kinetic measurements is to decide, if the reader should perform plate or well kinetics.

Plate Kinetics

If Plate Kinetics is selected, the user can add measurement operations to a kinetic operation and repeat this sequence as many times as wanted. In plate kinetics one operation is always performed on all the selected wells in the plate map first and after that the next operation in the list will start. The operations in the Plate Kinetics list are repeated as many times as it was defined by the user. This type of measurement order is also known as plate repeats. The plate kinetic start is marked with Plate kinetics – Plate start in the selected operations list. Plate kinetics – Plate end marks the end of the kinetic operation. Selected operations can include both, kinetic operations and non-kinetic operations. When adding operations to the kinetic protocol, first click Plate kinetics – Plate start. To insert an operation after the kinetic operation, first click Plate kinetics – Plate end.

Well K inetics

If Well Kinetics is selected the user can add measurement operations to be performed within a kinetic operation and repeat this sequence as many times as wanted. Well kinetics means that the whole kinetic operation list is performed for one well first and after that the whole operation list is performed on the next well and so on. This type of measurement order is also known as well repeats. The well kinetic start is marked with Well kinetics – Well start in the selected operations list. Well kinetics – Well end marks the end of kinetic operation. Selected operations can include both kinetic operations and non-kinetic operations. When adding operations to the kinetic protocol, first click Well kinetics – Well start. To insert an operation after the kinetic operation, first click Well kinetics – Well end.

Measurement operations (kinetic)

Measurement operations define the measurement technology or technologies used in a kinetic protocol. An operation can also include other functions than measurements – for example shaking. The list of available kinetic operations is as follows. Please note that the list will vary based on the purchased reader model and options installed. • FI-Kinetics (Fluorescence intensity in kinetic protocol) • FP-Kinetics (Fluorescence polarization) • TRF-Kinetics (Time-resolved fluorescence) • LUM-Kinetics (Luminescence) • ABS-Kinetics (Absorbance with spectrometer) • Filter ABS-Kinetics (Absorbance with optical filters) • Dispensing in Kinetics • Shake in Kinetics • Delay in Kinetics



FI Kinetics (Fluorescence intensity in k inetics)

Fluorescence intensity is one of the most common measurement technologies. Here a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths. The emission light is generated almost simultaneously with the excitation.

The amount of emission light is measured very precisely using a high sensitivity photo multiplier tube (PMT). The result is presented as RFU values (relative fluorescence units).

FP Kinetics (Fluorescence polarization in k inetics)

FP-Kinetics means that a fluorescence polarization measurement is performed within a kinetic protocol. Fluorescence polarization is a measurement technology where a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths, as in FI experiments (see above). The emission light is generated almost simultaneously with the excitation. The difference to fluorescence intensity is that in fluorescence polarization experiments the polarization of the light is taken into account. The excitation light is linearly polarized and exciting the sample in the so-called s-direction. The emitted light is measured twice, one time in s-direction, another time in p- The results of the S- and P-polarizations are presented as RFU values, but the final FP result is presented as Millipolarization (mP) value. The formula for calculating the mP value is as follows:

PSPSmP

+−

=

In the formula S represents the RFU value in S-polarization measurement and P represents the RFU value in P-polarization measurement.

In real experiments, optical elements can also influence the polarization of emission light. In order to take that into account, we recommend to do a re-calculation of the mP value. This step will also enable to calibrate the mP-results to the literature values referenced in an experiment. Therefore, a calibration factor G is introduced in the above formula, giving:

𝑚𝑚𝑚𝑚 = 𝑆𝑆 − 𝐺𝐺𝑚𝑚𝑆𝑆 + 𝐺𝐺𝑚𝑚

With G usually 0.5 < G < 1.5.

The G-factor has to be calculated experimentally based on a reference sample. For example, FITC has a polarization of 27mP in distilled water which can be used to evaluate the G-factor in a measurement.

TRF Kinetics (Time-resolved fluorescence in k inetics)

TRF-Kinetics means that a time-resolved fluorescence measurement is performed within a kinetic protocol. Time-resolved fluorescence is a measurement technology where a sample is excited with a certain excitation wavelength and then the sample emits the emission light with longer wavelengths. The difference to fluorescence intensity is that in TRF the lifetime of the emission signal is quite long (µsec, vs. psec in FI). The long lifetime of the emission signal makes it possible to use a delay time between sample excitation and emission light collection. During this delay time almost all unwanted auto fluorescence emerging from e.g. buffer, plate, etc. fades away and the real emission signal is very pure. This feature improves the sensitivity of TRF measurements greatly.

Due to the long emission lifetime and the resulting lower intensity compared to FI the TRF signal is measured with a photomultiplier tube using an extremely sensitive photon counting technology. The TRF results are presented as Counts, which represent single photons.



LUM Kinetics (Luminescence in k inetics)

LUM-Kinetics means that a luminescence measurement is performed within a kinetic protocol. Luminescence is a measurement technology where a sample emits light based on a chemical reaction without any excitation. The long signal collection time makes it possible to use extremely sensitive photon counting technology in luminescence. The luminescence results are presented as Counts.

ABS Kinetics (Spectrometer-based absorbance in k inetics)

ABS Kinetics means that a spectrometer based absorbance measurement is performed within a kinetic protocol. Absorbance is a very common measurement technology where the amount of light absorbed by the sample is measured. In spectrometer based absorbance it is possible to record the full spectrum between 230 nm – 1000 nm. The amount of light absorbed by the sample can then be presented in any wavelength. The absorbance values are presented as OD-values, which corresponds to the optical density of the sample. The formula for calculating the OD value is as follows:

0

logIIOD −=


Filter ABS K inetics (Filter-based absorbance in k inetics)

Filter ABS Kinetics means that a filter-based absorbance measurement is performed within a kinetic protocol. Absorbance is a very common measurement technology where the amount of light absorbed by the sample is measured. In filter-based absorbance an optical filter is used to select only the needed wavelength for measurement. The amount of light absorbed by the sample is then presented as OD value, which corresponds to the optical density of the sample. The formula for calculating the OD value is as follows:

0

logIIOD −=


Dispensing in Kinetics

Dispensing in kinetics means that a dispensing operation is performed within a kinetic protocol. Dispensing operations can be selected if a dispenser option is included in the reader. The dispenser allows dispensing liquids directly into the sample wells using one or two dispensing pumps. Using Dispensing in Kinetics the user can choose on which kinetic cycles the dispensing is performed.

Shake in Kinetics

Shake in kinetics means that a shaking operation is performed within a kinetic protocol. Shaking operations are useful in situations where a plate shaking step is needed while the measurement protocol is running. When the Shaking operation starts, the plate holder shakes the plate for a defined period of time and continues the protocol afterwards.



Delay in K inetics

Delay in kinetics means that a delay operation is performed within a kinetic protocol. Delay operations are used in situations where a certain delay time is needed while the measurement protocol is running. When the Delay operation starts, the reader waits for defined time and continues the protocol. The user can define, if the delay step is performed inside or outside the reader, indicated by the sub-operations “Delay in kinetics” and “Delay outside in kinetics”, respectively.

4.2.2.3 Selected operations list

The Selected operations list shows the content of the protocol (see picture below). One or more operations can be used in the protocol and are part of this list. In the Selected operations list the user can change the order of operations as needed, the protocol proceeds with the operations from top to bottom. One protocol can include a kinetic part and also non-kinetic operations.



4.2.2.4 Measurement settings

If the user selects any operation on the Selected operations list, the measurement settings of that operation appear on the right side of the screen (see picture below). In most cases there is no difference in the available measurement settings between non-kinetic or kinetic operations. When developing a new protocol, start with the default settings for the parameters and adjust them, if needed, during the assay development process.



Measurement settings – FI

The available measurement settings for fluorescence intensity are as follows (part of the parameters are shown only in Expert mode, which can be activated by checking the box on the top right corner): • Measurement type

o Single label (one measurement) o Dual excitation (two measurements with different excitation filters and same

emission filter) o Dual emission (two measurements with same excitation filter and different

emission filters) o Dual excitation/emission (two measurements with different excitation filters and

different emission filters) • Excitation filter (select a filter from the list) • Emission filter (select a filter from the list) • Dichroic mirror (separates the excitation and emission wavelengths, select a mirror

from the list) • Measurement direction (top or bottom side of the plate) • Measurement time (measurement time per well in milliseconds) • Z-Focus (measurement spot position in vertical direction, millimeters from the plate

holder surface) • Measurement spot size (select excitation and emission spot sizes from the list) • Flash energy (energy of the individual flash lamp pulse) • Instrument settings (high voltage of the photomultiplier tube – gain adjustment) • Measurement order (order of wells to be measured)

See picture below for the FI settings when Expert mode is checked.



Measurement settings – FP

The available measurement settings for fluorescence polarization are as follows (parts of the parameters are shown only in Expert mode, which can be activated by checking the box on the right top corner). When measuring fluorescence polarization, two individual emission filters with the same specifications are needed (one for S polarization and a second one for P polarization. Polarization filters are not needed; they are located inside the reader). • Excitation filter (select a filter from the list) • Emission filter S (select a filter from the list) • Emission filter P (select a filter from the list) • Dichroic mirror (separates the excitation and emission wavelengths, select a mirror

from the list) • Measurement direction (top or bottom side of the plate) • Measurement time (measurement time per well in milliseconds) • Z-Focus (measurement spot position in vertical direction, millimeters from the plate

holder surface) • Measurement spot size (select excitation and emission spot sizes from the list) • Flash energy (energy of the individual flash lamp pulse) • Instrument settings (high voltage of the photomultiplier tube – gain adjustment) • Measurement order (order of wells to be measured) See picture below for the FP settings when Expert mode is checked.



Measurement settings – TRF

The available measurement settings for time-resolved fluorescence are as follows (parts of the parameters are shown only in Expert mode, which can be activated by checking the box on the right top corner). • Measurement type

o Single label (one measurement) o Dual excitation (two measurements with different excitation filters and same

emission filter) o Dual emission (two measurements with same excitation filter and different

emission filters) o Dual excitation/emission (two measurements with different excitation filters and

different emission filters) • Excitation filter (select a filter from the list) • Emission filter (select a filter from the list) • Dichroic mirror (separates the excitation and emission wavelengths, select a mirror

from the list) • Time-resolved settings

o Delay time (time between sample excitation and emission counting in microseconds)

o Emission time (Emission signal counting time in microseconds) • Measurement direction (top or bottom side of the plate) • Measurement time (measurement time per well in milliseconds) • Z-Focus (measurement spot position in vertical direction, millimeters from the plate

holder surface) • Measurement spot size (select excitation and emission spot sizes from the list) • Flash energy (energy of the individual flash lamp pulse) • Measurement order (order of wells to be measured)

See picture below for the TRF settings when Expert mode is checked.



Measurement settings – LUM

The available measurement settings for luminescence are as follows (parts of the parameters are shown only in Expert mode, which can be activated by checking the box on the right top corner). • Measurement type

o Single label (one measurement) o Dual emission (two measurements with different emission filters)

• Emission filter (select a filter from the list). For luminescence measurements the filter “IR Blocker” is recommended to reduce the non-wanted background signal generated by most white plates.

• Measurement direction (top or bottom side of the plate) • Measurement time (measurement time per well in milliseconds) • Z-Focus (measurement spot position in vertical direction, millimeters from the plate

holder surface) • Measurement spot size (select emission spot size from the list) • Measurement order (order of wells to be measured)

See picture below for the LUM settings when Expert mode is checked.



Measurement settings – ABS

The available measurement settings for absorbance are as follows (parts of the parameters are shown only in Expert mode, which can be activated by checking the box on the right top corner). • Measurement mode

o Band (only one or multiple bands from the spectrum 230 nm – 1000 nm are measured)

o Whole spectrum (wavelengths between 230 nm – 1000 nm are measured) • Bandwidth (if measurement mode “Band” is selected, this defines the measured

wavelength range) • Wavelength selection (if measurement mode “Band” is selected, the user can add

several wavelength bands to be measured) • Ratio calculation (if measurement mode “Band” is selected, the user can define two

wavelengths where the ratio of absorbance values is calculated) • Measurement time (measurement time per well in milliseconds) • Measurement order (order of wells to be measured)

See picture below for the ABS settings when Expert mode is checked.



Measurement settings – Filter ABS

The available measurement settings for filter-based absorbance are as follows (parts of the parameters are shown only in Expert mode, which can be activated by checking the box on the right top corner). • Excitation filter (select a filter from the list) • Measurement time (measurement time per well in milliseconds) • Measurement order (order of wells to be measured)

See picture below for the Filter ABS settings when Expert mode is checked.



Measurement settings – Dispensing in non-kinetic protocol

Dispensing settings are different in non-kinetic protocols and in kinetic protocols. In the non-kinetic protocol each well selected in the plate map is filled according to the dispensing step parameters. Only after that the next operation in the protocol is started. The available settings for dispensing in non-kinetic protocols are as follows. • Reagent 1 (select whether to use dispensing pump 1 or 2 for dispensing the reagent). • Dispensing speed (select one of the three dispensing speeds. Generally speaking,

fluid with greater viscosity requires slower dispensing speed.) • Dispensing volume (define the dispensing volume in microliters).

Warning! Take care when defining the parameters for dispensing to avoid over filling the wells. This can cause spillage and contamination of the reader.

See picture below for the dispensing settings in a non-kinetic protocol.



Measurement settings – Dispensing in kinetic protocol

Dispensing settings are different in non-kinetic protocols and in kinetic protocols. The available settings for dispensing in kinetic protocols are as follows: • Reagent 1 (select whether to use dispensing pump 1 or 2 for dispensing the reagent). • Reagent 2 (select to dispense the second reagent with the other dispensing pump.

Reagent 2 selection becomes active, if more than one kinetic cycle is selected.) • Dispensing speed (select one of the three dispensing speeds. Generally speaking,

fluid with greater viscosity requires slower dispensing speed.) • Dispensing volume (define the dispensing volume in microliters). • Delay (select a certain delay time after dispensing the reagent). • Kinetic dispensing (select to dispense the first reagent on every kinetic cycle or only

on selected kinetic cycles. If a second reagent is selected, define the kinetic cycle for the dispense step when it is dispensed.)

Warning! Take care when defining the parameters for dispensing to avoid over filling the wells. This can cause spillage and contamination of the reader.

See picture below for the dispensing settings in a kinetic protocol.



Measurement settings – Warming On (non-kinetic)

The temperature control operation can be selected to be part of the protocol. It will insert a Warmin On and Warming Off step into the protocol. Warming of the reader can also be controlled separately from the protocols on the TEMPERATURE page. The temperature target given in the protocol will overwrite the target given on the TEMPERATURE page, but after the protocol is finished, the temperature target will return to the value set on the TEMPERATURE page.

The available settings in the Warming On operation are as follows: • Target temperature: select the target temperature for the temperature control. When

the warming operation starts, the temperature control starts to warm the reader to target temperature. If the box Wait until the target is reached is ticked, the following operation will start after the target temperature is reached. If the box is not ticked, the next operations will start immediately even if the reader is still warming.

Measurement settings – Warming Off (non-kinetic)

The temperature control can be selected to be part of the protocol. The Warming Off operation simply turns the temperature control off during the protocol. The following operation starts automatically. Please note that it will take time before the reader is cooled down.

Tip: Insert a Delay outside operation before the following operation, if you want to let the instrument cool down before proceeding.



Measurement settings – Shaking

It is possible to use the reader´s XY-conveyor for shaking the plate during a protocol. The available settings for the Shaking operation are as follows: • Shaking type

• Linear (long side) • Linear (short side) • Orbital • Double orbital

• Shaking speed • Slow (300 rpm) • Medium (600 rpm) • Fast (1200 rpm))

• Shaking time (define the shaking time in hours, minutes and seconds)

See picture below for the shaking settings.



Measurement settings – Delay

It is possible to have a delay time in the protocol using a delay operation. See the picture below for the delay settings. It is also possible to select Delay outside. Then the plate holder will come out during the delay operation.



4.3 Protocol handling

4.3.1 Copying a protocol If you want to modify an existing protocol, but still keep the original protocol unchanged, it is possible to make a copy of the original protocol. First, select the right protocol and then click Copy. A pop-up window to enter the name of the new protocol will open (see picture below). Type in the new name and click OK.

4.3.2 Editing a protocol If you want to modify an existing protocol, select a protocol and click Edit. Note that only an Admin user or Standard users with identical user names can edit custom protocols (marked with open green locks). The default protocols (marked with closed red locks) can’t be edited. When an existing protocol is edited, the protocol page is similar to when creating a new protocol (see chapter 4.2).

4.3.3 Deleting a protocol If you want to permanently delete an existing protocol, select a protocol and click Delete. A pop-up window for confirming the deletion will open (see picture below). Note that only an Admin user or a Standard user logged in with the identical user name can delete custom protocols (marked with open green locks).The default protocols (marked with closed red locks) can’t be deleted.



4.3.4 Exporting a protocol If you want to export an existing protocol to a different reader, select a protocol and click Export. A pop-up window showing the content of the protocol will open (see picture below). Select the whole text and copy it to the clipboard e.g. by pressing Ctrl+C. Open a text editor program (Notepad for example) and paste the content of the protocol by e.g. pressing Ctrl+V. Save the text file.



4.3.5 Importing a protocol If you want to import an existing protocol from a different reader, click Import. A pop-up window for protocol name and content of the protocol will open (see picture below). Enter a name for the new protocol and open the file that contains the data of the existing protocol (see chapter 4.3.4). Copy the whole content of the existing protocol from the text file and paste it to the new protocol by setting the mouse cursor into the Protocol data field and pressing Ctrl+V.

Measuring with the VICTOR Nivo


5 Measuring with the VICTOR Nivo



5.1 Plate load / unload To open the plate loading door: Click the OPEN TRAY icon in the upper right corner. The tray will come out. Place the plate so that its A1 position points to the A1 position indicated in the tray (see figure below).

To close the tray, click the CLOSE TRAY icon and the tray with the plate will be inserted into the reader and the door will close.



5.2 Active / non-active protocols Protocols in the protocol list can be either labeled as Active or Not Active. The status of the protocol is shown in parenthesis after the protocol name in the status bar once you have selected it. • “Active” means that the protocol is ready to be run. • “Not Active” means that the protocol cannot be run.

There is a reason why the protocol is not active (displayed in the upper right corner of the status bar): In the example below, there are missing optical components that the user should insert into the reader before the protocol can be started (see chapter 8 for handling the optical components).

Missing optical components can mean the following: • The filter used in the protocol is not installed in the reader. Open the protocol and

select the Parameters tab. Make sure that the filter used in the protocol is not grayed out. Either select a filter that is installed in the reader or load the filter into the reader. Please note that only Admin users can load filters.

• A mirror or mirror slide used in the protocol is not installed in the reader. Open the protocol and select the Parameters tab. Make sure that the mirror used in the protocol is not grayed out. Either select a mirror that is installed in the reader or load the right mirror into the reader. Please note that only Admin users can edit mirror slides.

• The filter or mirror might be present, but the name given to the filter/mirror does not match the name of the filter/mirror used in the protocol. Either load the filter/mirror with the correct name or change the name of the used filter/mirror.

Other reason for a protocol being non-active: • Selected protocol uses dispensers that are not primed (see chapter 9.2 for dispenser

priming).



5.3 Starting a measurement If the measurement protocol is active, the measurement can be started by clicking the START button. Select the protocol you want to measure and start the measurement by clicking START. Start buttons are located at the end of each protocol bar or in the upper right corner next to the OPEN TRAY button. If you want to edit, for example, the plate map before running the protocol, select Edit.

Note: You can only edit protocols labeled with green open locks. The ones with a red lock cannot be changed. If you want to use locked protocols as a template, make a copy of a locked protocol by selecting Copy and then edit it freely.



5.3.1 Adding a comment to measurement data After starting the protocol, a window will pop-up asking: Do you want to start measurement? At this point it is possible to add a comment to the measurement. This comment can be edited after the measurement was finished and is also exported with the measurement results. It is possible to start the protocol without entering any comments.

5.4 Showing the results When a measurement protocol is started, results will be shown on the screen during the measurement process when the measurement proceeds. The headline above the plate map displays the protocol type and the time that has passed after starting the protocol. From the panel on the left, the user can select different options to view the results from the drop-down menus. The possible choices depend on the protocol type that is run.

• The first drop-down menu shows the results in the plate map either as a value or as a

color, which is dependent on the signal level. • The second drop-down menu shows the results either by plate or by well. By well

selection is possible, for example, when measuring an absorbance spectrum.



• The third drop-down menu shows the results for the selected measurement operation in the protocol that contains several measurement operations.

• The fourth drop-down menu shows the results for different filters when measuring dual label or fluorescence polarization protocols.

• The fifth drop-down menu shows the results of different kinetic cycles when a kinetic protocol is run.

5.5 Exporting the results When the measurement is finished, the results can be seen on the screen (figure below). The data is saved automatically in the instrument.

The results can be exported by clicking Export. A new window will open showing the results in text form (figure below).

The results can then be exported for further analysis in e.g. a spreadsheet calculation software by clicking Export XLSX data. A Microsoft Excel formatted file with the results will be generated and opens automatically, if respective software is installed on the PC. The export file name can be changed by entering a name into the File name field. Its default file name is [protocol name_date_time]. A measurement comment can be added



or edited (see section 5.3.1) by typing the comment to the Measurement Comment field and pressing the Update measurement comment button.

A second export option is Export CSV data, which will save the data in the csv format. The user can select the delimiter of the cells to be comma, tab or semicolon. Also, the decimal separator can be selected (point or comma).

5.6 Log out When measurements are done and results are exported, if needed, the user should log out from the instrument and free it for the next user. This can be done by clicking the Log out – button (figure below).

Select Yes and the following page will appear. After successful log out, the browser window can be closed.

Temperature Control


6 Temperature Control

Temperature Control


6.1 Temperature Control Overview The temperature inside the measurement chamber can be controlled within a range of ambient + 3° C up to 65°C in two ways: from the main menu by clicking the TEMPERATURE icon or by adding the warming operation to the protocol as a protocol step. When the warming is on, the indicator Temp Ctrl status on the upper left of the screen will change from OFF to show the target temperature value. A red temperature target value means that the reader is still warming. When the target temperature is reached, the color of the target value will change to white. Remember to turn the warming off when it is not needed any more.

Please note that cooling down the reader can take several hours depending on reader temperature and room temperature.

6.2 Temperature control from the main menu Select TEMPERATURE from the main menu:

This function has only one parameter: Target temperature/°C, where you can set the desired temperature (in the example, 37 °C). When the warming is on, the target temperature will appear at the upper bar below the current temperature display.

6.3 Warming within the protocol Warming can be added as a step in a protocol via the Parameters tab - Available operations – Temperature – Warming On). The target temperature can be set under Warming Parameters on the right of the screen. Please note that the selected operations will be performed in the order in which they are listed. Warming Off will stop the warming. This should be added to the Selected operations list, if the temperature control needs to be turned off after measuring the protocol.

Please note that cooling down the reader can take several hours depending on reader temperature and room temperature.

Temperature Control


Shaker


7 Shaker

Shaker


7.1 Shaker Operation overview The Shaker operation can be used if plate shaking is needed between different measurements. The shaking amplitude and frequency are defined automatically depending on the plate format, but the user can still define the shaking type, speed, interval and intensity. As for Warming, the Shake operation can be set in two ways: from the main menu by clicking the SHAKER icon or by adding the shaking to the protocol as a protocol step.

7.2 Shaking from the main menu Select the SHAKER icon from the main menu. The picture below shows the shaking parameters.

• Linear (long side): The plate is shaken in one dimension (along the long side of the

plate). • Linear (short side): The plate is shaken in one dimension (along the short side of the

plate). • Orbital: The plate is shaken in circular orbit. • Double orbital: The plate is shaken in a “number eight” orbit. • Slow is a very careful shaking with larger amplitude (suitable for cells). • Medium is a medium speed shaking. • Fast is a shaking type with very efficient mixing. • Shake Time allows the user to select the duration of shaking. Maximum shaking time

is 24 hours.

You can start the shaking by selecting Start Shake and stop it by selecting Stop Shake.

Warning! Shaking of a plate with overfilled wells or at too high speed can cause spilling and contamination of the reader.

Shaker


7.3 Shaking in the protocol Shaking can be added as a step in a protocol (Parameters – Available operations – Shaker – Shaking). You can select the shake type, speed and time (picture below). Please note that all operations in Selected operations will be performed in the order in which they are listed.

Shake Type • Linear (long side): The plate is shaken in one dimension (long side of the plate). • Linear (short side): The plate is shaken in one dimension (short side of the plate). • Orbital: The plate is shaken in circular orbit. • Double orbital: The plate is shaken in a “number eight” orbit. Shake Speed • Slow is a very careful shaking with larger amplitude (suitable for cells). • Medium is a medium speed shaking. • Fast is a shaking type with very efficient mixing. • Shake Time allows the user to select the duration of shaking. Maximum shaking time

is 24 hours.

Handling optical components


8 Handling optical components



8.1 Optical components and user level The user can handle the optical components of the reader (mirrors and optical filters) depending on the user level settings (see chapter 10 (Settings)).

8.2 Dichroic mirrors Click the OPTICS icon on the main menu. There you can handle the dichroic mirrors that are installed in the reader. • As a Standard user, you can change the mirror slides. • As an Admin user, you can change the mirror slide, but also change the order of the

mirrors, remove mirrors or add new mirrors to a slide.

8.2.1 Changing a mirror slide While on the OPTICS page, click Unload Slide. The lid at the front will open and the mirror slide will be ejected (see figures below).

Remove the mirror slide and replace it with a different slide. Push the slide in so that all mirrors in the slide are inside the reader. In the software click the Load Slide button of the slide you want to insert into the reader. The slide will start moving inside the reader. Close the optics loading door.



8.2.2 Creating a new mirror Admin users can create new mirrors by clicking the New Mirror button. A new window will open where the user can enter the mirror name and the parameters. • Click Save and Exit to create this new mirror and add it to the mirror list. • Clicking the Exit button will bring you back to the OPTICS page without creating a

new mirror.

Please note: If you plan to use a new mirror with an existing protocol, make sure, that you name the mirror exactly the same way as it is named in the protocol. Otherwise, the software will not be able to match the protocol with the mirror. It will then refuse to start the protocol and indicate that an optical component is missing.



8.2.3 Editing a mirror slide Admin users can edit mirror slides by selecting the slide from the drop-down menu and clicking Edit. The following window will open:

From the drop-down menus mirror positions can be edited. Click Done to save the changes or Cancel to exit to the OPTICS page without saving the changes.

Please note: It is recommended to name the slides on their labels and to use the same name in the software.

8.3 Optical filters Click the OPTICS icon on the main menu. There you can handle the optical filters that are installed in the reader. • As a Standard user, you can see the optical filters that are installed in the reader. • As an Admin user, you can also load the optical filters, unload the filters or create new

optical filters.

Filters in the filter list are labeled depending on their presence in the instrument. • Filters currently present in the instrument are shown on a bright background. • Filters that have been defined already, but are currently not in the instrument are

shown on a dark background.

Additionally, default filters cannot be edited and are labeled with a closed, red lock. Custom filters can be edited and deleted and are indicated by an open, green lock.



8.3.1 Loading a filter

Notice Be careful when loading and unloading filters. Follow the software’s instructions closely. Otherwise it can happen that the filter library will get mixed up, potentially resulting in instrument malfunctions.

Pick a filter on dark background from the list that you want to load inside the reader. If there is no suitable filter in the list, see chapter 8.3.3.

Once you have found a suitable filter, select it and click Load filter. The instrument will turn the loading position to the user and open the optics loading door. When this process is finished the instrument will ask you to place the filter into the loading position (see below).



Place the new filter in the loading position. Make sure that the letters on the filter’s label can still be read and are not facing upside down (figure below).

Notice Always use the filter loading position to insert filters into the filter storage! Never place a filter into any other position. The loading position is presented by the instrument following the filter loading process in the software. The filter loading position is identifiable by its low frame for easy filter access. All other positions contain a high frame and must not be used for loading.

Close the optics loading door and click Done, if you are ready. The instrument moves the filter into the filter storage system. Now the filter should be visible in the filter list as a loaded filter on a bright background.

8.3.2 Unloading a filter Select a present filter (indicated by a bright background) from the list and click Unload filter (in the example below, the 260/10 nm filter was selected).



The instrument will automatically move the filter to the loading position and open the loading door. When this process is finished the instrument will ask you to “Unload now the filter” (see picture below). Once you have taken out the filter, click Done. If you do not wish to remove the filter, leave the filter in the loading position and click Cancel.

8.3.3 Creating a new filter If the filter name cannot be found in the drop-down menu, you can create new filters by clicking Create Filter. The filter name and filter parameters can be inserted in the software dialogue. Please note: If you plan using a new filter with an existing protocol, make sure, that you name the filter exactly the same way as it is named in the protocol. Otherwise, the software will not be able to match the protocol with the filter, will refuse starting the protocol and indicate that an optical component is missing.

Clicking Save and Exit will create this new filter and add it to the filter list. The Exit button will bring you back to the OPTICS page without creating a new filter.



Filters created by users are marked with a green lock and can be edited or deleted. However, you can only delete such a filter if it is not currently used in a protocol.

Dispenser and gas connectors


9 Dispenser and gas connectors



9.1 Dispenser option A dispenser with two pumps is provided as an optional feature. The photo below shows a two-pump dispenser installed on the reader. Please note that it is not recommended to touch the dispenser pumps while they are moving. Also, the user should take extra care to ensure that the bottles containing the liquids to be dispensed are stable and do not topple.

Before and after dispenser operation, it is recommended to routinely wash the dispenser pumps and tubing (see chapter 9.3).

Naming of the dispenser pumps in the software: • Left pump unit: Pump 1 • Right pump unit: Pump 2



9.2 Prime Before using the dispenser in your measurements, the pumps need to be primed with the liquid you plan to dispense (e. g., water, a buffer, a suspension, etc.). To do this, select DISPENSER from the main menu (figure below):

Remove the dispenser tip from the reader and place it into a waste container. Place the injection tubes into the liquid you are using for your experiment and click Wash/Prime. Priming needs to be performed until the liquid is flowing consistently from the tip. This will appear on the screen (after you have primed only the left pump 1):

Once this is done, repeat it for the right pump 2 (figure below) if needed.



Put the dispenser tip back into the reader. After this has been done, the dispenser will be active and ready to use.

Please note: Priming can also be performed by using the buttons located at the dispenser unit. • The PUMP SEL button is used to select the pump. Green light will indicate the

selected pump. • The WASH/PRIME button will start the priming of the pump. Priming does not start if

the dispensing tip is inside the reader. Press the WASH/PRIME button as many times as needed to confirm that the liquid flows consistently from the tip.

• The EMPTY button will start emptying the syringe.



9.3 Wash Before and after utilization, the pump(s) should be thoroughly washed. You can do this, depending on the reagents being dispensed, by washing it several times with pure water and ethanol:

1. Three times with water

2. Three times with ethanol

3. Three times with water (or more, to be sure to remove any ethanol left).

For cleaning after the dispense operations, empty the dispenser by priming with air using 2-3 more Prime operations, this time with the reservoir container removed. You do this by going back to the main menu selecting DISPENSER and then clicking Wash/Prime the number of times needed.

Note: • Careful and thorough routine washing will ensure a clean dispenser and ideal working

conditions. If in doubt, tend to clean with more liquid and more often, than cutting back on this maintenance step.

• If you are only dispensing water or a light buffer, you do not need to follow the above washing protocol each time you are dispensing. It is enough to wash it 2 or 3 times with water (in case of a buffer), and remove the remaining water afterwards. Still, if the dispenser is used regularly, a weekly wash following the protocol should be performed.

• Solutions which contain proteins will compromise your microplate dispenser performance over time. In that case we recommend rinsing the system daily with an enzyme detergent solution followed by pure water. On a weekly or monthly basis, flush with 0.1-0.5N sodium hydroxide (NaOH), followed by neutralization with an equivalent normality (0.1-0.5N) of hydrochloric acid (HCl). Rinse with pure water to remove the HCl.

• Remove the stainless steel weights on the input tubings as well during the cleaning process.



Please note: Washing can also be performed by using the buttons located at the dispenser unit. • The PUMP SEL button is used to select the pump. Green light will indicate the

selected pump. • The WASH/PRIME button will start the priming of the pump. Washing does not start if

the dispensing tip is inside the reader.

9.4 Empty syringe After dispensing, the remaining liquids can be returned to the bottle or to the waste container by clicking the Empty syringe button.

Please note: Emptying the syringes can also be performed by using the buttons located at the dispenser unit. • The PUMP SEL button is used to select the pump. Green light will indicate the

selected pump. • The EMPTY button will start emptying the syringe.



9.5 Gas connectors

The reader is equipped with gas connectors at the back of the reader. External gas control option is available for VICTOR Nivo readers. This digital CO2-O2 controller allows to control (and log) the O2 and CO2 concentrations by infusing pure CO2 and pure N2. The CO2 concentration is adjustable in the range of 0-20 %, the O2 concentration in the range of 1-20 %. The user should read carefully and follow the instructions provided with the gas control option.

The input and output of the gas control option can be connected either way to the reader’s two connectors. Best results are usually obtained by connecting the gas sensor input to the lower connector and the gas sensor output to the upper connector. The lower connector will guide the gas to the front end of the reader’s measurement chamber. The gas flows back to the gas controller through the upper connector from the back end of the measurement chamber.

Recommended output connector

Recommended input connector

Settings


10 Settings

Settings


On the SETTINGS page, the user can check the serial number of the reader and the reader configuration, change the user level by giving the correct password, create custom plates using the Plate Wizard or lock the instrument for transportation.

10.1 User Levels There are two user levels: Standard and Admin. The differences between these user levels are the right to delete protocols and filters and handle the optical components. The default log in mode is as Standard user. The Admin mode can be entered by either clicking Show optional password setting button when logging in or clicking Enter Admin mode -button on the SETTINGS page and entering the right password.

A Standard user can: • Edit or delete protocols created while logged in with the identical user name. • Unload and load the mirror slides.

An Admin user can: • Edit or delete any custom-made protocol. Only default protocols (marked with a

red lock) cannot be edited or deleted. • Change the mirror positions in the mirror slide and generate new mirrors. • Load and unload filters. • Delete custom filters or create new filters. Only default filters (marked with red

lock) cannot be edited or deleted. They can be loaded or unloaded however. • Change the Admin password (this option becomes visible after the Admin

password is entered).

Notice The default Admin password is “Advanced”. It is recommended to change this password after the first login.

Settings


10.2 Plate Wizard A user can set default plate types or create new plates by clicking the Plate Wizard -button. The following window will open:

The drop-down menus can be used to select the plate format and plate type. Clicking Use As Default will use the selected plate type as a default plate when creating new protocols. New plates can be created by clicking Create New. The following window will open:

• The plate name can be freely chosen. • The plate format can be selected from the list. • Well coordinates for the A1 well can be freely given as well as the distance between

middle points of the well (from center of the A1 well to the center of the A2 well). • The Well Volume is an important factor if the dispenser is used, since based on this

parameter the reader can try to prevent overfilling of the wells.

Settings


10.3 Lock instrument for transport Before transporting the reader to a different location, make sure that there is no plate inside the reader.

Notice Before transporting the reader, it must have been locked for transport. The locking process locks the XY-conveyor and the optical filter storage and prevents uncontrolled movements inside the reader.

The reader will be locked automatically if you turn off the instrument. If you want to lock the reader manually, go to the SETTINGS page and click Lock instrument (see picture below). The reader will be locked for transportation. Switch off the instrument right after this step and close the browser window. When starting the instrument in the new location, the locks will open automatically.

Maintenance


11 Maintenance

Maintenance


Warning! All maintenance procedures other than those mentioned in this manual must be performed by authorized service personnel.

11.1 Dust removing The plate holder must be kept clean and dust free to avoid the contamination of the reader.

Dust can be removed by using canned air intended for optics cleaning or very clean compressed air.

11.2 Spillage cleaning Spillage on the plate holder can be cleaned using mild detergent or alcohol and microfiber cloth. For Europium spillage, it is recommended to use Europium enhancement solution to clean it, followed by mild detergent or alcohol.

11.3 Cleaning optical components Optical components must be kept free of dust and finger prints. Dust can be removed by using canned air intended for optics cleaning or very clean compressed air. Finger prints can be removed by using pure ethanol and soft microfiber cloth. It is recommended that the optical filters are always kept in the instrument’s own filter storage.

Maintenance


11.4 Changing the air filter On the back of the instrument there is an air filter which is used to filter the room air flown into the reader (see picture below). The air filter is attached with four screws. The recommended changing period for the air filter is once a year.

Specification of filter material: • 92 mm air filter made of polyurethane foam • 45 pores per inch (ppi)

Warning! Switch off the instrument and unplug from mains before changing the air filter.

To change the air filter, remove these

four screws

Maintenance


11.5 Dispenser maintenance After utilization, the pump(s) should be thoroughly washed. You can do this by washing several times with pure water and ethanol (70 %):

1. Three times with water

2. Three times with ethanol

3. Three times with water (or more, to be sure to remove any ethanol left)

4. Three times with air (to empty the syringes and tubes)

You do this by going back to the main menu, selecting DISPENSER and then clicking Wash/Prime the number of times needed.

Note: • If you are only dispensing water or a light buffer, you do not need to follow the above

washing protocol each time you are dispensing. It is enough to wash it 2 or 3 times with water (in case of a buffer). Still, if the dispenser is used regularly, a weekly wash following the protocol should be performed.

• Solutions which contain proteins will compromise your microplate dispenser performance over time. We recommend rinsing the system daily with an enzyme detergent solution followed by pure water. On a weekly or monthly basis, flush with 0.1-0.5N sodium hydroxide (NaOH), followed by neutralization with an equivalent normality (0.1-0.5N) of hydrochloric acid (HCl). Rinse with pure water to remove the HCl.

Reader specifications


12 Reader specifications



GENERAL

Measurement technologies

Fluorescence (FI) Fluorescence polarization (FP) Time-resolved fluorescence (TRF) Luminescence (LUM) Absorbance (ABS)

Measurement direction Top, bottom

Measurement mode End-point, Kinetic, Well bottom scan, Z-focus scan, Spectrum scan (ABS)

Plate types Plates with 1, 6, 12, 24, 48, 96, 384 or 1536 wells

Plate height 4-21 mm (without dispenser) 12-21 mm (with dispenser)

Wavelength range FI 230 – 850 nm

Wavelength range FP 400 – 850 nm

Wavelength range TRF 230 – 850 nm

Wavelength range LUM 230 – 850 nm

Wavelength range ABS 230 – 1000 nm

Temperature control Ambient +3°C up to +65°C

Z-focus range 0 – 16 mm

Shaking modes Linear, orbital, double orbital

Number of optical filters Up to 32

Number of dichroic mirrors Up to 5 per slide

Instrument connection Ethernet

Dispenser unit (optional) 2 injectors

DIMENSIONS (w/o dispenser) Width 200 mm / 7.9 ”

Height 268 mm / 10.6 ”

Depth 495 mm / 19.5 ”

Weight 13 kg / 29 lb INSTRUMENT MODELS (Technology Options) VICTOR Nivo 3F Abs Filter, FI, Lum

VICTOR Nivo 3S Abs Spectrometer, FI, Lum

VICTOR Nivo 5F Abs Filter, FI, Lum, FP, TRF

VICTOR Nivo 5S Abs Spectrometer, FI, Lum, FP, TRF



PERFORMANCE

FI detection limit (FITC) Top < 0.01 fmol/well (384 well plate) Bottom < 0.06 fmol/well (384 well plate)

FI dynamic range (FITC) 5 orders of magnitude

FP sensitivity (FITC) < 3 mP (384 well plate)

TRF detection limit (Europium) < 0.5 amol/well (384 well plate)

TRF dynamic range 5 orders of magnitude

LUM detection limit (ATP) < 50 amol (96 well plate)

LUM dynamic range 6 orders of magnitude ENVIRONMENTAL CONDITIONS

Room conditions

Indoor use only, not exposed to direct sunlight. The instrument should not be located where the temperature varies strongly up and down. This might affect the results.

Altitude Up to 2000 m

Operation conditions, temperature range +15 °C – +35 °C (ideal: 22 °C)

Operation conditions, relative humidity 80 % max. at 31 °C, decreasing linearly to 50 % at 40 °C

Transportation and storage conditions, temperature range -20 °C – +50 °C

Transportation and storage conditions, relative humidity 5 % – 90 %

ELECTRICAL REQUIREMENTS Component VA Max. Supply Voltage Freq. (f) VICTOR Nivo (temperature control on) 120 W 110-240 V 50/60 Hz

Laptop (optional) 45 W 110-240 V 50/60 Hz

Wireless Router 12 W 110-240 V 50/60 Hz

One individual power socket is required for each system component (see above).

PerkinElmer Schnackenburgallee 114 22525 Hamburg Germany Internet: http://www.perkinelmer.com email: [email protected]

Documents

VICTOR NIVO MULTIMODE PLATE READERIntroduction 6 VICTOR Nivo – User Manual 1.1 Measurement technologies The VICTOR Nivo Multimode Plate Reader can be used to quantitatively measure