Cell MicroManipulation and Nanoscale Probing Techniques

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Cell Micro-Manipulation and Nanoscale Probing
Techniques

• Terrence Dobrowsky
• February 12, 2007
• IGERT/HHMI Journal Club

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Different Experimental Techniques and Mechanical
Models for Cells

• Method used to observe will dictate response
• Experimental observations lead to different
  models
• Linking mechanical signals to biological signals
• How cells mechanically respond under physical
  loads

Lim, CT. et al. Journal of Biomechanics, 39.
2006
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Different Experimental Techniques and Mechanical
Models for Cells
Lim, CT. et al. Journal of Biomechanics, 39.
2006
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Optical TrappingA Hands Off Manipulation of Cells

• Trapping a particle in the focal point of a
  single beam of light
• Implications for biological applications apparent
  from the beginning
• Discussion Focused Topics
• Modifications necessary for adaptation to live
  cell technique

Ashkin, A. et al. Optic Letters, 11.5, 1986
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Optical TrappingA Hands Off Manipulation of Cells

• Varying levels of complication
• Newtonian explanation

Ulanowski, ZJ, et al. New Physics. 2006
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Optical TrappingA Hands Off Manipulation of Cells

• Experimental Setup
• Objective used to converge laser
• External lens on 3D mount
• Leveled beam splitters and blocking filters

Askin, A. et al. Letters To Nature, vol. 330,
769. 1987
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Atomic Force MicroscopyMultiple Levels of
Information

• Multiple applications for cellular probing
• Classical, Imaging live and fixed cells for a
  total 3D representation of adherent cellular
  structure
• Cellular Elastography, Measuring the local
  viscoelastic properties of live cells
• Molecular Force Probe, Measuring the dissociation
  kinetics of surface receptors through single
  molecule manipulation

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Atomic Force MicroscopyMultiple Levels of
Information

• AFM Basic Setup and principles
• Surface mediated cantilever deflection
• Photodetector measuring reflected laser
  displacement
• Measure surface deformation with 10pm resolution

Panorchan, P. et al. Journal of Cell Science,
119. 2006
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Atomic Force MicroscopyMultiple Levels of
Information

• AFM Cantilevers tips are equipped with a specific
  geometry for function

Costa, KD. Disease Markers, 19. 2003, 2004
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Atomic Force MicroscopyMultiple Levels of
Information

• Viscoelastic studies using AFM probing techniques
• Quantifying local cellular elastic properties
  with regard to the cytoskeleton

Costa, KD. Disease Markers, 19. 2003, 2004
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Molecular Force ProbeAFM without the Microscopy

• Functionalizing Cantilevers as a live biological
  substrate
• Receptor binding in native environment observed
• Theoretical models are used to extract unstressed
  kinetic values
• Bells Model

Panorchan, P. et al. Journal of Cell Science,
119. 2006
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Quantum DotsAs Applied to Biological Problems

• Unique optical and spectroscopic properties
• Broad adsorption
• Narrow/Tunable emission
• Photobleaching resistance
• Long Luminescent lifetimes

Zhou, M. et al. Biopolymers. Dec. 2006
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Quantum DotsAs Applied to Biological Problems

• Hydrophobic inorganic surfactants
• Stepped through
• Maintaining optical yield while producing
  hydrophilic surface
• Loss of stability, releasing heavy atoms into
  medium
• Recently, di-block copolymers used

Zhou, M. et al. Biopolymers. Dec. 2006
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Quantum DotsAs Applied to Biological Problems

• Examples of current QD uses in biology

Zhou, M. et al. Biopolymers. Dec. 2006
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Quantum DotsAs Applied to Biological Problems
Zhou, M. et al. Biopolymers. Dec. 2006

• RGD peptide Tumor related Integrin Antagonist

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Multiple-Particle-Tracking Microrheology

• Operates with understanding that physical contact
  alters mechanical properties of cells and cannot
  be used to measure those properties
• In Vivo assay using nanoparticles to observe
  cellular elasticity and diffusion within cytoplasm

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Multiple-Particle-Tracking Microrheology

• Upstream introduction before egg development
• Downstream analysis during cellular division

Daniels, B. et al. Biophysical Journal, 90. 2006
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What We Cant Measureand How It is Always Present

• Future applications

Everett, WN, et al. Biophysical Journal, 92. 2007
Fernandez, GE. et al. Langmuir, 23. 2007
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Conclusions

• There are several methods to manipulate and probe
  cellular characteristics
• Models are developed through those assays
• Application of assays not originally designed for
  biological applications may hold the most promise
• Quantitative values for comparison
• By observing we alter the sample, how ever
  delicate we may be