Optical and Acoustic Detection of Laser-Generated Microbubbles in Single Cells IEEE Transactions on Ultrasonics, Ferroelectronics, and Frequency Control Vol. 53, No. 1, pp , 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 Meas. Sci. Technol Vogel et. al Chem. Rev

Laser Induced Optical Breakdown Vogel et. al Chem. Rev

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/cm 2

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 (a)–(d) Selected movie frames from optical detection of nondestructive photodisruption in a single cell, with laser fluence 9 J/cm 2 and total laser exposure time 2.6 ms, at times t = 0 ms, 33 ms, 66 ms, and 100 ms, respectively. 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. ***10 pulses

Optical Detection in the Nondestructive Regime

Optical Detection in the Destructive Regime (a)–(d) Selected movie frames from optical detection of destructive photodisruption in a single cell, with laser fluence 5 J/cm 2 and total laser exposure time 2.6 s, at times t = 0 s, 3 s, 4 s, and 5 s, respectively. 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. ***10,000 pulses

Optical Detection in the Destructive Regime

Location of the focal spot: In the Nucleus (a)–(d) Selected movie frames from optical detection of photodisruption in the cell nucleus, with laser fluence 9 J/cm 2 and total laser exposure time 263 ms, at times t = 0 ms, 533 ms, 667 ms, and 833 ms, respectively. 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. ***1,000 pulses

Location of the focal spot: In the Nucleus

Location of the focal spot: In the Cytosol (a)–(d) Selected movie frames from optical detection of photodisruption in the cell cytosol, with laser fluence 9 J/cm 2 and total laser exposure time 263 ms, at times t = 0 ms, 533 ms, 667 ms, and 833 ms, respectively. (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. ***1,000 pulses

Location of the focal spot: In the Cytosol

Acoustic Detection Acoustic wavefield plot of a nondestructive bubble induced in a cell, with laser fluence 5 J/cm 2 and total laser exposure time 2.6 ms. Acoustic wavefield plot of a destructive bubble induced in a cell, with laser fluence 46 J/cm 2 and total laser exposure time 26 ms. A faint echo is visible after laser exposure ends, indicating a longerlived residual bubble.

Cell Viability Results Viability data for cells exposed to a varying number of laser pulses at low (5–9 J/cm 2 ), intermediate (14–23 J/cm 2 ), and high (41– 55 J/cm 2 ) laser fluences. Error bars represent standard error at each set of parameters. Viability data for cells exposed to a varying number of laser pulses at low (5–9 J/cm 2 ) laser fluence, where the intracellular focal spot was positioned in either the nucleus or the cytosol of 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/cm 2 ) 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/cm 2 in water and 1.5 J/cm 2 in tissue

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