Cell Manipulator Laser Micromanipulation System

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Cell ManipulatorLaser Micromanipulation System
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MMI the Company

• Established 1998
• Head Office Glattbrugg, Zürich,
  Switzerland
• Branches MMI GmbH, Germany
• MMI Inc., USA
• Represented in more than 65 Countries
• Microscope Partner OLYMPUS NIKON
• Vision
• Be the preferred provider for
  micromanipulation solutions

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mmi Product range
mmi CellManipulator
mmi CellCut Plus
mmi ApplicationSupport
mmi CellEctor
mmi Service Support
mmi SmartCut Plus
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Topics to be addressed

• How optical tweezers work?
• How force measurement works?
• What options can be combined with the mmi
  CellManipulator?
• What are the applications for optical tweezers?
• Laser Safety
• How to set up the MBPS quadrant detector
• Hands on machine

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mmi CellManipulator Movie
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How optical tweezers work Forces created by
photons
Change of momentum of light due to
refraction Difference in refraction indes of
beads n1 and surrounding solution n2 N1 gt N2
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How optical tweezers work forces created by
light gradients
The change of momentum by refraction results in
a force towards the highest light intensity
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How optical tweezers work How to select the
right laser
NdYAG at 1064nm is optimal for most biological
applications
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How optical tweezers work Optical setup of MMI
CellManipulator
1 YAG Laser 1064 nm, 3 or 8 Watt 2 Focusing
lens 3 Galvo scanners 2 kHz 4 Objective with high
NA 5 Cells, particles in solution
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How optical tweezers work traditional single
level setup
MMI is using this setup with Zeiss microscopes
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How optical tweezers work advanced dual level
setup

• Using the infinite microscope optics
• to raise the stage
• Separated light path for tweezers
  
  
• laser and epi fluorescence
• Standard fluorescence filter cubes
• useable

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How optical tweezers workWhat objects I can
trap?

• Polystyrene beads (p. ex. Polysciences
  Polybead Microspheres 4.50µm)
• Silica beads
• Biological cells
• Intracellular vacuoles
• .

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How optical tweezers workWhat objects I can
trap?

• Necessary condition for trappable objects
• Objects must have a higher optical refraction n1
  index than surrounding medium n2
• Objects must be transparent for infrared light
• Objects must have a relative smooth surface
• Particle sizes 200nm - 20µm

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How force measurement works Alternative
technologies

• Threshold measurements
• How fast can I move a bead until I loose it?
• How fast can I move the stage until I loose the
  bead?
• Bead position measurements
• Is there an external force pulling the bead out
  of the trap center?
• How far the force can pull the bead out of the
  trap center?
• Do we observe a retardation of the bead movement
  when the trap moves fast?

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How force measurement works How fast can I move
a bead until I loose it?

• Use the galvoscanners and let the bead swing
• The maximum force can be easily calculated from
  the maximum oscillation frequency ? with which
  the trap can hold the bead
• F 6p2µrA?

Amplitude A Bead radius r Hydrodynamic
viscosity µ
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Operation of the mmi CellManipulator

• Fully controlled with well known mmiCellTools
  software
• Live Image
• Camera control
• Image saving for documentation and publication
• Creation of up to 10 traps with simple mouse
  click
• Moving each trap separetly
• Moving traps in groups
• Rotation, stretching
• Laser power control
• Extremely precise position control
• Stage with 75nm resolution
• Trap position with 50nm resolution
• Trap oszillation experiments

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector

• The detector will be mounted on a camera port of
  the microscope
• The detector analyses the image of the bead
• Additional zoom optics magnifies the bead image
  by a factor of 13x
• The bead image must be on the same size than one
  quadrant of the detector

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector

• The better and clearer the image the better the
  signal
• Increasing the image contrast by
• DIC imaging
• Phase contrast
• Fluorescence (high light intensity by FluoBeads)

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector

• Getting voltage signals by changing the bead
  position slightly

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector
Voltages observed when moving the bead image over
the detector area
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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector the
oszillation method

• Voltages observed when oscillating the trap
  slowly (1Hz)

? The bead can follow the trap, same position at
same time
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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector the
oszillation method

• Voltages observed when oscillating the trap
  faster (gt100Hz)

• The bead cant follow the trap the bead is
  delayed and will not show the full amplitude of
  the trap oscillate with

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector the
oszillation method

• The phase shift is caused by external forces on
  the bead
• Viscous forces from surrounding water
• Molecules attached to the bead
• The stronger external forces pull on the bead
• the bigger the phase shift
• the smaller the amplitude
• Trap stiffness can easily be estimated by
  oscillating beads in water
• When trap stiffness is known molecular forces can
  be calculated from the phase shift and the
  amplitude

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector
Browns motion method

• Each bead fluctuates caused by Browns motion

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How force measurement worksMeasuring bead
positions with the MBPS quadrant detector
Browns motion method

• Fourier-Transformation of the measured data
• The stronger the trap catches the bead, the less
  oszillations will be observed ? fc will be shifted

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How force measurement worksThe full system setup
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Modularisation of the mmiCellManipulator

• Fully compatibel with MMI modules
• mmi CellCut
• mmi Cellector
• MBPS (Multibead positioning detector)
• Quadrant detector to measure bead positions with
  nanometer resolution
• Mounted on microscope camera port (80 light to
  MBPS, 20 to mmi CellCamera required)
• Force measurement support via software
• Data acquisition via software
• DIC and phase contrast
• Combinated systems available with
• TIRF (Total internal reflection microscopy)
• Laser scanning
• Spinning disk
• Piezo z-drive for objective
• Further options on request

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Applications biological
Cell-based Studies Cell fusions and
cell-to-cell interactions Implant studies
Intracellular manipulations Study of neuronal
networks Drug effects on cells Ca2-channel
studies
Measurements of Binding Forces DNA studies
Viscosity measurements Antibody, antigen
binding forces Bacterial adhesion studies
Virus to cell adhesion studies
Molecular Motor Studies Actin, Myosin
interactions Kinesin Motors Dynein Motors
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Applications others

• Piconewton force measurements
• Viscosity measurments
• In the fields of
• Material science
• Colloid science
• Chemical surfaces
• ...
• REVIEW OF SCIENTIFIC INSTRUMENTS 78, 074302 2007
• Using optical tweezers for measuring the
  interaction forces between human bone cells and
  implant surfaces System design and force
  calibration
• Martin Andersson, Stefan Niehren et al.

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Laser safety

• mmi CellManipulator is a Class 4 laser product!
• The 8W infrared laser is harmfull for skin and
  eyes
• AVOID EYE OR SKIN EXPOSURE TODIRECT OR SCATTERED
  RADIATION

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Laser safety

• Always use laser safety eyewear
• Never open the cover of the laser box or unmount
  parts
• Never start the laser when an objective position
  in the objective turret is not covered
• Laser safety eyewear must be specified for 1064nm
  / 3W

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MPBS calibrationPreparation of a test slide

• Dilute 1 drop of 1µm beads in 5ml water
• Remove membrane from a membrane slide
• Fix a coverslip on the metal frame with superglue
• Pipette 1 drop of diluted beads on slide and let
  it dry

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MPBS calibrationAdjust parfocality

• Hook up a small observation camera instead of one
  detector head
• Loose the first ring (thorlabs mount) behind the
  c-mount adapter
• Adjust the tube length so that both images from
  mmi CellCamera and on the observation camera are
  in focus
• Lock the thorlabs mount

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MPBS calibrationFind the active position of the
detector head

• Adjust the microscope observation method to get a
  clear bright bead on relative dark background.
  Best results are observed by DIC illumination.
• Move the xy-stage, so that a well recognizable
  bead (best a double or triple) is visible in the
  center of the observation camera
• Set the QD-marker in mmi CellTools on the
  observed bead by the menu item

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MPBS calibrationfine tuning of the active
position

• Move a single bead to the active position (QD
  marker on screen)
• Move the stage in tiny steps (75nm)
• Observe the voltages on the MPBS output
  connectors
• You must observe a signal like the black line in
  the diagram when you move the bead over the
  detector

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