Power Supply 110 - 230 V; 50 / 60 Hz; 240 VA

Regulations CE, UL, CSA

Temperature Storage: 0° - 40° C

Range Operation: 15° - 35° C

Humidity 10 – 85%, not condensating

Dimensions 472 x 494 x 374 mm (W x D x H),

incl. injectors

Weight 52 – 54 kg (depending on configuration)

Measurement Luminescence

Technologies Top-reading Fluorescence

Bottom-reading Fluorescence (option)

Fluorescence Polarisation (option)

Absorbance (option)

AlphaScreen™ (option)

Detection Unit Low-noise photomultiplier tube in single photoncounting mode

Excitation Sources Halogen lamp, 75 W, 340 to 700 nm;

Laser, 680 nm (option)

Excitation Filters 355 nm, 485 nm; 405 nm, 450 nm, 490 nm,

620 nm (with Absorbance option)

Emission Filters 460 nm, 535 nm; 400 nm, 515 nm, 480 nm,

530 nm (with BRET option);

568 nm (with AlphaScreen™ option)

Performance

Luminescence: detection limit <20 amol ATP

Fluorescence: detection limit <20 fmol FITC (top reading)

Absorbance: linear range 0 – 2 OD

Fluorescence

Polarisation: c.v. 4.4 % with 1 nM Fluorescein

AlphaScreen™: detection limit <12.5 ng standard beads

Dynamic Range >6 orders of magnitude

Crosstalk Low crosstalk through crosstalk reduction design:5 x 10-6

Injection Unit Up to 4 injectors (variable volume: 10 – 100 µl),

JET Injection technology

Injection into 384 well plates

Plate Formats All 96 and 384 well microplates with outer dimen-sions: 86.0 x 128.2 (W x L); tolerance in plateheight: 13.9 – 15.1 mm

With plate height adjustment option:

All 6 well to 1536 well microplates with outerdimensions: 86.0 x 128.2 mm (W x L); tolerance inplate height: 8.0 – 22.2 mm

Petri dishes, Terasaki Plates, filters with respectiveadapters

Plate Height Optional: automatic detection and adjust-

Adjustment ment to varying plate heights (8.0 to 22.2 mm)

Temperature Control Optional: +5°C above RT to 37 °C

Includes cooling unit for PMT

Operation Modes Integral measurement 0.1 – 600 sec

Kinetics measurement (total time up to 24 h)

Repeated measurement (total time up to 24 h)

Plate repeats (up to 999)

Delay (up to 600 sec)

Scanning (up to 100 individual measurementpoints)

Dispensing with 4 independently controlled vari-able volume injectors

Shaking (3 modes, variable amplitude and speed)

BRET and BRET2 assays

Dual-Luciferase® Reporter Gene assays

Readit™ parameter file

AlphaScreen™ settings

FRET settings

Ratiometric assays

Stacker Stacker20 for 20 microplates (option)

Stacker100 for 100 microplates (option) Robotic Integration Optional Interface Serial RS232, 9 pin PC Operating System Win 98, Win 2000, Win NT, Win XP PC Requirements Pentium processor, 500 MHz (or better), CD ROM

drive, display 1024x768 (or better), serial port,parallel port

Software Windows® application BERTHOLD TECHNOLOGIES MikroWin 2000

Order Information Order NumberMithras LB 940 incl. MikroWin 2000 Lite 940-38099-10Mithras LB 940 with temperature controlincl. MikroWin 2000 Lite 940-38099-15 Injector 1 pre-position L, 10 – 100 µl 940-37772-11Injector 2 measurement position L, 10 – 100 µl 940-37772-12Injector 3 measurement position L, 10 – 100 µl 940-37772-13Injector 4 measurement position F, 10 – 100 µl 940-37772-14 Waste pump (for injectors 1 to 3) 940-38883 Plate Height adjustment 940-39364 Robot integration module 940-39354

BERTHOLD TECHNOLOGIES GmbH & Co. KGCalmbacher Str. 22 • D-75323 Bad Wildbad, GermanyPhone +49 7081 177-0 • Fax +49 7081 177-100info@BertholdTech.com • www.BertholdTech.com

Mithras LB 940 Specifications and ordering informations

LB 9

40 ·

042

003

· I

d.N

r. 8

1425

PR2

Rev

02

DLR, DLReady logo and Readit are trademarks of Promega Corporation. Dual-Luciferase and Stop & Glo areregistered trademarks of Promega Corporation. AlphaScreen and DeepBlueC are trademarks of PerkinElmerInc. Windows is a registered trademark of Microsoft. To run certain applications on this product may requirelicenses from others.

PMT cooling unit 940-39337Bottom-reading Fluorescence 940-39348 BRET package 940-39350 AlphaScreen™ package 940-39359 Absorbance option 940-39360 Fluorescence Polarisation option 940-39351 Stacker20 940-40056 Stacker100 940-40057 MikroWin 2000 Advanced I 37854-003 MikroWin 2000 Advanced II 37854-006

A Multilabel ReaderBeyond Expectations

Mithras LB 940

BIO

AN

AL

YT

ICA

L I

NS

TR

UM

EN

TS

Today’s needs to perform Many different assaysWith Multiple labels

Are now met by One readerWith No compromise in terms of sensitivity.

The Mithras LB 940 operates with dedicated optical paths to ensure thebest available performance for each technology. In consequence, theimplemented detection technologies can be read with the performanceof dedicated instruments. Those consist of luminescence, fluorescence,absorbance and in addition the homogeneous methods BRET, FRET,AlphaScreen™ as well as Fluorescence Polarisation.

Ancient legend ...... says that on December 25th, Mithras was conceived in the

shade of a sacred tree beside a sacred stream, holding a knifeand a torch. A raven from the sun god Sol then gave him a

message that told him to slay the mysterious white bull.Mithras obeyed and tracked down the bull, accompanied by two minor deities, Cautes and Cautophes. When he finally

met up with the mystic bull, he did slay it, using the knife hewas born with. The white bull then became the moon and

Mithras’ vicorious cape became the sky. Supposedly, this evolved into the alteration of night and day. The legend endwith Mithras’ climbing into Sol’s golden chariot and flashing

across the sky in glory.

• Absorbance• AlphaScreen™• Fluorescence Polarisation• Fluorescence intensity• FRET capability• Dual excitation, dual emission

The highlight of any laboratoryMithras LB 940

• Bottom reading fluorescence• Optimised BRET/BRET2 reading• Flash luminescence• Glow luminescence• Multi-wavelength luminescence• Dual luminescence

• Shaking• Scanning• Small footprint• Dynamic Range adaption (DRA)• Temperature control• Robot accessibility• Stackers• 21CRF part 11 compliancy

detect and identify

• Reading of 1 to 1536 well plates• Fast kinetics• Slow kinetics• Live kinetic monitoring• Up to 4 reagent injectors• Dispensing in 6 to 384 well

plates• Fast filter change enabling ratio-

metric assays

The highlight of any laboratoryMithras LB 940

2

Optics

The proprietary Dedicated Optical Path System

(DOPS) guarantees non-compromised perform-

ance for the individual measurement technolo-

gies luminescence, top reading fluorescence,

bottom reading fluorescence, fluorescence

polarisation, absorbance and AlphaScreen™. As

each technology has its own demands on the

optical system only separated light paths

ensure that the needs for high sensitivity and

wide dynamic range in each technology are

met equally.

Excitation & Emission Filters

Software-driven filtercarriers provide quick

change of emission and excitation filters within

one reading sequence especially important for all

applications with a ratiometric readout, e.g.

Indo-1 and Fura 2. If the measurement requires

several wavelength measurements this is possible

with the flexible protocol definition software.

The changing time for adjacent emission filters is

less than 130 ms providing rapid ratio kinetic

measurements.

Selected high quality interference filters are

mounted in the required positions and additional

filters can easily be added by the user if required.

4

3

Plate Formats

Any kind of microplate regardless of its colour

and format can be measured. All microplate

formats from 6 wells up to 1536 wells are sup-

ported. Additionally Petri dishes and Terasaki

plates can be loaded with adapters. There is no

need for any mechanical adjustment – just

select the appropriate plate type in the operat-

ing software.

The plate height adjustment module detects

the height of the plate used and automatically

adjusts the measurement optics for it.

Even filter mats can be handled in the instru-

ment. A specific filter adapter has been devel-

oped for dot blots especially taking care for

low cross talk while maintaining high counting

efficiency.

5

Reagent Dispensing

Up to 4 independently controlled injectors with

variable volume give entire freedom in the selec-

tion of assay sequence. The injectors are located

in measurement and pre-measurement positions

of the luminescence and the fluorescence path

respectively.

JET injection technology stands for precision and

accuracy – better than 98% – and the only reli-

able way of proper mixing of the added

reagents. Tuneable injection speed covers the dif-

ferent demands of viscosities, cells or filling

heights.

The volume is adjustable from 10 to 100 µl – in

increments of 1 µl. This is perfectly fitting the

requirements for reagent addition into 96 and

384 well plates.

Dispensing can even be performed within a run-

ning kinetic measurement, e.g. to watch the

effects of added agonists / antagonists.

detect and identify

Any type of ratio calcula-

tion, e.g. DLR, or other

mathematical formulas can be linked to every

well individually.

Standard curve fitting and calculation of

unknown samples is available for those users

looking for quantification of their results

(Advanced I and II).

All data can be exported in

EXCEL® or ASCII formats.

Instrument set-

tings, user signa-

tures and file

information are stored with the results.

The password system and the audit trail function

of the Advanced I

and II versions are

important tools

for data security.

The highlight of any laboratoryMithras LB 940

MikroWin 2000

The Windows® based software combines

operation and definition of instrument settings

as well as data reduction and evaluation.

The wells for measure-

ment and injection can be

selected inde-

pendently

Various opera-

tions can be

picked and placed in any order to accommodate

for all different requirements of the assays to be

performed.

Kinetic data

reduction and

graphical display

of the respective

curves help the

user to judge

the results.

6

21 CFR part 11 statementSystem access is controlled by log-on routines where unique combinations of user name and password are required to enter

operation levels. Unauthorized access is denied and documented. Electronic signatures are provided by user ID and date/time

stamps in data files. Files can be viewed in electronic and printable form. Audit trails are generated. System has been

developed, tested and documented according to ISO 9001 regulations.

3

Stacker & Robotic integration

Specially designed for, but not restricted to the

use in HTS departments of drug discovery com-

panies, the robot access module allows for

uncomplicated integration of the Mithras into

any type of lab automation system. Software

and hardware are easy to integrate into exist-

ing robotic or liquid handling systems.

A barcode reader is available on

request for positive plate identification.

The stacker units provide an of-the-

shelf solution for labs facing moderate

to advanced throughput requirements.

Two pre-configured models are available

Stacker20 to handle up to 20 microplates

Stacker100 to handle up to 100 microplates

Based on the BUTLER plate handling robot the

stackers are characterized by the option of

integrating additional equipment at any time.

The stacker operates in an efficient, reliable

and fast manner. Microplates are securely

gripped due to built-in sensors. There is no

plate loss after power failure. Low noise step-

per motor technology ensures fast and precise

plate handling.

Throughput is even further opti-

mised by the integration of the

unique High Throughput

Transfer & Loading (HTTL) sta-

tion minimising the time loss of

transferring read plates back to the stackers.

Plate stacks are

individually

removable and

can be exchanged

by the user.

7

detect and identify

The highlight of any laboratoryMithras LB 940

Intracellular Ca++ levels have become important indicators for the acti-

vation state of ion channels and G-protein coupled receptors as well as

for the phases of apoptosis and cell injury. Though the respective kinetics

and the absolute amounts of the Calcium levels are different for each of

these physiological processes there are common ways for monitoring

them. Luminescent labels like Aequorin as well as fluorescent ones are

versatile and widely used solutions for microplate assays. Fura 2 and

Indo-1 provide a ratiometric readout thereby reducing effects caused by

leaking or bleached dyes or varying assay conditions. OregonGreen

offers a fast single wavelength readout especially suited for high

throughput environments.

These assays can easily and reliably be performed with the use of

Mithras´s variable reagent injectors.

8

340 nm360 nm

380 nm

Fluorescence resonance energy transfer (FRET)

Fluorescence Resonance

Energy Transfer (FRET)

has for long been known

to be a smart method for

visualization of molecular

interactions of proteins

and nucleic acids. The discovery and further

development of the Fluorescent Protein family as

well as the Cy dyes has led to an increased use in

microplate assays. Another popular donor/accep-

tor combination is FITC and Rhodamine.

FRET is based on the fact that a donor dye can

transfer a part of this energy and excite an accep-

tor dye. Its emission can be detected as soon as

both dyes are in close proximity.

Applications

GPCRs / 7TM receptorsReporter gene assays

Dual-Luciferase® reporter gene assays (DLR™)Ca++ determination

Protein quantificationATP monitoring

Immunoassays (LIA, FIA)Enzyme kinetics

Readit™ SNP determinationDNA probe assays

Receptor binding studiesCell proliferation

Cell viabilityMembrane potential

Cytokine analysisIon channel studies

Nucleic acid quantificationCellular luminescence / Phagocytosis

Protein-protein interactionsKinase activity

Proteases, e.g. MMPsChemotaxisCytotixicityApoptosis

cAMPNADH

Calcium monitoring

9

480 nm

480 nm

480 nm530 nm

detect and identify

Homogeneous functional GPCR assay using BRET detectionRenilla luciferase emits blue

light at 480 nm upon addi-

tion of its substrate

coelenterazine.

Activation of receptor (con-

formation change, attach-

ment of G-protein) due to

binding of

ligand.

β-arrestin / eYFP fusion pro-

tein attaches to receptor

enabling energy transfer

between Renilla luciferase

and the eYFP moiety result-

ing in a rising peak of green

light at about 530 nm.

G-protein coupled receptors, also referred to as

7-transmembrane (7TM) receptors, comprise the

largest and most diverse superfamily of proteins

known. To a minor part only the ligands are

known.

Especially in the field of G-protein coupled recep-

tor research, the BRET technology offers the

opportunity for the establishment of a homoge-

neous and universal functional assay, taking

advantage of the fact that β-arrestin (which is

naturally playing a role in the desensitisation of

the receptors) binds to the intracellular part of

the activated receptor.

BRET is based on the fact that the energy derived

from a Renilla luciferase reaction can be used to

excite a fluorescent protein molecule if the latter

is in close proximity to the luciferase enzyme.

There are several advantages of BRET over other

methods. It is a non-radioactive and homoge-

neous technology. The ratiometric signal mini-

mizes interferences from assay conditions and

keep the time management non critical. There is

no auto-fluorescence coming from compounds or

cell and buffer components as no light source is

required.

Effect of agonist AVP on COS7#41 cells expressing VP2-Rlucand β-arrestin-GFP2

The highlight of any laboratoryMithras LB 940

AlphaScreen™ technology for secondary messenger screening

AlphaScreen™ relies on hydrogel coated Donor

and Acceptor beads providing functional

groups for conjugation to biomolecules. The

beads come in close proximity when the bio-

molecules interact by molecular binding, e.g.

antibody-antigen or receptor-ligand. Laser exci-

tation at 680 nm of a photosensitiser present

on the Donor bead results in the production of

singlet oxygen. The singlet oxygen migrates to

react with chemiluminescers on the Acceptor

bead. The chemiluminescers then activate fluo-

rophores emitting light at 520 - 620 nm.

AlphaScreen™ has proven to be a reliable and

sensitive method in detection assays for second-

ary messengers like cyclic AMP or IP3 activity.

Kinase assays based onFluorescence Polarisation

Fluorescence polarisation (FP) is ideal to meas-

ure the binding of a small molecule to a much

larger one. Unbound fluorescent tracers will

depolarise light during emission resulting in

low polarisation, because the unbound mole-

cules tumble more rapidly than bound tracers.

In contrast, bound fluorescent labelled mole-

cules keep the polarisation orientation as their

rotation is inhibited.

Besides cAMP determination and receptor–lig-

and binding assays a variety of kinase assays

has been established. After the kinase assay has

been performed in its usual way a defined

amount of fluorescent labelled phosphorylated

peptide is added together with the phospho-

specific antibody. The fluorescent tracer com-

petes with the phosphorylated kinase substrate.

Polarisation will decrease inversely proportional

to the kinase activity.

10

11

detect and identify

Readit™ SNP determination

SNPs are the most common type of human vari-

ability promising to play key roles in the deve-

lopment of diseases and the response to thera-

peutic treatments. The Readit™ test is an accu-

rate, reliable and convenient affordable ap-

proach which uses a light emitting luciferase

reaction triggered by ATP. The ATP itself is

derived from pyrophosphorolysis and a subse-

quent phosphorylation of ADP upon matched

probe hybridisation.

DNA probe assaysSeveral diagnostic DNA probe

assays based on acridinium ester

labelled oligo-nucleotides are

commonly used providing the most sensitive

detection and diagnosis of infectious diseases.

Reporter Gene Assays

In basic research of gene regulation as well as

in drug discovery the use of luciferases, ß-galac-

tosidases, ß-glucuronidases and secreted alkali-

ne phosphatases have become a standard tool

offering the highest sensitivity.

Especially the dual luminescence type assays,

e.g. Dual-Luciferase® Reporter Gene Assay,

have become a favourite means as they provide

an internal control for transfection

efficiency or general

expression level.

ATP determination

A detection limit of less than 20 attomoles of ATP

per well makes the Mithras to one of the best

suited microplate instruments for the determina-

tion of cell viability, e.g. in tumor chemo-

sensitivity assays, cell proliferation, antibiotic

susceptibility testing or hygiene monitoring.

Luminescent immunoassays(LIA, ILMA)

By exchanging colorimetric substrates of horse-

radish peroxidase or phosphatases with lumi-

nescent ones an 100-fold increase in sensitivity

can usually be achieved. MikroWin software

with the curve fitting option adds convenient

and extensive data evaluation capabilities to

the superb instrument performance.