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					      Optical and Acoustic Detection of
Laser-Generated Microbubbles in Single Cells
       IEEE Transactions on Ultrasonics, Ferroelectronics,
                     and Frequency Control
            Vol. 53, No. 1, pp. 117-125, January 2006

          Marwa J. Zohdy, Christine Tse, Jing Yong Ye,
                   and Matthew O’Donnell




                        Presented by:
                        Thomas Steen
                           5/22/06
Outline

 • Introduction
 • Laser Induced Optical Breakdown
 • Experimental Setup
      • Laser Irradiation and Optical Detection
      • Acoustic Detection
 • Cell Viability
 • Results
      • Destructive and Nondestructive Bubble Formation
      • Effect of the Location of the Focal Spot
                     • Nucleus
                     • Cytosol
      • Acoustic Detection Results
      • Cell Viability Results
 • Applications
 • Conclusions
Introduction

 • Laser induced optical breakdown (LIOB), or photodisruption,
 can be used to generate microbubbles within individual cells.

 • Can be spatially confined by tightly focusing laser beam

 • With femtosecond laser pulses, the generation threshold can be
 obtained with low total energy delivery (few nJ/pulse)
     • Virtually no collateral thermal and mechanical effects to out
     of focus areas

 • Laser generated bubbles can be clearly detected and monitored
 with high-frequency ultrasound do to their excellent acoustic
 reflectivity
Laser Induced Optical Breakdown

   • Optical breakdown in water is similar to transparent biological tissue
   • Electrons in liquid are either bound to a particular molecule or “quasi-free”
         • Possess sufficient kinetic energy to move without being captured by local
         molecular energy potentials
   • Treat water as an amorphous semiconductor
         • “Free electrons” means “quasi-free electrons”
         • “Ionization” means “excitation into the conduction band”
   • For optical breakdown to occur, a nonlinear absorption mechanism must
   deposit laser energy into the material by promoting electrons from the valence
   band to the conduction band
         • Photoionization and avalanche (cascade) ionization
   • If enough energy is deposited into the material by these nonlinear absorption
   mechanisms, a self-absorbing plasma is created
   • This rapidly expands and vaporizes the material
   • The expansion of gaseous products produced during ablation creates a bubble
   in the liquid surrounding the ablation site.


Schaffer et. al. 2001 Meas. Sci. Technol. 12 1784
Vogel et. al. 2003 Chem. Rev. 103 577
Laser Induced Optical Breakdown




Vogel et. al. 2003 Chem. Rev. 103 577
Outline

 • Introduction
 • Laser Induced Optical Breakdown
 • Experimental Setup
      • Laser Irradiation and Optical Detection
      • Acoustic Detection
 • Cell Viability
 • Results
      • Destructive and Nondestructive Bubble Formation
      • Effect of the Location of the Focal Spot
                     • Nucleus
                     • Cytosol
      • Acoustic Detection Results
      • Cell Viability Results
 • Applications
 • Conclusions
Laser Irradiation and Optical Detection
• Chinese hamster ovary cells in vitro
• Short axis diameter of approx. 10 m
• Microbubbles were generated in individual cells with:
    • 100fs pulses
    • 793nm Ti:Sapphire laser
    • Repetition rate = 3.8kHz
• 40x 0.6 N.A. objective
    • Diffraction-limited spot size = 1.6 m

• Minimum intensity to produce a
microbubble visible using the optical
microscope with 100 pulses
was 4.6 J/cm2
Acoustic Detection

• Single element ultrasonic transducer
          • 50 MHz center frequency
          • 4.1mm focal length
          • 3mm diameter
• Acoustic probe pulses were synchronized with laser pulsing at
3.8 kHz
• Transducer was triggered approx. 2s after each pulse
Cell Viability

 • 0.4% trypan blue was used as a colorimetric
 live/dead exclusion assay
     • Cells with intact membranes could exclude the
     dye and were considered viable
     • Cells that were nonviable took up the dye and
     became darkly stained
 • Still images were recorded before laser exposure
 and again after laser exposure and staining
 • 1-2 hours after laser exposure
Optical Detection in the Nondestructive Regime




                                                 Images of the same cell before laser
                                                 exposure (left panel) and after laser
                                                 exposure and live/dead staining
                                                 (right panel). In the right panel,
                                                 though, there is some morphology
                                                 change, the target cell remains
                                                 unstained following irradiation,
                                                 indicating viability.
(a)–(d) Selected movie frames from optical detection of
nondestructive photodisruption in a single cell, with        ***10 pulses
laser fluence 9 J/cm2 and total laser exposure time 2.6
ms, at times t = 0 ms, 33 ms, 66 ms, and 100 ms,
respectively.
Optical Detection in the Nondestructive Regime
Optical Detection in the Destructive Regime




                                                     Images of the same cell before laser
                                                     exposure (left panel) and after laser
                                                     exposure and live/dead staining (right
                                                     panel). In the right panel, the target
                                                     cell is darkly stained, indicating cell
                                                     death.

(a)–(d) Selected movie frames from optical
                                                               ***10,000 pulses
detection of destructive photodisruption in a single
cell, with laser fluence 5 J/cm2 and total laser
exposure time 2.6 s, at times t = 0 s, 3 s, 4 s, and 5
s, respectively.
Optical Detection in the Destructive Regime
Location of the focal spot: In the Nucleus




                                                    Images of the same cell before laser
                                                    exposure (left panel) and after laser
                                                    exposure and live/dead staining (right
                                                    panel). In the right panel, the target cell
                                                    is darkly stained, indicating cell death.




(a)–(d) Selected movie frames from optical
                                                                 ***1,000 pulses
detection of photodisruption in the cell nucleus,
with laser fluence 9 J/cm2 and total laser
exposure time 263 ms, at times t = 0 ms, 533
ms, 667 ms, and 833 ms, respectively.
Location of the focal spot: In the Nucleus
Location of the focal spot: In the Cytosol




                                                (e) Images of the same cell before
                                                laser exposure (left panel) and after
                                                laser exposure and live/dead staining
                                                (right panel). In the right panel,
                                                though, there is some morphology
                                                change, the target cell remains
                                                unstained following irradiation,
                                                indicating viability.
(a)–(d) Selected movie frames from optical
detection of photodisruption in the cell cytosol,             ***1,000 pulses
with laser fluence 9 J/cm2 and total laser
exposure time 263 ms, at times t = 0 ms, 533 ms,
667 ms, and 833 ms, respectively.
Location of the focal spot: In the Cytosol
Acoustic Detection




 Acoustic wavefield plot of a            Acoustic wavefield plot of a
 destructive bubble induced in a cell,   nondestructive bubble induced in a
 with laser fluence 46 J/cm2 and total   cell, with laser fluence 5 J/cm2 and
 laser exposure time 26 ms. A faint      total laser exposure time 2.6 ms.
 echo is visible after laser exposure
 ends, indicating a longerlived
Cell Viability Results




Viability data for cells exposed to a varying   Viability data for cells exposed to a
number of laser pulses at low (5–9 J/cm2),      varying number of laser pulses at low
intermediate (14–23 J/cm2), and high (41–       (5–9 J/cm2) laser fluence, where the
55 J/cm2) laser fluences. Error bars            intracellular focal spot was positioned
represent standard error at each set of         in either the nucleus or the cytosol of
parameters.                                     each target cell. Error bars represent
                                                standard error at each set of
                                                parameters.
Applications

 • Laser-induced microbubbles can potentially be used as
 therapeutic agents by selectively destroying single cells without
 damaging cells in the immediate vicinity.
 • Acoustic microscopy for detection of LIOB induced
 microbubbles
     • Photodisruption through the entire thickness of human
     scleral tissue for ophthalmic surgery applications.
     • Not optically transparent
     • Ultrasonic microscopy can clearly detect the microbubbles
     through the thickness
Conclusions

 • LIOB to generate localized acoustically detectable bubbles within
 single cells
 • Exploit high precision of tightly focused fs pulses
 • Acoustic detection had not been previously explored
 • By controlling laser fluence and exposure duration, LIOB can produce
 intracellular bubbles with a broad range of sizes and lifetimes
      • Can act as minimally invasive diagnostic markers or, if desired,
      destructive agents for selective cell killing.



 • Long term effects need to be examined
 • Minimum pulse fluence used in these studies (4.6 J/cm2) was fluence
 needed to create a bubble that could be optically detected within
 limitations of the optical microscopy system.
      • Actual threshold fluence for breakdown with 100 fs pulses is
      approx. 1J/cm2 in water and 1.5 J/cm2 in tissue
Questions?

				
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