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MICROMANIPULATION

DSA Application days LSRSept., 15th -18th 09

Werner Wittke

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The Leitz Labovert MärzhäuserMicromanipulator 1981

The Leica DMIRE2 Leica Micromanipulator 2004

The Leica ASTP 2004

LeicaMicromanipulation

HistoryThe Leitz Micromanipulator1952

The Leica AM6000 2006

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Content

• Success story IMSI– ESHRE – IVF

• Micromanipulators• Applications

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Optical magnification of sperms via light microscopy in order to correlate morphology with quality

IMSIIntracytoplasmic morphology selected sperm injected

IMSI System

–DMI microscope or AM6000

–Micromanipulator

–Objective 100x, DIC

– + 2x Mag changer

– + vario1.6x Zoom c-mount

– + camera/monitor factor (50)

= 16000X magnification at the monitor

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„Standard Image“

ICSI

20X objective

IMSIIntracytoplasmic Morphology selected Sperm Injected

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Correlation of morphology

with quality -

HOW DOES A

„GOOD“ SPERM

LOOKS LIKE ? vacuoles

IMSIIntracytoplasmic Morphology selected Sperm Injected

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Since 2007

Approx. >30 IMSI systems sold

ANSP: around 100k€

EU, J, Asia, Afrika

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Living up to LifeNew IVF brochure

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New IVF brochuresupports Leica IVF portfolio:

Inverted microscope

Upright microscopes

Stereo microscopes

and accessories in IVF

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Our IMSI/IVF Reference lab

• Prof Stephane Viville, Strassbourg, FrancePossibilty to visit this lab with customers

– He and his coworker (Dr. Christiane Wittemer) will give lectures during IVF congresses

– Give application support– IMSI courses

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Manipulators

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MICROMANIPULATION

AM 6000

Narishige Co-operation

• New Narishige manipulators• Narishige peripheral equipment

Eppendorf Co-operation

• Leica AM6000• Epp. Peripheral equipment

Leicamanipulator

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Mechanic MicromanipulatorsLeicaLeica

Hydraulic MicromanipulatorsNarishigeNarishige

Electronic MicromanipulatorsEppendorfEppendorf

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Features:

Continuously selectable gear reduction

Tension control

Translation, directly and without any backlash

3 separate gears (fine and coarse) for x,y and z

1 tilting gear ( 0° - 15° )

1 multidirection hanging joystick

LeicaLeica Mechanic Micromanipulators

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Features:

No movements of inertia to overcome when moving to specimen details

precise and straight movements of the micro-instruments give optimum results

maximum tool positioning accuracy even at high magnification

Smallest travel range approx. 0,25 µm


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Features:

Reliable functioning

rapid pre-alignment of the micro tools with the individually adjustable instrument holders, -saving valuable time

excellent user convenience – hands rest in a comfortable position on the base plate

can carry weight around 200 g


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Features:

no vibration : e.g. by motorsno delay : hydraulic, electronicfeeling : “you fell the touch”perfect : precise mechanic

Applications:mechanical advantages others than mechanicalslow but precise work routine worksimple holding function more sophisticatedsingle cells or objects adherent cell cultureooccyte/ blastocyt injection large quantity


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Accessories:

Single instrument holder

Double instrument holder

Instrument sleeves to hold the tips

Spacer for DMÍ-Series to hold the instruments

Base plate for DM IL or DMI-Series


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Versions:

Micromanipulator L (for the left site of Microscope)

Micromanipulator R(for the right site of Microscope)

(to achieve the right turn direction for the Z-axis)


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Capillary movement

Capillary movement

Capillaries

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Leica DMI 3000/4000/6000

NL-11 : 1 unitMounting Adaptor

MOM-202D : 2 unitsThree-axis Oil Hydraulic Joystick-controlled Micromanipulator (integrated with Motorized CoarseManipulator)

IM-300 : 1 unitMotorized Microinjector

Application Set up: ‘TRANSGENICMotorized‘

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Leica DMI3000/4000/6000


MON-202D : 2 unitsThree-axis Oil Hydraulic Joystick-controlled Micromanipulator(integrated with Manual CoarseManipulator)

IM-300 : 1 unitMotorized Microinjector (requires separate pressure source)

Application Set up: ‘TRANSGENICManual‘

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Leica DMI 3000


MOM-202D : 2 unitsThree-axis Oil Hydraulic Joystick-controlled Micromanipulator (integrated with Motorized CoarseManipulator)　　IM-9B : 1 unitMicroinjector (for Injection):

IM-9C : 1 unitInjector (for Holding)

Application Set up: ‘ICSI motorized‘

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Leica DMI 3000


MON-202D : 2 unitsThree-axis Oil Hydraulic Joystick-controlled Micromanipulator (integrated with Manual CoarseManipulator)

IM-9B : 1 unitMicroinjector (for Injection)

IM-9C : 1 unitInjector (for Holding)

Application Set up: 'ICSI manual‘

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Manipulators and Control Panel

• ‘Enhancement of Eppendorf NK2‘

• ‘Special Leica Edition of NK2’

• Big brother of NK2

• Microscope and micromanipulator functions are controlled & operated from one control element, simultaneously

Leica AM6000

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Leica AM6000“Chimeric” Control Panel

• Blue buttons control onlyEppendorf NK2 functions

• Red buttons control onlyLeica AM6000 functions

• Blue/Red buttons control Eppendorf or Leicafunctions

Objective changer

Microscope focus

Manipulator/mot stagecoarse/fine

selector

Light intensity

3 memory buttonsfor

object/contrast/magChg

Mode

Manipulator/mot stagecontrol

AM6000

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• Microscope and micromanipulator are controlled & operated from one control element, simultaneously!

• Important functions within a fingertip

- Convenient working with less stress

- Easy finding the control elements

- ‘Blind’ switching between microscope and manipulator functions

AM6000 Handling

PATENTED!

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• Microscope and micromanipulator are controlled & operated from one control element, simultaneously!

- Ergonomic, time saving faster manipulation via workflow automation

- Ideal for teaching and multi-user environment, very short ‘learning curve’

AM6000 Handling

PATENTED!

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Control Panel• The following functions are integrated

- All manipulator functions +

- Important microscope functions

Focusing

Objective change

Light setting

Speed adjustment of microscope stage

- Quick and easy switching between microscope and micromanipulator functions

AM6000 HandlingPATENTED!

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• Automated adjustment of micromanipulator movements to the microscope magnification!

- While changing the magnification, the sensitivity of manipulator is automatically adjusted

• Motorized 3-plate stage in combination with electronic micromanipulators and digital microscope

AM6000 Handling

PATENTED!

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One Supplier of Electronics, Optics and Mechanics

• Care and maintenance are well defined and are in the responsibility of one company

• In case of service an exchange of the panel will re-instate both the microscope and the micromanipulator

• Fast and complete update in case of system improvements

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• 3 motorized axis

• Precise vibration free movement of tips

• Proportional movement of manipulators

• Automated, pre-defined movements possible

• One button move to focus or any other position

The AM6000 Micromanipulator

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Setting of thresholds

• Limit in z Saves the specimenEnsures no damage of pipettes

• y off Saves the specimen

• Home, Clean One button press: moving back to defined ‘home’ resp. ‘clean’ positions

• Pos. 1-3 Up to three positions programmable


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Home and Clean Function

• Moves manipulators per click in a save end-switch position or in a user defined area to change or clean the pipettes

• Fast and save re-finding of pipette after cleaning or changing pipettes


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Special Features

Application related improvements

Smoothen the workflow –improves the results!

Benefits• 3 positions to save objectives together with

contrasting methods +focus, e.g.

5X PH 40x DIC 63x Fluo

or

5x IMC 20x IMC 100x DIC

• Auto-adjustment of best image, e.g:

– Prisms

– Analyzer and polarizer

– Light Manager, aperture and field diaphragms

– Phase rings

– TL-lamp and fluo lamp

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Special Features

• 3 Positions to save objectives together with contrasting methods and magnificationchanger, e.g.

100X DIC M 20X IMC 20X DIC

• M = 1.6x magnification

total magnification 160x

• Changing magnification without touching the microscope

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• Leica Stereo microscopes and manipulators

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Narishige manipulators with Leica stereo microscopes

11532641 adapter NL-A (M125, M165C, M205)

11532598 adapter NL –16 (MZ with basis TL, ST, HL)

11532640 1X Manual coarse manipulator MMN-5

1X Single axis oil hydraulicac micromanipulator MMO-220A

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11532640 1X Manual coarse manipulator MMN-5

1X Single axis oil hydraulic micromanipulator MMO-220A


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MN-153 manipulator with adapter NR2 (11101317) is a cheaper and pragmatic solution

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M-152 manipulator with magnetic stand GJ-1 and iron plate IP is the cheapestsolution

11531557 M-152

11531564 GJ-1

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Applications

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Micromanipulation

Adherent cell injection

ICSI IMSI

Animal Injection

Egg

Organisms

TransgenicsES cell Injection

P/N Injection

Cloning

IVF

Stereomicroscopes/inverted

„Manual“ inverted research microscope

Inverted research microscope Inverted research microscope

Applications

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Micromanipulation

Suspended cells (a)

Transgenics, Cloning, IVF

• Technique: Microinjection

• Injected materials: DNA, ES-cells, Sperm

• Market segments: Life Science Research, Cell Bio, Neuroscience, IVF…

<10°

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Micromanipulation

Adherent cells

• Technique: Microinjection

• Injected materials: DNA, RNA, antibodies, fluorochromes …

• Market segments: Life Science Research, Cell Bio, Neuroscience …

• Manipulators and equipment: Eppendorf NI 2, Eppendorf FemtoJet, Narishige MMO-220A, Narishige IM-300 Injector, Leica Manipulator

>40°

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Micromanipulation

Application requirements:

•Working distance

•~ 2 Magnifications

•Convenient handling

•Temp. Control

•Stability (Vibrations)

•Periphery (Laser

•Camera (Documentation) Single manipulator >40°

Two manipulators <10°

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Micromanipulation

Application requirements:

•Optical performance

•Transgenics: DIC (PH) IVF: IMC IMSI: DIC

IMC

PH

DIC

Quality of sperms/small vacuoles

3D/ depth of focus formulti cell embryo

3D/relief impressionfor Pronucleus injection

DIC

FLUORESCENCE – GFP organisms/Enucleation

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IVF

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Assisted Fertilization ART

IVF In Vitro FertilizationIn vitro –the opposite of –in vivo

add spermatozoids to an oocyte inside a petri dish

ICSI Intracytoplasmic Sperm Injection

Microinjection

IMSI Intracytoplasmic Morphology selcted Sperm Injection

Optical magnification and selection of sperm before ICSI

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PZD

ICSI

SUZI

HistoryAssisted Fertilizations

1. Partial Zona Dissection2. Sub-Zonal Insertion – SUZI3. Intracytoplastic Sperm Injection - ICSI

PB

Oolema

PB - Polar BodyZP - Zona Pellucida

Zona Pellucida

Ooplasma

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ICSI: The customer

The customer may be in an assisted human reproduction clinic (IVF lab) at a hospital or university, Andrology, dermatology, women´s hospital

or they may be working with animal oocytes as a part of a research project or breeding program.

Specialties:

Human fertility is the most common application. In research setting, rats or mice are sometimes used. Breeding programs often work with valuable livestock of exotic species preservation. Doing ICSI on some animal species requires the addition of a Piezo device, e.g. Prime Tech TMM-150FU piezo drive. However this is not used for the human procedure

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ICSI: The equipment

Microscope:

ICSI is done on a research inverted microscope (DMI or Leica AM6000).

Contrasting method is Modulation Contrast or DIC (or BF/AD).

Dry Objectives: 10x(BF), 20x(BF), 40x(DIC or IMC) LWD.

Three plate stage (manual or motorized) with 37°C insert

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ICSI: Intracytoplasmic Sperm Injection

Oocytes and spermatozoons

Zona pelucida

Oolemma

Ooplasma

The application

Sperm head Sperm tail

The oocytes are round, between 50 and 200 microns in diameter, suspended or free-floating.

HumanRat

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Oocytes Sperms

BufferOil

Oocyte MetaPhase II

with first polar bodySperms

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The operator presses the tail of the spermatozoon with the injection pipette against the bottom of the dish to immobilized the sperm and to open the cytoplasm. The spermatozoon is then aspirated, tail first, into the injection pipette.

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The metaphase II oocyte is immobilized by slight negative pressure exerted on the holding pipette a 9 o’clock. The polar body is held at 6 o’clock

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The micropipette containing a single spermatozoon is pushed through the zona pellucida at 3 o’clockThe oolemma is stretched by the tip and relaxes when the pipette moves into the ooplasma

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The injection pipette is gently withdrawn and the injected oocyte is released from the holding pipette

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After about 16 to 18 h incubation, the oocytes are observed for presence of pronuclei and polar bodies. Fertilization is considered normal when 2 clearly distinct pronuclei containing nucleoli are present.

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Cultivation before reimplantation

Co-culture of a human embryo with human granulosa/cumulus cells. Co-culture appears to benefit embryo growth by providing growth factors and/or removing embryo-toxic factors from the environment

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The embryo division of 2PN-oocytes is evaluated after further 24 h of in-vitro culture. Embryo replacement is usually done 48 h after the microinjection procedure (law).

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Transgenics

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Why Transgenics ?

• Gene studies• Better understanding of diseases and their treatment• Quality improvement • Better resistance against diseases

Random integrationIntegration site and copy number not predictable

What is Transgenics ?

• Introduction of foreign DNA • Insertion of extra copies of an existing gene• Insertion of exogenous DNA fragments

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• Pronucleus Injection

• ES cell Transfer

Two different techniques are used:

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• Workflow:

Pronucleus injection

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• Microinjection of linear DNA into the pronucleus

• Pronucleus Injection

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Pronuclear injection

Pronuclear injection is the injection of a small volume of fluid containing your gene of interest into a pronucleus of a zygote.

The foreign DNA will be integrated stable at a random chromosomal position of the host genome

An overproduction of the gene product is caused

Fields of application:

- Tissue and developmental specific gene regulation

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Isolation of Zygotes

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Microinjection chamber atlow 5x magnification, providing a good overviewFor embryo sorting

Microinjection chamber athigh 40x magnification, providing a detailed view for injection

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Technical Equipment PN-Injection

• Two micromanipulators• DIC or Modulation Contrast• 5x and 40x magnification objectives• Holding capillary (Vacu Tip)• Injection capillary (0.5 µm) inner diameter (Femtotip II)• Injector (FemtoJet)

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Microinjection chamber/petri dish glass bottom/slide

• Drop of media for embryos

• Media covered with oil to prevent evaporation

• Holding capillary at one side

• Injection needle at the other side

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• Transfer of embryonic stem cells intoblastocysts

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Blastocyst Injection

Injection of embryonic stem cells (ES) into the blastocoel of mouse blastocysts

ES cells will integrate into the inner cell mass (ICM) and form the embryo proper

leads to a knock-out of gene function

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Technical Equipment –ES cell transfer

• Two micromanipulators

• Phase contrast optics

• 5x and 20x objectives

• Holding capillary (VacuTip)

• Injection needle, inner diameter 8-12 µm (TransferTips)

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Injection needle with loaded ES-cells

• Pick up cells as tight as possible

• Do not allow to mix oil with cells

• Use only perfectly clean needle

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• Capture the blastocyst and hold firmly• Set the focus to the midplane of the blastocyst• Align the injection needle to the sharply focused position• Target the blastocyst right at the middle• Try to find a junction between two trophectoderm cells

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• Insert the injection needle into the blastocyst• Allow overpressure to equilibrate• Inject the ES-cells

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• Withdraw the injection needle slowly• If necessary allow overpressure to equilibrate again• Release the blastocyst from the holding pressure

immediately

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ES-Cells

Phase contrast – round cells, clear ‘Halo‘

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Chimeric mice and their offspringA chimeric mouse is created by mixing cells from a host embryo with embryonic stem cells (ES-cells). This means: the host embryo has a different genotype then the ES-cells. The level of chimerism and the outcoming offspring is directly related to the input of host and “guest cells”.

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Relevant Market Segments Micromanipulation

• Biology InstitutesCell Bio, Molecular Bio., Biochemistry ...

• Biotechnology• Pharmacology• Agricultural• Oncology• Genetics/Genomics

• Neuroscience• Embryology• Transgenic Services• Many more

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Cloning

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Enucleation/Nuclear transfer• Remove the nucleus from an

oocyte (blunt end capillary –laser drilling,

• Inject the nucleus of interest into the enucleated cell

Cloning

Laser drilling:•e.g. Hamilton Thorne:

•Laser drilling objective •e.g. Octax

•Laser encoupled via fluo axis

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Cloned species:1952Tadpole1963 Carp1996 Sheep 97 Dolly2000: Rhesus monkey2001: Cattle2003: Cat2003: Horse...

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LEICAMicro-

manipulationNo 1!

Bud

get €

Functionality

30 K €

50 K €

75 K €

20 K €

Microscopes DM IL DMI3000 B DMI4000 B

Manipulators Leica Man Narishige Man Narishige Mot Eppendorf NK2AM6000

micromanipulation - meyer instruments · pdf fileliving up to life content • success...

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