MICROMANIPULATION - Meyer Instruments · PDF fileLiving up to Life Content • Success...
Transcript of MICROMANIPULATION - Meyer Instruments · PDF fileLiving up to Life Content • Success...
Living up to Life
MICROMANIPULATION
DSA Application days LSRSept., 15th -18th 09
Werner Wittke
Living up to Life
The Leitz Labovert MärzhäuserMicromanipulator 1981
The Leica DMIRE2 Leica Micromanipulator 2004
The Leica ASTP 2004
LeicaMicromanipulation
HistoryThe Leitz Micromanipulator1952
The Leica AM6000 2006
Living up to Life
Content
• Success story IMSI– ESHRE – IVF
• Micromanipulators• Applications
Living up to Life
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
Living up to Life
„Standard Image“
ICSI
20X objective
IMSIIntracytoplasmic Morphology selected Sperm Injected
Living up to Life
Correlation of morphology
with quality -
HOW DOES A
„GOOD“ SPERM
LOOKS LIKE ? vacuoles
IMSIIntracytoplasmic Morphology selected Sperm Injected
Living up to LifeESHRE Poster 07-09
Living up to Life
Since 2007
Approx. >30 IMSI systems sold
ANSP: around 100k€
EU, J, Asia, Afrika
Living up to LifeIMSI
Living up to LifeNew IVF brochure
Living up to Life
Living up to Life
Living up to Life
New IVF brochuresupports Leica IVF portfolio:
Inverted microscope
Upright microscopes
Stereo microscopes
and accessories in IVF
Living up to Life
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
Living up to Life
Manipulators
Living up to Life
MICROMANIPULATION
AM 6000
Narishige Co-operation
• New Narishige manipulators• Narishige peripheral equipment
Eppendorf Co-operation
• Leica AM6000• Epp. Peripheral equipment
Leicamanipulator
Living up to Life
Mechanic MicromanipulatorsLeicaLeica
Hydraulic MicromanipulatorsNarishigeNarishige
Electronic MicromanipulatorsEppendorfEppendorf
Living up to Life
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
Living up to Life
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
LeicaLeica Mechanic Micromanipulators
Living up to Life
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
LeicaLeica Mechanic Micromanipulators
Living up to Life
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
LeicaLeica Mechanic Micromanipulators
Living up to Life
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
LeicaLeica Mechanic Micromanipulators
Living up to Life
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)
LeicaLeica Mechanic Micromanipulators
Living up to Life
Capillary movement
Capillary movement
Capillaries
Living up to Life
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‘
Living up to Life
Leica DMI3000/4000/6000
NL-11 : 1 unitMounting Adaptor
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‘
Living up to Life
Leica DMI 3000
NL-11 : 1 unitMounting Adaptor
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‘
Living up to Life
Leica DMI 3000
NL-11 : 1 unitMounting Adaptor
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‘
Living up to Life
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
Living up to Life
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
Living up to Life
• 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!
Living up to Life
• 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!
Living up to Life
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!
Living up to Life
• 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!
Living up to Life
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
Living up to Life
• 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
Living up to Life
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
The AM6000 Micromanipulator
Living up to Life
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
The AM6000 Micromanipulator
Living up to Life
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
Living up to Life
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
Living up to Life
• Leica Stereo microscopes and manipulators
Living up to Life
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
Living up to Life
Living up to Life
11532640 1X Manual coarse manipulator MMN-5
1X Single axis oil hydraulic micromanipulator MMO-220A
Narishige manipulators with Leica stereo microscopes
Living up to Life
Narishige manipulators with Leica stereo microscopes
MN-153 manipulator with adapter NR2 (11101317) is a cheaper and pragmatic solution
Living up to Life
Narishige manipulators with Leica stereo microscopes
M-152 manipulator with magnetic stand GJ-1 and iron plate IP is the cheapestsolution
11531557 M-152
11531564 GJ-1
Living up to Life
Narishige manipulators with Leica stereo microscopes
Living up to Life
Applications
Living up to Life
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
Living up to Life
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°
Living up to Life
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°
Living up to Life
Micromanipulation
Application requirements:
•Working distance
•~ 2 Magnifications
•Convenient handling
•Temp. Control
•Stability (Vibrations)
•Periphery (Laser
•Camera (Documentation) Single manipulator >40°
Two manipulators <10°
Living up to Life
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
Living up to Life
IVF
Living up to Life
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
Living up to Life
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
Living up to Life
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
Living up to Life
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
Living up to Life
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
Living up to Life
Oocytes Sperms
BufferOil
Oocyte MetaPhase II
with first polar bodySperms
Living up to Life
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.
Living up to Life
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
Living up to Life
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
Living up to Life
The injection pipette is gently withdrawn and the injected oocyte is released from the holding pipette
Living up to Life
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.
Living up to Life
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
Living up to Life
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).
Living up to Life
Transgenics
Living up to Life
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
Living up to Life
Living up to Life
• Pronucleus Injection
• ES cell Transfer
Two different techniques are used:
Living up to Life
• Workflow:
Pronucleus injection
Living up to Life
• Microinjection of linear DNA into the pronucleus
• Pronucleus Injection
Living up to Life
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
Living up to Life
Isolation of Zygotes
Living up to Life
Microinjection chamber atlow 5x magnification, providing a good overviewFor embryo sorting
Microinjection chamber athigh 40x magnification, providing a detailed view for injection
Living up to Life
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)
Living up to Life
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
Living up to Life
• Transfer of embryonic stem cells intoblastocysts
Living up to Life
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
Living up to Life
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)
Living up to Life
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
Living up to Life
• 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
Living up to Life
• Insert the injection needle into the blastocyst• Allow overpressure to equilibrate• Inject the ES-cells
Living up to Life
• Withdraw the injection needle slowly• If necessary allow overpressure to equilibrate again• Release the blastocyst from the holding pressure
immediately
Living up to Life
ES-Cells
Phase contrast – round cells, clear ‘Halo‘
Living up to Life
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”.
Living up to Life
Relevant Market Segments Micromanipulation
• Biology InstitutesCell Bio, Molecular Bio., Biochemistry ...
• Biotechnology• Pharmacology• Agricultural• Oncology• Genetics/Genomics
• Neuroscience• Embryology• Transgenic Services• Many more
Living up to Life
Cloning
Living up to Life
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
Living up to Life
Cloned species:1952Tadpole1963 Carp1996 Sheep 97 Dolly2000: Rhesus monkey2001: Cattle2003: Cat2003: Horse...
Living up to Life
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