UltraMicroscope Blaze™User manual

Copyright © 2021 Miltenyi Biotec and/or its affiliates. All rights reserved.

No part of this user manual may be reproduced, stored in a retrieval system, transmitted, published, or distributed in any form or by any means,electronically, mechanically, by photocopying, microfilming, recording, or otherwise, without the prior written consent of Miltenyi Biotec; however,notwithstanding the foregoing, the owners of the UltraMicroscope Blaze™ may make copies solely for purposes of training personnel in the use andservicing of the unit within their business or organization. Maximal care has been taken by Miltenyi Biotec in the preparation of this user manual. However,Miltenyi Biotec shall not be liable for any technical or editorial errors or omissions contained herein or for incidental or consequential damages inconnection with the furnishing, performance, or use of this document. The information in this document is provided “as is” without warranty of any kind,and is subject to change without notice. Blaze, MACS, and the Miltenyi Biotec logo are either registered trademarks or trademarks of Miltenyi Biotec and/orits affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used foridentification purposes only. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeuticor diagnostic use.

UltraMicroscope Blaze™User manual

Original instructions

Issued: 2021-08

212526.01

Miltenyi Biotec B.V. & Co. KG

Friedrich-Ebert-Straße 68

51429 Bergisch Gladbach

Germany

Phone +49 2204 8306-0

Fax +49 2204 85197

macsde@miltenyi.com

www.miltenyibiotec.com

Read the chapter Important safety information and all

other information in this user manual before using the

instrument

Before using the UltraMicroscope Blaze, read the chapter Important safety information and all other

information contained in this user manual, including any safety and operating instructions. Pay special

attention to all warnings displayed on the instrument. Failure to read and follow these guidelines could lead to

improper or incorrect usage and result in damage to the instrument. Improper usage could also cause severe

personal injury, death, unpredictable results, instrument malfunction, and premature wear to components

shortening the lifetime of the instrument. Such actions may void your warranty. Keep the user manual and any

other safety and operating instructions provided with the instrument in a safe place accessible to all users for

future reference.

If you have a serious concern regarding the safe use of your instrument, contact your authorized Miltenyi Biotec

service provider or call Miltenyi Biotec Technical Support.

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Content

1 Important safety information 7

2 Introduction 17

2.1 Introduction 17

2.2 Intended use 17

3 Installation 21

3.1 Components 21

3.2 Accessories 22

3.3 Consumables 22

3.4 Unpacking and installation 23

4 Switching on and off 25

4.1 Switching on the instrument 25

4.2 Switching off the instrument 25

4.3 The interface of the UltraMicroscope Blaze 25

5 Preparing the instrument 27

6 Laser alignment 29

6.1 Light sheet alignment 29

6.2 Setting the beam path 29

6.3 Adjustment of the lateral beams on the right-hand side 34

6.4 Adjustment of the center beam on the left-hand side 34

6.5 Adjustment of the lateral beams on the left-hand side 35

7 Setting up an experiment 37

7.1 Capturing a single-color z-stack 37

7.1.1 Aligning the sample 37

7.1.2 Optimizing the measurement settings 39

7.1.3 Capturing an image 40

7.2 Capturing a multi-color z-stack 40

7.3 Capturing a mosaic image 42

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8 Maintenance 45

8.1 Servicing 45

8.2 Cleaning the instrument 45

8.3 Cleaning the cuvette 45

9 Technical data and specifications 47

9.1 Specifications 47

9.2 EC / EU Declaration of Conformity 49

10 Technical support 51

11 Legal notes 53

11.1 Limited warranty 53

11.2 Trademarks 54

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1Important safety information

General safety instructions

THIS CHAPTER DESCRIBES POTENTIALLY HAZARDOUS SITUATIONS ASSOCIATED WITH THIS INSTRUMENT AND

PROVIDES IMPORTANT SAFETY INFORMATION TO MINIMIZE THE RISKS AND PROTECT YOURSELF AND OTHERS.

The instrument can be used safely, if the following is ensured:

The instrument is installed correctly.

The instrument is only operated by professional laboratory personnel.

General safety practices are followed.

All instructions in this user manual are followed.

• Use this instrument only as indicated in this user manual to avoid injury to personnel and damage of the

equipment.

• Keep this user manual in a place that is always accessible to all users.

• Follow local working area safety instructions and laboratory policies and standards for health, safety, and

prevention of accidents.

• Ensure safe working conditions and provide for sufficient light.

• Ensure that the power switch and the plug of the power cable of the instrument are easily accessible.

• To entirely disconnect the instrument from the power supply, unplug the power cable.

• Ensure that the power cable plug is close to the user.

• If it is necessary to cut the power supply, unplug the cable from the power outlet and contact an authorized

Miltenyi Biotec service provider or Miltenyi Biotec Technical Support.

• Read the instructions in the chapter Important safety information before using the instrument.

• Check the instrument before each use for visible damage.

• In case of severe accidents, damages to the instrument, or if smoke or flames appear, cut the power supply

immediately.

• Do not use the instrument if it has been dropped or is damaged. Contact Miltenyi Biotec Technical Support

immediately.

Hazard levelsSignal words are used to warn against hazardous situations and property damages. The following signal words

are used in this user manual.

or WARNING! indicates a hazardous situation that could result in serious injury or death.

or CAUTION! indicates a hazardous situation that could result in minor or moderate injury. It is also used

to warn against unsafe practices.

or NOTICE indicates information considered important but not hazard related (e.g. messages relating to

property damage).

Safety symbols

SAFETY WARNING: THE DOCUMENTATION MUST BE CONSULTED IN ALL CASES WHERE THIS SAFETY

SYMBOL IS USED ON THE INSTRUMENT

ELECTRICITY

MOVING PARTS

SHARP ELEMENTS

HAZARDOUS LASER RADIATION

BIOLOGICAL HAZARD

CRUSH HAZARD

ERGONOMIC HAZARD

TRIP HAZARD

WEEE (WASTE OF ELECTRICAL AND ELECTRONIC EQUIPMENT)

READ THE USER MANUAL BEFORE USING THE INSTRUMENT

ON (POWER ON)

OFF (POWER OFF)

WEIGHT

SERIAL NUMBER

TYPE NUMBER

ORDER NUMBER

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MANUFACTURER

EUROPEAN CONFORMITY APPROVAL

Safety labelsNotice the hazard points and safety symbols of the instrument.

• Keep safety labels and safety markings clean and legible.

• Inspect the safety labels and safety markings regularly and replace them if they are not legible or identifiable

from a safe viewing distance.

• Contact Miltenyi Biotec for replacement labels.

Figure 1.1: Hazard areas and safety symbols on the front side of the instrument.

Figure 1.2: Hazard areas and safety symbols on the rear side of the instrument.

Figure 1.3: Hazard areas and safety symbols on the left-hand side of the instrument.

Figure 1.4: Hazard areas and safety symbols on the right-hand side of the instrument.

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Electricity and fire hazards

ELECTRICITY

Electric devices bear the risk of electric shock, short circuits, overheating, fire, and explosion.

This may lead to burns, severe personal injury, or death.

Protective housingThe housing of the instrument reduces the risk of an electric shock and short circuits.

• Do not disassemble the instrument.

• Do not use the instrument if it has been dropped or is damaged.

Cables and power suppliesThe instrument has a three-wire electrical grounding plug with a third pin for grounding. Using the instrument

with other than the supplied cables is potentially hazardous.

• Only use the included power cable.

• Do not use the instrument if the power cable is damaged.

• Only insert the plug into a grounded power outlet. Do not force the plug into an incorrect electrical outlet.

• Operate the instrument only from a power source that meets the electrical specifications mentioned on the

marking plate. If you have questions about the type of power source to use, contact the Miltenyi Biotec

Technical Support or your local electricity supplier.

Liquids inside the instrumentLiquids inside the instrument can cause short circuits.

• Do not use the instrument if liquids have entered the inside of it.

• Protect the instrument against spilled liquids.

• Do not allow liquids to enter the inside of the instrument.

• Clean up spilled liquids immediately.

• Unplug the instrument before cleaning it.

• Use only small amounts of cleaning agents on a soft cloth to wipe the instrument. Do not spray or pour liquid

cleaning agents onto or into the instrument.

Humid and dusty environmentsA change in the environmental temperature may cause condensation inside the instrument. Humidity,

condensation, or dust inside the instrument can cause short circuits.

• Use the instrument only indoors.

• Do not use the instrument in a wet location or areas with high humidity or condensation.

• After moving the instrument from a cold environment to room temperature, wait for the instrument to

dehumidify before using it.

Mechanical hazards

MOVING PARTS

Risk of crushing or cutting.

Motorized turretThe motorized turret moves while the instrument is in operation.

• Keep away from the motorized turret while the instrument is in operation.

• Do not obstruct the movement of the motorized turret.

SHARP ELEMENTS

Risk of cutting.

UltraMicroscope Blaze

• Do use caution while handling the instrument.

• Provide sufficient lighting and workspace.

Optical radiation hazards

LASER BEAM

Exposure to the laser may cause eye injury.

Laser beam combinerThe instrument is equipped with a Class 3B laser product (Laser BC) or Class 4 laser product (Supercontinuum

WLL) per standard IEC 60825-1:2014.

• Do not remove the protective housing.

• Do not look directly at laser or reflected laser radiation from a mirrored surface.

• Do not insert reflective objects into the cuvette.

• Switch off immediately, if there is any indication of laser radiation outside the instrument.

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Biological hazards

BIOLOGICAL HAZARD

Contamination or infection may lead to severe personal injury or death, depending on the used

material.

Biological materialAll biological material must be considered potentially infectious.

• Wear personal protective equipment (such as gloves, safety glasses, etc.) as indicated in the documentation

for the particular substance.

• If hazardous or potentially infectious material has been spilled or leaked from the system, decontaminate the

affected area.

• Operate the instrument in a biological safety cabinet suitable for the used specimen if hazardous or potentially

infectious materials are processed.

• Take care when handling samples and reagents.

• Provide sufficient lighting and workspace.

WasteAll liquid and solid waste must be considered hazardous.

• Follow local regulations regarding waste disposal.

Equipment damageDefective or inadequate equipment can cause a biological hazard. Some decontamination methods may

damage the instrument.

• If parts of the instrument are damaged, unplug and do not use the instrument until damaged parts are

replaced. Contact Miltenyi Biotec Technical Support, if necessary.

• Follow the instructions in the datasheet for each consumable.

• Use only 70% ethanol or isopropyl alcohol for disinfection.

Chemical hazards

CHEMICAL HAZARD

Substances and reagents can be hazardous.

Hazardous substances and reagents

• All safety measures in section Biological hazards also apply to any hazardous substances and reagents that

may be present in the sample.

• Use any substances and reagents only as stated in the respective data sheet.

• Operate the instrument in a fume hood or with a fume extraction arm if hazardous substances and reagents

are processed.

Ergonomic hazards

CRUSH HAZARD

Risk of crushing.

Heavy instrument

• Only use tables that support a weight of 250 kg.

• Do not install the instrument yourself.

• Only trained Miltenyi Biotec personnel or personnel authorized by Miltenyi Biotec is allowed to install the

instrument.

ERGONOMIC HAZARD

Risk of tearing or straining muscles.

Heavy instrument

• Do not move the instrument yourself.

• Only trained Miltenyi Biotec personnel or personnel authorized by Miltenyi Biotec is allowed to transport the

instrument.

Environmental hazards

TRIP HAZARD

Tripping can cause injuries.

Uncovered cables

• Provide sufficient lighting and workspace.

• Check the environment around the instrument before each use for trip hazards such as cables lying on the

floor.

Servicing and transportation

ServicingImproper servicing or repair of the instrument or use of unauthorized parts can cause malfunction of or damage

to the instrument. This can cause hazards to users. Unless otherwise specifically noted in this user manual or

other Miltenyi Biotec documentation, do not service the instrument yourself. Servicing and repair must be

performed by Miltenyi Biotec certified and qualified service personnel. If the instrument needs servicing,

decontaminate the instrument to remove any hazardous material. If you have questions regarding proper

decontamination or shipment, contact Miltenyi Biotec Technical Support for assistance.

TransportationThe instrument should be transported with care in packaging specified by Miltenyi Biotec. Internal damage can

occur if the instrument is subjected to excessive vibration or if it is dropped. If the instrument needs to be

shipped back to the manufacturer for service, contact Miltenyi Biotec for instructions and packaging materials.

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Disposal

WASTE OF ELECTRICAL AND ELECTRONIC EQUIPMENT (WEEE) CUSTOMER INFORMATION

Dispose of your end-of-life Miltenyi Biotec products in accordance with the applicable WEEE and hazardous

waste disposal legislation, which may differ by country or region. Electrical equipment may contain hazardous

substances that may have a serious detrimental effect on the environment and/or human health. All equipment

must be specifically collected and treated by designated waste facilities and by qualified WEEE compliance

schemas. By ensuring that you dispose of your unwanted electrical and electronic equipment according to the

applicable WEEE and hazardous waste disposal legislation, you are helping to preserve our natural resources and

protect human health. Miltenyi Biotec is committed to protecting the environment. Miltenyi Biotec offers

product end-of-life return programs in many countries, and partners with licensed WEEE compliance schemes

throughout the world. Miltenyi Biotec takes back your end-of-life Miltenyi Biotec equipment for recycling free of

charge. The terms and availability of this offer vary by geography because of differences in regulatory

requirements. Note that, depending on the type and use of your equipment, additional requirements may

apply. Before shipping the instrument back to the manufacturer for disposal, decontaminate the instrument to

remove any hazardous material. For more information, or if you wish to dispose of your end-of-life Miltenyi

Biotec equipment, contact your local Miltenyi Biotec representative or Miltenyi Biotec Technical Support.

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2Introduction

2.1 Introduction

The UltraMicroscope Blaze is a fully automated light sheet microscope for imaging cleared tissue samples with a

subcellular resolution.

2.2 Intended use

The UltraMicroscope Blaze is a laboratory equipment for fluorescence microscopic analysis of cleared samples

up to a size of 35 mm × 60mm × 17 mm. The sample may only be examined in the supplied cuvette.

Unauthorized modifications of the cuvette or the instrument as a whole are not permitted for safety reasons.

The cuvette may only be filled with MACS Imaging Solution or another microscopy solution approved by

Miltenyi Biotec. The operating, maintenance, and servicing instructions described in this manual must be

observed.

The UltraMicroscope Blaze is for research use only in a laboratory environment. It is to be used by laboratory

professional users only.

Figure 2.1: Front view of the UltraMicroscope Blaze.

Figure 2.2: Rear view of the UltraMicroscope Blaze.

Figure 2.3: Detail view of the I/O ports and power switch.

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Figure 2.4: Jog wheel formoving the sample arm in 3dimensions (x, y, z) and sensitivity.

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3Installation

3.1 Components

The Ultramicroscopes Blaze Beam Combiner and White Light Laser are delivered with lasers, lenses, and

emission filters, as specified when ordering the instrument (Table 3.1). Additional accessories (Table 3.2) and

consumables (Table 3.3) developed specifically for the system are available separately. For details visit

www.miltenyibiotec.com or contact your local sales representative.

Components included in the delivery

UltraMicroscope

Blaze Beam

Combiner

(# 150-001-651)

UltraMicroscope

Blaze White Light

Laser

(# 150-001-733)

UltraMicroscope

Blaze Beam

Combiner 4.2

(# 150-001-760)

UltraMicroscope

Blaze Beam

Combiner 5.5

(# 150-001-761)

Light sources up to 5 lasers ofchoice*

white light laser 405 nm, 488 nm, 561nm, 639 nm, and785 nm lasers

405 nm, 488 nm, 561nm, 639 nm, and785 nm lasers

Objective lenseswith dippingcaps

up to 3 objectivelenses*

up to 3 objectivelenses*

objective lenses1.1× MI PLAN, 4× MIPLAN, 12× MI PLAN

objective lenses1.1× MI PLAN, 4× MIPLAN, 12× MI PLAN

Emission filters(Ø 43 mm)

up to 7 filters ofchoice*

up to 7 filters ofchoice*

5 filters 5 filters

Excitation filters(Ø 12,5 mm)

- up to 8 filters ofchoice*

- -

Cameras 4.2 Mpx sCMOS or5.5 Mpx sCMOScamera*

4.2 Mpx sCMOS or5.5 Mpx sCMOScamera*

4.2 Mpx sCMOScamera

5.5 Mpx sCMOScamera

Sample chamber × × × ×

Jog wheel × × × ×

Power supply forinstrument

× × × ×

Workstation PC × × × ×

Keyboard × × × ×

Mouse × × × ×

Monitor × × × ×

Table 3.1: Components included in the delivery (* The delivery of these items depend on the ordered configuration).

3.2 Accessories

Accessories Order no.

Objective lenses with dipping caps

Objective lens 1.1× NA 0.1 MI PLAN with two dipping caps for organic/aqueous buffers 150-000-493

Objective lens 4× NA 0.35 MI PLAN with dipping cap for water 150-001-654

Objective lens 4× NA 0.35 MI PLAN with dipping cap for aqueous buffers 150-001-655

Objective lens 4× NA 0.35 MI PLAN with dipping cap for organic solvents 150-001-656

Objective lens 12× NA 0.53 MI PLAN with dipping cap for water 150-000-495

Objective lens 12× NA 0.53 MI PLAN with dipping cap for aqueous buffers 150-000-496

Objective lens 12× NA 0.53 MI PLAN with dipping cap for organic solvents 150-000-497

Dipping caps

Dipping cap water for objective lens 4× MI PLAN 150-001-660

Dipping cap aqueous buffers for objective lens 4× MI PLAN 150-001-658

Dipping cap organic solvents for objective lens 4x MI PLAN 150-001-657

Dipping cap water for objective lens 12× MI PLAN 150-000-500

Dipping cap aqueous buffers for objective lens 12× MI PLAN 150-000-499

Dipping cap organic solvents for objective lens 12× MI PLAN 150-000-498

Cameras

4.2 Mpx sCMOS camera 150-000-552

5.5 Mpx sCMOS camera 150-000-551

Various accessories

Alignment tool for UltraMicroscope Blaze 150-001-813

Sample holder set for UltraMicroscope Blaze 150-001-814

Cuvette for UltraMicroscope Blaze 150-001-673

Table 3.2: Optional accessories available for the UltraMicroscope Blaze.

3.3 Consumables

Consumables Order no.

MACS Clearing Kit 130-126-719

MACS Imaging Solution 130-126-335

Table 3.3: Consumables that have to ordered separately.

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3.4 Unpacking and installation

Protect the instrument from shock and vibration. Do not place vibrating devices on (e.g. vortex mixers) on

the same table as the UltraMicroscope Blaze. This could impair the quality of the results.

Only Milteny Biotec service personnel is allowed to unpack and install the instrument. Please contact Miltenyi

Biotec Technical Support for further information.

Before installation, ensure that your site is properly prepared. The instrument should be installed on an optical

table with vibration isolation that can carry a weight of at least 250 kg. Allow a minimum of 15 cm free space to

the rear side and to both sides of the instrument. Allow a minimum of 100 cm free space in front of the

instrument. All sides of the instrument must be accessible for service and maintenance. Due to vibrations, an

adjacent desk is recommended for the laser source and the workstation PC setup.

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4Switching on and off

4.1 Switching on the instrument

1 Make sure that all components are connected to the power supply.

2 Switch on the external laser module. The key switch is located on the laser module (see manufacturer

documentation).

3 Switch on the UltraMicroscope Blaze. The power switch is located on the rear side of the instrument (Figure

2.2).

4 Switch on the workstation PC and log in with your user credentials.

5 Start the ImSpectorPro Software by double clicking on the program icon. The switched on devices are

registered and checked.

4.2 Switching off the instrument

1 Close the ImSpectorPro Software.

2 Shut down Windows.

3 Switch off the UltraMicroscope Blaze. The power switch is located on the rear side of the instrument (Figure

2.2).

4 Switch off the external laser module. The key switch is located on the laser module (see manufacturer

documentation).

5 Disconnect the camera from the power supply.

4.3 The interface of the UltraMicroscope Blaze

Name Function Location

Start measurement Starts the defined measurement Menu bar

Stop measurement Stops the defined measurement Menu bar

Save settings Saves all settings Menu bar

Start measurement Starts the defined measurement Measurement Wizard

Start batch measurement Starts the defined batch measurement Measurement Wizard

Start live preview Starts live video in the Live Window Measurement Wizard

Name Function Location

Adjust window/live Adjusts the contrast based on the regionof interest in the Live Window

Live Window

ROI for active profile Defines a region of interest in the LiveWindow

Live Window

Light sheet focus Turns on the light sheet focus in the LiveWindow

Live Window

Show crosshairs Turns on the crosshairs in the LiveWindow

Live Window

Move objective tomeasurement position

Moves the objective down Settings 1 window

Move objective to parkposition

Moves the objective up Settings 1 window

Select left light sheet Activates the selected left-hand lightsheet

Settings 1 window

Select all light sheets Activates the selected light sheets on bothsides

Settings 1 window

Select right light sheet Activates the selected right-hand lightsheet

Settings 1 window

Block all beams Blocks all laser beams Settings 1 window

Enable only the central beam Enables only the central laser beam Settings 1 window

Disable the central beam Disables only the the central laser beam Settings 1 window

Enable all three beams Enables all laser beams Settings 1 window

Table 4.1: ImSpectorPro Software icons.

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5Preparing the instrument

BIOLOGICAL HAZARD

Contamination or infection may lead to severe personal injury or death, depending on the used

material.

• Take care when handling samples and reagents.

• To avoid spilling of liquids, wipe all parts of the instrument (e.g. objectives, cuvette) with a soft, lint-free

disposable tissue.

• Wear personal protective equipment (such as gloves, safety glasses, etc.) as indicated in the documentation

for the particular substance.

1 Click Objective Lens Focus > Move Objective to Park Position in the Settings 1 window. Make

sure the motorized turret is in the resting position.

2 Pull out the frontal drawer as far as it will go to lower the cuvette (Figure 5.1).

Figure 5.1: Open drawer to remove the cuvette.

3 Remove the sample arm by loosening the thumb screw (Figure 5.1).

4 Remove the cuvette by lifting it from its magnectic holders.

5 Fill the cuvette about three quarters with the selected imaging buffer.

6 Place the cuvette on its magnetic holders again. Be careful not to spill any buffer.

7 Make sure that the cuvette is securely placed on the magnetic holders.

8 Reattach the sample arm by tightening the thumb screw again.

9 Use a disposable syringe to fill the cuvette with more imaging buffer up to the notch on the side (Figure

5.2).

Figure 5.2: Fill the cuvette up to the notch.

10 Press the button on the drawer to move it up again.

11 Push the drawer to close it.

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6Laser alignment

6.1 Light sheet alignment

To generate high-contrast images with the best z-resolutions, all light sheets must be perfectly aligned to the

same z-plane. A set of adjustable precision screws at the bottom of the instrument can be used to align the

individual beams creating the light sheet (Figure 6.1): Screw z1 is used for aligning the left-hand sheet in the z-

plane. Screws z2 and z4 are used for aligning the three beams of each light sheet.

Figure 6.1: Bottom view of the UltraMicroscope Blaze with the positions of the adjustable precision screws.

6.2 Setting the beam path

1 Switch on the instrument as described under Switching on the instrument on page 25.

2 Pull out the drawer out as far as possible, to lower the cuvette.

3 Place the alignment tool on the sample arm.

4 Raise the cuvette by pressing the button on the drawer. Close the drawer (Figure 6.2).

Figure 6.2: UltraMicroscope Blaze with open drawer.

5 Select the excitation and emission wavelengths for the alignment tool (488 nm or 561 nm excitation) under

Channel Setup in the Settings 1 window. Set Laser Transmission Control between 5–10%.

6 Click the Select right light sheet button under Light Sheet Selection.

7 Click the Start live preview button in the Measurement Wizard to start the preview mode.

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8 Make sure the cuvette is in a centered position. Move the alignment tool until the beams on the right side

pass through the tool and form three thin lines on the other side of the pinhole foil.

9 Select the 1.1× objective lens (LVBT 1×) with 1.0× zoom and the medium used in Optical Setup.

10 Adjust the Laser Transmission Control until the three beams passing the pinhole can easily be detected

while the other side of the pinhole is not saturated.

11 Activate the crosshairs at the left-hand side of the Live Mode window and line up the alignment tool in the

center.

12 Activate the Light Sheet Focus and place the Rayleigh length into the middle of the field of view.

13 Set the Light Sheet Properties > Sheet NA to the maximum.

14 Set the Light Sheet Properties > Sheet Width to 100%.

15 Select the Advanced tab in the Settings 1 window. Always start with the center beam on the right-hand

side. It is the reference beam and cannot be adjusted by the user. Click Light Sheet Configuration >

Enable only the central beam to activate the center beam.

16 If two laser beams are visible passing the mask (Figure 6.3; A), lower or raise the alignment tool with the

mouse wheel. You are moving in the correct direction if the beams move closer to each other. The aim is to

align the center beam to the pinhole located in the middle of the mask in order to align all beams to this

position (Figure 6.3; B).

Figure 6.3: Laserbeams in the alignment tool.

If it is not possible to align the beams by lowering or raising the alignment tool, the actual position of the

horizontal focus does not match with the position displayed in the ImSpectorPro software. Align the central

beams as well as possible before adjusting the horizontal focus (Adjusting the horizontal focus on the

facing page).

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Adjusting the horizontal focus

1 To adjust the horizontal focus, select the Calibration tab on the bottom of the Settings 1 window.

2 Move the slider Light Sheet Focus > Offset

Calibration until only one beam passes through the

pinhole. Ensure the horizontal focus is always in the

center and the Light Sheet Properties > Sheet

NA is at the maximum. If necessary, readjust the

height.

3 If a clearly recognizable, single beam passes through the pinhole in the center of the mask, increase the

intensity of this beam by moving the alignment tool in z direction. The central beam is now adjusted to the

highest intensity. Repeat this procedure until no more improvement is possible.

4 Click on Save settings in the menu bar to fix the adjusted horizontal focus.

After the settings have been saved, the alignment tool must not be moved again.

6.3 Adjustment of the lateral beams on the right-hand side

To adjust the lateral beams on the right-hand side, the horizontal focus must not be changed.

1 Click Light Sheet Configuration > Disable the central beam in the Advanced tab to activate the

lateral beams.

2 Adjust the height of the lateral laser beams. Start with the rear laser beam on the right-hand side (Figure

6.1; z2). Use the adjustment screw below the system to adjust the laser beam to the center of the pinhole in

the mask.

3 Repeat the procedure with the front laser beam on the right-hand side (Figure 6.1; z4).

4 Click Light Sheet Configuration > Enable all three beams in the Advanced tab to activate all

beams. Now all three laser beams pass through the pinhole in the center of the mask of the alignment tool.

6.4 Adjustment of the center beam on the left-hand side

To adjust the center beam on the left-hand side, follow the instructions below.

1 Click Light Sheet Selection > Select left light sheet inn the Settings 1 window.

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2 Click Light Sheet Configuration > Enable only the central beam in the Settings > Advanced tab

to activate the center beam.

3 Ensure that the slider for the horizontal focus is still in the center. If necessary, adjust the position.

4 Adjust the height with the adjustment screw (Figure 6.1; z1). If it is not possible to get only one beam to

pass through the pinhole, follow the instructions under Adjusting the horizontal focus on page 33.

6.5 Adjustment of the lateral beams on the left-hand side

To adjust the lateral beams on the left-hand side, the horizontal focus must not be changed:

1 Click Light Sheet Configuration > Disable the central beam in the Advanced tab to activate the

lateral beams.

2 Adjust the height of the lateral beams. Start with the rear beam on the left-hand side (Figure 6.1; z2). Use

the adjustment screw below the instrument to adjust the laser beam to the center of the pinhole in the

mask.

3 Repeat the procedure with the front laser beams on the left side (Figure 6.1; z4).

4 Click Light Sheet Configuration > Enable all

three beams to activate all beams in the Advancedtab. Now all three laser beams pass through the

pinhole in the center of the mask of the alignment

tool.

If the alignment of the left-hand side to the right-hand side is not successful, check the positioning of the

cuvette again.6

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7Setting up an experiment

7.1 Capturing a single-color z-stack

7.1.1 Aligning the sample

1 Start the ImSpectorPro software.

2 Select Instrument Mode > Ultramicroscope in the Measurement Wizard window.

3 Select Measurement Mode > Acquisition for measuring a z-stack.

4 Select xyz-Table Z in the Devices list. Check Split only if you want to generate seperate files for each

color.

5 Click the Autosave button at the end of the row under AS. It will turn green.

6 Click the Autosave Settings to select the storage location.

7 Select the appropriate objective Optical Setup > Objective, the zoom Optical Setup > Zoom and the

imaging buffer Optical Setup > Liquid in the Settings 1 window.

8 Select the excitation and emission wavelengths for your sample under Channel Setup. Move the slider

under Laser Transmission Control while the filter is selected to set the laser power.

9 Select the light sheet setup you want to use under Light Sheet Selection.

10 Click the Start live preview button in the Measurement Wizard to start the video mode.

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11 Click and hold the Objective Lens Focus > Move Objective to Measurement button to move down

the motorized turret. The objective will automatically stop.

12 Click the Show crosshairs button on the right side of the live window to activate the crosshairs.

13 Use the jog wheel to move the sample inside the cuvette until the surface of the sample is visible in the

center of the crosshairs.

7.1.2 Optimizing the measurement settings

1 Click the ROI for active profile button on the right side of the live window and draw a rectangle over a

light and/or a dark region by holding the left mouse button.

2 Click the Adjust window/live button to achieve a better contrast.

3 Click the Light sheet focus button on the right side of the live window to show the horizontal focus.

Move the horizontal focus in the picture until you have the optimal contrast for your region of interest.

4 Optional: Move the slider under Light Sheet Properties > Sheet NA to reduce the numerical aperture

of the light sheet. This will create a more homogeneous light sheet.

5 Optional: Move the slider under Light Sheet Properties > Sheet Width to reduce the sheet width of

the light sheet. This will result in more light being focused on a smaller region of the sample.

The settings described may need to be adjusted when the zoom settings are changed.

7.1.3 Capturing an image

1 Move in the z-direction to your starting position.

2 Click Current Position > Set as Zero and Scan Range > Start > Take in the Settings 2 window.

3 Move through the sample until you reach the end of the z-stack and click Scan Range > End > Take.

4 Define the increment by entering it in Scan Range > StepSize.

5 Check the Autosave Settings in the Measurement Wizard again.

6 Click the Start measurement button in the Measurement Wizard to start the measurement.

7.2 Capturing a multi-color z-stack

1 Select Measurement Mode > Multi Color 3D in the Measurement Wizard.

2 Select xyz-Table Z and Blaze Filter in the Devices list.

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3 Click the Autosave button under AS. It will turn

green. Check Split only if you want to generate

seperate files for each color.

4 Select the right excitation and emission wavelengths for your sample under Filter for Measurement in

the Settings 1 window. Move the slider under Laser Transmission Control while the filter is highlighted

to set the laser power for each individual filter.

5 Select the shortest emission wave length and create a sharp picture.

6 Optional: Correct the different focus lengths for different emission wave lengths using the Chromatic

Correction, starting with the next longer wave length

7 After correcting for all emission wavelengths click Save settings in the menu bar.

8 To start the measurement, proceed as described under Capturing an image on the previous page.

7.3 Capturing a mosaic image

1 Select Measurement Mode > Mosaic Aquisition in the Measurement Wizard.

2 Click the Mosaic bar in the xyz-Table Visual XY window.

3 Double-click the Green Square representing the field of view. The square will turn grey.

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4 Click and drag the Black Dots at the edges to create a mosaic.

5 Enter a percentage value to set an Overlap.

6 To start the measurement, proceed as described under Capturing an image on page 40

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8Maintenance

8.1 Servicing

Service the instrument every 12 months. Contact Miltenyi Biotec for support.

8.2 Cleaning the instrument

ELECTRICITY

Electric devices bear the risk of electric shock, short circuits, overheating, fire, and explosion.

This may lead to burns, severe personal injury, or death.

Unplug the instrument before cleaning it. Use only small amounts of cleaning agents on a soft cloth to wipe

the instrument. Do not spray or pour liquid cleaning agents onto or into the instrument.

1 To disinfect, wipe accessible surfaces with 70% ethanol or isopropanol.

2 If necessary wipe the instrument with a damp tissue.

8.3 Cleaning the cuvette

Clean the cuvette with a cleaning solution suitable for the microscopy solution used. Be careful not to damage

the windows of the cuvette.

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9Technical data and specifications

9.1 Specifications

The safety of the instrument may be compromised if used outside its specifications.

• Do not use the instrument outside its specifications.

UltraMicroscope Blaze specifications

General information

Dimensions (w × d × h) 67 cm × 52.5 cm × 91 cm

Weight 98 kg (w/o laser source)

Nominal input voltage 24 V provided by external power supply

Working conditions

Temperature 15–27 °C

Humidity max. 70% relative humidity, non-condensing

Storage conditions

Temperature 4–55 °C

Humidity max 70% relative humidity, non-condensing

Transport conditions

Temperature 4–55 °C

Humidity max 70% relative humidity, non-condensing

Cuvette

Imaging solutions Aqueous buffers and organic solvents

Sample travel range (x, y, z) 24 mm, 50 mm, 23 mm

Chamber size (inside dimensions w × d × h) 51 mm ×129 mm × 64 mm

Refraction index matching 1.33-1.56

UltraMicroscope Blaze specifications (continued)

Dectection optics / Objective lenses 1.1× 4× 12×

Zoom | Magnification changer 0.66-2.75 2.4–10 7.2–30

Resolution at detector 4.8–19.6 μm 1.3–5.4 μm 0.44–1.8 μm

Numerical aperture 0.1 0.35 0.53

Working distance ≤17 mm ≤16 mm ≤10.9 mm

FOV diagonal (5.5 Mpx sCMOS camera) 7.9–33 mm 2.2–9.1 mm 0.73–3 mm

Emission filters Seven filters Ø 43 mm

Chromatic correction Dynamic 400-850 nm

Sheet optics

Illumination Uni– and bidirectional

Number of light sheets 1–6

Thickness 4–24 μm

Width 1.5–20 mm

Numerical aperture 0.0135–0.135

Focus positioning Dynamic

Table 9.1: UltraMicroscope Blaze specifications

Camera specifications

Detector 4.2 Mpx sCMOS camera 5.5 Mpx sCMOS camera

Active pixels (w × h) 2048×2048 2560×2160

Pixel size 6.5 μm × 6.5 μm 6.5 μm × 6.5 μm

Sensor size 13.3 mm × 13.3 mm; 18.8 diagonal 16.6 mm × 14 mm; 21.8 mm diagonal

Readout noise 0.8 med e- 1 med e-

Maximal frame rates 100 fps 100 fps

Maximum quantum efficiency 82% 60%

Table 9.2: Camera specifications

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9.2 EC / EU Declaration of Conformity

This declaration of conformity is issued under the sole responsibility of the manufacturer:

LaVision BioTec GmbH - A Miltenyi Biotec Company

Astastraße 14

33617 Bielefeld

Germany

The declaration of conformity refers to the machinery identified as follows:

Description: Laboratory equipment

Model: UltraMicroscope Blaze

The machinery complies with all essential requirements of the following directives:

2006/42/EC Machinery

2014/30/EU Electromagnetic compatibility

2014/35/EU Low Voltage

The machinery is in conformity with the following harmonized standards:

EN 12100-03:2011

EN 14121-02:2013

EN 60825-1:2015

Person authorized to compile the relevant technical documentation:

Heinrich Spieker

LaVision BioTec GmbH - A Miltenyi Biotec Company

Astastraße 14

33617 Bielefeld

Germany

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10Technical support

For technical support, contact your local Miltenyi Biotec representative or Miltenyi Biotec Technical Support at

Miltenyi Biotec headquarters:

Miltenyi Biotec B.V. & Co. KG

Friedrich-Ebert-Straße 68

51429 Bergisch Gladbach

Germany

Phone: +49 2204 8306 3803

Fax +49 2204 8306-89

Email: technicalsupport@miltenyi.com

Visit www.miltenyibiotec.com for local Miltenyi Biotec Technical Support contact information.

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11Legal notes

11.1 Limited warranty

Except as stated in a specific warranty statement which may accompany this product or as otherwise agreed in

writing by an authorized representative of Miltenyi Biotec, Miltenyi Biotec’s warranty to you, the original

purchaser and end user (“you” or “your”), with respect to the product accompanied by this limited warranty shall

be subject to the following provisions and the general terms and conditions of sale of the company within the

Miltenyi Biotec group which supplied the product in effect at the date of purchase. Those terms and conditions

of sale may vary by country and region. Nothing in this document should be construed as constituting an

additional warranty.

Miltenyi Biotec warrants that this product will operate or perform substantially in conformance with Miltenyi

Biotec's published specifications and be free from material defects in material and workmanship, when

subjected to normal, proper, and intended usage by properly trained personnel, for the period of time set forth

in the product documentation or package inserts accompanying the product (the “Warranty Period”).

Miltenyi Biotec agrees, during the Warranty Period, to repair or replace, at Miltenyi Biotec's option, the defective

product so as to cause the same to operate in substantial conformance with said published specifications;

provided that you shall (a) promptly notify Miltenyi Biotec in writing upon the discovery of any nonconformity

or defect, which notice shall include the product model and serial number (if applicable) and details of the

warranty claim; and (b) return the nonconforming or defective product to Miltenyi Biotec, freight prepaid, only

after receipt of a Return Material Authorization (“RMA”) from Miltenyi Biotec, which may include biohazard

decontamination procedures and other product-specific handling instructions, if applicable.

Miltenyi Biotec shall have no obligation to make repairs, replacements, or corrections to the product or any

component thereof required, in whole or in part, as the result of (i) normal wear and tear, (ii) improper handling,

installation, operation, storage, service, maintenance, or repair, (iii) failure to follow the instructions, cautions,

warnings, and notes set forth in the product documentation provided with the product or provided by Miltenyi

Biotec from time to time, (iv) abnormal use, misuse, neglect, abuse, mishandling, misapplication, modification,

or alteration of the product, (v) use of the product in a manner for which it was not designed, (vi) causes

external to the product such as, but not limited to, power failure or electrical power surges, (vii) use of the

product in combination with equipment, accessories, consumables, or software not supplied or approved by

Miltenyi Biotec, or (viii) accident, disaster, or acts of God. ANY INSTALLATION, MAINTENANCE, REPAIR, SERVICE,

OR ALTERATION TO OR OF, OR OTHER TAMPERING WITH, THE PRODUCT PERFORMED BY ANY PERSON OR

ENTITY OTHER THAN MILTENYI BIOTEC AUTHORIZED PERSONNEL WITHOUT MILTENYI BIOTEC’S PRIOR WRITTEN

APPROVAL, OR ANY USE OF REPLACEMENT PARTS NOT SUPPLIED BY MILTENYI BIOTEC, SHALL IMMEDIATELY

VOID AND CANCEL ALL WARRANTIES WITH RESPECT TO THE AFFECTED PRODUCT.

Miltenyi Biotec’s warranty does not cover products sold AS IS or WITH ALL FAULTS, or which had its serial

number defaced, altered, or removed, or any consumables or parts identified as being supplied by a third party.

Miltenyi Biotec must be informed promptly if a claim is made under this limited warranty. If a material or

manufacturing defect occurs within the Warranty Period, Miltenyi Biotec will take the appropriate steps, at

Miltenyi Biotec's option, to make repairs, replacements, or corrections to the product or any component thereof

as may be required to restore the full usability of your product. If Miltenyi Biotec determines that a product for

which you have requested warranty services is not covered by the warranty hereunder, you shall pay or

reimburse Miltenyi Biotec for all costs of investigating and responding to such request at Miltenyi Biotec’s then

prevailing time and materials rates. If Miltenyi Biotec provides repair services or replacement parts that are not

covered by this warranty, you shall pay Miltenyi Biotec therefor at Miltenyi Biotec’s then prevailing time and

materials rates.

THE OBLIGATIONS CREATED BY THIS WARRANTY STATEMENT TO REPAIR OR REPLACE A DEFECTIVE PRODUCT

ARE EXCLUSIVE AND SHALL BE THE SOLE REMEDY OF BUYER IN THE EVENT OF A DEFECTIVE PRODUCT. EXCEPT

AS EXPRESSLY PROVIDED IN THIS WARRANTY STATEMENT, MILTENYI BIOTEC HEREBY DISCLAIMS ALL OTHER

WARRANTIES, WHETHER EXPRESS OR IMPLIED, ORAL OR WRITTEN, WITH RESPECT TO THE PRODUCT, INCLUDING

WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR

PURPOSE. MILTENYI BIOTEC DOES NOT WARRANT THAT THE PRODUCT IS ERROR-FREE OR WILL ACCOMPLISH

ANY PARTICULAR RESULT.

UNDER NO CIRCUMSTANCES WILL MILTENYI BIOTEC BE LIABLE FOR ANY LOSS OF USE, INTERRUPTION OF

BUSINESS, OR ANY INDIRECT, SPECIAL, INCIDENTAL, PUNITIVE, OR CONSEQUENTIAL DAMAGES OF ANY KIND

(INCLUDING LOST PROFITS) REGARDLESS OF THE FORM OF ACTION WHETHER IN CONTRACT, TORT (INCLUDING

NEGLIGENCE), STRICT PRODUCT LIABILITY, OR OTHERWISE, EVEN IF MILTENYI BIOTEC HAS BEEN ADVISED OF THE

POSSIBILITY OF SUCH DAMAGES. IN NO EVENT SHALL THE TOTAL LIABILITY OF MILTENYI BIOTEC HEREUNDER

EXCEED THE GREATER OF 100.00 EUROS OR THE AMOUNT YOU ACTUALLY PAID FOR THE PRODUCT GIVING RISE

TO SUCH LIABILITY, REGARDLESS OF THE CAUSE OF ACTION, IN CONTRACT, TORT, STRICT LIABILITY, OR

OTHERWISE.

NOT ALL JURISDICTIONS ALLOW SUCH LIMITATIONS OF DAMAGES SO THE FOREGOING LIMITATIONS MAY NOT

APPLY TO YOU. This warranty statement gives you specific legal rights and you may have other rights, which

may vary from jurisdiction to jurisdiction.

11.2 Trademarks

Blaze, MACS, and the Miltenyi Biotec logo are either registered trademarks or trademarks of Miltenyi Biotec

and/or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the

property of their respective owners and are used for identification purposes only.11

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Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact.

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. Blaze, MACS, and the Miltenyi Biotec logo areregistered trademarks or trademarks of Miltenyi Biotec and/or its affiliates in various countries worldwide. All other trademarks mentioned in this publication are the property of theirrespective owners and are used for identification purposes only.Copyright © 2021Miltenyi Biotec and/or its affiliates. All rights reserved.

Germany/AustriaMiltenyi Biotec B.V. & Co. KGFriedrich-Ebert-Straße 6851429 Bergisch GladbachGermanyPhone +49 2204 8306-0Fax +49 2204 85197macsde@miltenyi.com

USA/CanadaMiltenyi Biotec Inc.2303 Lindbergh StreetAuburn, CA 95602, USAPhone 800 FOR MACSPhone +1 866 811 4466Fax +1 877 591 1060macsus@miltenyi.com

AustraliaMiltenyi BiotecAustralia Pty. Ltd.Unit 11, 2 Eden Park DriveMacquarie Park NSW 2113AustraliaPhone +61 2 8877 7400Fax +61 2 9889 5044macsau@miltenyi.com

BeneluxMiltenyi Biotec B.V.Sandifortdreef 172333 ZZ LeidenThe Netherlandsmacsnl@miltenyi.comCustomer serviceThe NetherlandsPhone 0800 4020120Fax 0800 4020100Customer service BelgiumPhone 0800 94016Fax 0800 99626Customer service LuxembourgPhone 800 24971Fax 800 24984

ChinaMiltenyi Biotec Technology &Trading (Shanghai) Co., Ltd.Room 401No. 1077, Zhangheng RoadPudong New Area201203 Shanghai, P.R. ChinaPhone +86 21 6235 1005-0Fax +86 21 6235 0953macscn@miltenyi.com

FranceMiltenyi Biotec SAS10 rue Mercoeur75011 Paris, FrancePhone +33 1 56 98 16 16macsfr@miltenyi.com

Hong KongMiltenyi Biotec Hong Kong Ltd.Unit 301, Lakeside 1No. 8 Science Park West AvenueHong Kong Science ParkPak Shek Kok, New TerritoriesHong KongPhone +852 3751 6698Fax +852 3619 5772macshk@miltenyi.com

ItalyMiltenyi Biotec S.r.l.Via Paolo Nanni Costa, 3040133 BolognaItalyPhone +39 051 6 460 411Fax +39 051 6 460 499macsit@miltenyi.com

JapanMiltenyi Biotec K.K.NEX-Eitai Building 5F16-10 Fuyuki, Koto-ku,Tokyo 135-0041, JapanPhone +81 3 5646 8910Fax +81 3 5646 8911macsjp@miltenyi.com

Nordics and BalticsMiltenyi Biotec Norden ABMedicon VillageScheeletorget 1223 81 LundSwedenmacsse@miltenyi.comCustomer service SwedenPhone 0200 111 800Fax +46 280 72 99Customer service DenmarkPhone 80 20 30 10Fax +46 46 280 72 99Customer serviceNorway, Finland, Icelandand Baltic countriesPhone +46 46 280 72 80Fax +46 46 280 72 99

SingaporeMiltenyi Biotec Asia Pacific Pte Ltd438B Alexandra Road, Block BAlexandra Technopark#06-01Singapore 119968Phone +65 6238 8183Fax +65 6238 0302macssg@miltenyi.com

South KoreaMiltenyi Biotec Korea Co., Ltd.Arigi Bldg. 8F562 Nonhyeon-roGangnam-guSeoul 06136, South KoreaPhone +82 2 555 1988Fax +82 2 555 8890macskr@miltenyi.com

SpainMiltenyi Biotec S.L.C/Luis Buñuel 2Ciudad de la Imagen28223 Pozuelo de Alarcón(Madrid)SpainPhone +34 91 512 12 90Fax +34 91 512 12 91macses@miltenyi.com

SwitzerlandMiltenyi Biotec Swiss AGGibelinstrasse 274500 SolothurnSwitzerlandPhone +41 32 623 08 47Fax +49 2204 85197macsch@miltenyi.com

United KingdomMiltenyi Biotec Ltd.Almac House, Church LaneBisley, Surrey GU24 9DR, UKPhone +44 1483 799 800Fax +44 1483 799 811macsuk@miltenyi.com

www.miltenyibiotec.com

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