S. Denis Courvoisier, N. Saklayen, M. Huber, J. Chen, E. D. Diebold, L. Bonacina, J. Wolf, and E. Mazur. 2015. “Plasmonic Tipless Pyramid Arrays for Cell Poration.” Nanoletters, 15, Pp. 4461–4466. Publisher's VersionAbstract
    Improving the efficiency, cell survival, and throughput of methods to modify and control the genetic expression of cells is of great benefit to biology and medicine. We investigate, both computationally and experimentally, a nanostructured substrate made of tipless pyramids for plasmonic-induced transfection. By optimizing the geometrical parameters for an excitation wavelength of 800 nm, we demonstrate a 100-fold intensity enhancement of the electric near field at the cell−substrate contact area, while the low absorption typical for gold is maintained. We demonstrate that such a substrate can induce transient poration of cells by a purely optically induced process.
    S. H. Chung, A. Schmalz, R. Clarissa Ruiz, C. V. Gabel, and E. Mazur. 2013. “Femtosecond Laser Ablation Reveals Antagonistic Sensory and Neuroendocrine Signaling that Underlie C. elegans Behavior and Development.” Cell Reports, 4, Pp. 316–326. Publisher's VersionAbstract
    The specific roles of neuronal subcellular compo- nents in behavior and development remain largely unknown, even though advances in molecular biology and conventional whole-cell laser ablation have greatly accelerated the identification of contrib- utors at the molecular and cellular levels. We system- atically applied femtosecond laser ablation, which has submicrometer resolution in vivo, to dissect the cell bodies, dendrites, or axons of a sensory neuron (ASJ) in Caenorhabditis elegans to determine their roles in modulating locomotion and the develop- mental decisions for dauer, a facultative, stress- resistant life stage. Our results indicate that the cell body sends out axonally mediated and hormonal sig- nals in order to mediate these functions. Further- more, our results suggest that antagonistic sensory dendritic signals primarily drive and switch polarity between the decisions to enter and exit dauer. Thus, the improved resolution of femtosecond laser ablation reveals a rich complexity of neuronal signaling at the subcellular level, including multiple neurite and hormonally mediated pathways depen- dent on life stage.
    J. Brugués, V. Nuzzo, E. Mazur, and D. Needleman. 2012. “Nucleation and Transport Organize Microtubules in Metaphase Spindles.” Cell, 149(3):554-64, Pp. –. Publisher's VersionAbstract
    Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and find that microtubules are shortest near poles and become progressively longer towards the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by non-uniform microtubule nucleation and the local sorting of microtubules by transport.
    S. H. Chung and E. Mazur. 2009. “Femtosecond laser ablation of neurons in C. elegans for behavioral studies.” Appl. Phys. A, 96, Pp. 335–341. Publisher's VersionAbstract
    Femtosecond laser ablation selectively dissects subcellular components of the C. elegans neuronal circuit with submicrometer precision for studying the neuronal origins of behavior. We describe the theoretical basis for the high precision of femtosecond laser ablation in the target bulk. Next, we present the experimental setup and a worm rotation technique to facilitate imaging and surgery. We describe the damage caused by different pulse energies on cell bodies and neuronal fibers. Finally, we discuss the regrowth of neuronal fibers after surgery and its impact on behavioral study.
    S. H. Chung and E. Mazur. 2009. “Surgical applications of femtosecond lasers.” J. Biophoton., 2, Pp. 557–572. Publisher's VersionAbstract
    Femtosecond laser ablation permits non-invasive surgeries in the bulk of a sample with submicrometer resolution. We briefly review the history of optical surgery techniques and the experimental background of femtosecond laser ablation. Next, we present several clinical applications, including dental surgery and eye surgery. We then summarize research applications, encompassing cell and tissue studies, research on C. elegans, and studies in zebrafish. We conclude by discussing future trends of femtosecond laser systems and some possible application directions.
    M. Zhang, S. H. Chung, C. Fang-Yen, C. Craig, R. A. Kerr, H. Suzuki, A. D. T. Samuel, E. Mazur, and W. R. Schafer. 2008. “A self-regulating feed-forward circuit controlling C. elegans egg- laying behavior.” Curr. Biol., 18, Pp. 1445–1455. Publisher's VersionAbstract
    Background Egg laying in Caenorhabditis elegans has been well studied at the genetic and behavioral levels. However, the neural basis of egg-laying behavior is still not well understood; in particular, the roles of specific neurons and the functional nature of the synaptic connections in the egg- laying circuit remain uncharacterized. Results We have used in vivo neuroimaging and laser surgery to address these questions in intact, behaving animals. We have found that the HSN neurons play a central role in driving egg-laying behavior through direct excitation of the vulval muscles and VC motor neurons. The VC neurons play a dual role in the egg-laying circuit, exciting the vulval muscles while feedback-inhibiting the HSNs. Interestingly, the HSNs are active in the absence of synaptic input, suggesting that egg laying may be controlled through modulation of autonomous HSN activity. Indeed, body touch appears to inhibit egg laying, in part by interfering with HSN calcium oscillations. Conclusions The egg-laying motor circuit comprises a simple three-component system combining feed-forward excitation and feedback inhibition. This microcircuit motif is common in the C. elegans nervous system, as well as in the mammalian cortex; thus, understanding its functional properties in C. elegans may provide insight into its computational role in more complex brains.
    A. Heisterkamp, J. Baumgart, I. Zaharieva Maxwell, A. Ngezahayo, E. Mazur, and H. Lubatschowski. 2007. “Fs-Laser Scissors for Photobleaching, Ablation in Fixed Samples and Living Cells, and Studies of Cell Mechanics.” In Laser Manipulation of Cells and Tissues, edited by Michael Berns and Karl Greulich, Pp. 293–307. Academic Press. Publisher's VersionAbstract
    The use of ultrashort laser pulses for microscopy has steadily increased over the past years. In this so-called multiphoton microscopy, laser pulses with pulse duration around 100 femtoseconds (fs) are used to excite fluorescence within the samples. Due to the high peak powers of fs lasers, the absorption mechanism of the laser light is based on nonlinear absorption. Therefore, the fluorescence signal is highly localized within the bulk of biological materials, similar to a confocal microscope. However, this nonlinear absorption mechanism can not only be used for imaging but for selective alteration of the material at the laser focus: The absorption can on one hand lead to the excitation of fluorescent molecules of fluorescently tagged cells by the simultaneous absorption of two or three photons or on the other hand, in case of higher order processes, to the creation of free-electron plasmas and, consequently, plasma-mediated ablation. Typical imaging powers are in the range of tens of milliwatts using 100-fs pulses at a repetition rate of 80-90 MHz, while pulse energies needed for ablation powers are as low as a few nanojoules when using high numerical aperture microscope objectives for focusing the laser radiation into the sample. Since the first demonstration of this technique, numerous applications of fs lasers have emerged within the field of cellular biology and microscopy. As the typical wavelengths of ultrashort laser systems lie in the near infrared between 800 and 1000 nm, high penetration depth can be achieved and can provide the possibility of imaging and manipulating the biological samples with one single laser system.
    A. Samuel, S. H. Chung, D. A. Clark, C. V. Gabel, C. Chang, V. Murthy, and E. Mazur. 2006. “Femtosecond laser dissection in C. elegans neural circuits.” In . SPIE Photonics West. Publisher's VersionAbstract
    The nematode C. elegans, a millimeter-long roundworm, is a well-established model organism for studies of neural development and behavior, however physiological methods to manipulate and monitor the activity of its neural network have lagged behind the development of powerful methods in genetics and molecular biology. The small size and transparency of C. elegans make the worm an ideal test-bed for the development of physiological methods derived from optics and microscopy. We present the development and application of a new physiological tool: femtosecond laser dissection, which allows us to selectively ablate segments of individual neural fibers within live C. elegans. Femtosecond laser dissection provides a scalpel with submicrometer resolution, and we discuss its application in studies of neural growth, regenerative growth, and the neural basis of behavior.
    I. Zaharieva Maxwell. 2006. “Application of femtosecond lasers for subcellular nanosurgery”. Publisher's VersionAbstract
    This dissertation o ers a study of femtosecond laser disruption in single cells. Cells and tissues do not ordinarily absorb light in the near-IR wavelength range of femtosecond lasers. However, the peak intensity of a femtosecond laser pulse is very high and material disruption is possible through nonlinear absorption and plasma generation. Because the pulse duration is very short, it is possible to reach the intensity of optical breakdown at only nanojoules of energy per pulse. The low energy deposition and the high spatial localization of the nonlinear absorption, make femtosecond laser pulses an ideal tool for minimally disruptive subcellular nanosurgery. We show definitively that there can be bulk ablation within a single cell by studying the disrupted region under a transmission electron microscope. The width of the ablated area can be as small as 250 nm in diameter at energies near the ablation threshold. We also studied the e ect of the laser repetition rate on the subcellular disruption threshold. We compared the pulse energies for kHz and MHz pulse trains, and found that in the MHz regime heat accumulation in the focal volume needs to be accounted for. For this repetition rate the minimum pulse energy necessary for disruption depends on the laser irradiation time. We used femtosecond laser nanosurgery to probe tension in actin stress fibers in living endothelial cells. By severing an individual stress fiber and visualizing its retraction, we showed that actin carries prestress in adherent, non-contractile cells. By plating the cells on softer, more compliant substrates, we measured the deflection of the substrate and extrapolated the force contribution of a stress filament on total amount of force exerted by the cell.
    S. H. Chung, D. A. Clark, C. V. Gabel, E. Mazur, and A. Samuel. 2006. “The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation.” BMC Neuroscience, 7, Pp. 30–. Publisher's VersionAbstract
    Background: Caenorhabditis elegans actively crawls down thermal gradients until it reaches the temperature of its cultivation, exhibiting what is called cryophilic movement. Implicit in the worms ability to actively bias its movements down thermal gradients is an ability to detect thermal gradients, and implicit in regulating the display of cryophilic bias is the ability to compare current ambient temperature with a stored memory of cultivation temperature. Several lines of evidence link the AFD sensory neuron to thermotactic behavior, but its exact role is not yet known. A current model contends that AFD is part of a thermophilic mechanism which biases movement up thermal gradients that counterbalances a cryophilic mechanism which biases movement down thermal gradients. Results: We used tightly-focused femtosecond laser pulses to dissect the AFD neuronal cell bodies and the AFD sensory dendrites in C. elegans to investigate their contribution to biased cryophilic movement. We establish that femtosecond laser ablation can exhibit submicrometer precision allowing the severing of individual AFD nerve fibers without causing collateral damage. Severing AFD dendrites in young adult worms permanently abolishes their sensory contribution without functional regeneration. We show that thermosensory input to the AFD neuron is required to activate a mechanism for generating cryophilic bias, but we find no evidence that AFD laser surgery reduces a putative ability to generate thermophilic bias. In addition, although disruption of the AIY interneuron causes worms to exhibit cryophilic bias at all temperatures, we find no evidence that disruption of the AIZ interneuron causes thermophilic bias at any temperature. Conclusions: We conclude that laser surgical analysis of the thermotactic circuit does not support a current model in which AFD opposes cryophilic bias by generating thermophilic bias. Our data supports a model in which a mechanism for generating cryophilic bias is gated by the AFD neurons.
    S. Kumar, I. Zaharieva Maxwell, A. Heisterkamp, T. R. Polte, T. Lele, M. Salanga, E. Mazur, and D. E. Ingber. 2006. “Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics.” Biophys. J., 90, Pp. 3762–3773. Publisher's VersionAbstract
    Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.
    A. Heisterkamp, I. Zaharieva Maxwell, E. Mazur, J. M. Underwood, J. A. Nickerson, S. Kumar, and D. E. Ingber. 2005. “Pulse energy dependence of subcellular dissection by femtosecond laser pulses.” Opt. Express, 13, Pp. 3690–3696. Publisher's VersionAbstract
    Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high- numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 m away.
    A. Heisterkamp, I. Zaharieva Maxwell, S. Kumar, J. M. Underwood, J. A. Nickerson, D. E. Ingber, and E. Mazur. 2005. “Nanosurgery in live cells using ultrashort laser pulses.” In . SPIE Photonics West. Publisher's VersionAbstract
    We selectively disrupted the cytoskeletal network of fixed and live bovine capillary endothelial cell using ultrashort laser pulses. We image the microtubules in the cytoskeleton of the cultured cells using green fluorescent protein. The cells are placed on a custom-built inverted fluorescence microscope setup, using a 1.4 NA oil-immersion objective to both image the cell and focus the laser radiation into the cell samples. The laser delivers 100-fs laser pulses centered at 800 nm at a repetition rate of 1 kHz; the typical energy delivered at the sample is 15nJ. The fluorescent image of the cell is captured with a CCD-camera at one frame per second. To determine the spatial discrimination of the laser cutting we ablated microtubules and actin fibers in fixed cells. At pulse energies below 2 nJ we obtain an ablation size of 200 nm. This low pulse energy and high spatial discrimination enable the application of this technique to live cells. We severed a single microtubule inside the live cells without affecting the cells viability. The targeted microtubule snaps and depolymerizes after the cutting. This nanosurgery technique will further the understanding and modeling of stress and compression in the cytoskeletal network of live cells.
    I. Zaharieva Maxwell, S. H. Chung, and E. Mazur. 2005. “Nanoprocessing of subcellular targets using femtosecond laser pulses.” Med. Laser Appl., 20, Pp. 193–200. Publisher's VersionAbstract
    In this paper we review the work done in our laboratory on femtosecond laser dissection within single cells and living organisms. Precise dissection of biological material with ultrashort laser pulses requires a clear understanding of the pulse-energy dependence of the onset and extent of plasma-mediated ablation (i.e., the removal of material). We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and in nuclei of fixed endothelial cells using femtosecond, near- infrared laser pulses focused through a high- numerical aperture objective lens (1.4 NA). We performed laser nanosurgery in live cells, where we ablated a single mitochondrion and severed cytoskeletal filaments without compromising the cell membrane or the cells viability. We also cut dendrites in living C. elegans without affecting the neighboring neurons. This nanoprocessing technique enables non-invasive manipulation of the structural machinery of cells and tissues down to several-hundred- nanometer resolution.
    N. Shen, D. Datta, C. B. Schaffer, P. LeDuc, D. E. Ingber, and E. Mazur. 2005. “Ablation of cytoskeletal filaments and mitochondria in cells using a femtosecond laser nanoscissor.” Mechanics and Chemistry of Biosystems, 2, Pp. 17–26. Publisher's VersionAbstract
    Analysis of cell regulation requires methods for perturbing molecular processes within living cells with spatial discrimination on the nanometer- scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub- micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies. Keywords: Nanoscissor; nanosurgery; femtosecond laser; photodisruption; cytoskeleton; mitochondria
    N. Shen. 2003. “Photodisruption in biological tissues using femtosecond laser pulses”. Publisher's VersionAbstract
    Transparent materials do not ordinarily absorb visible or near-infrared light. However, the intensity of a tightly focused femtosecond laser pulse is great enough that nonlinear absorption of the laser energy takes place in transparent materials, leading to optical breakdown and permanent material modification. Because the absorption process is nonlinear, absorption and material modification are confined to the extremely small focal volume. Optical breakdown in transparent or semi-transparent biological tissues depends on intensity rather than energy. As a result, focused femtosecond pulses induce optical breakdown with significantly less pulse energy than is required with longer pulses. The use of femtosecond pulses therefore minimizes the amount of energy deposited into the targeted region of the sample, minimizing mechanical and thermal effects that lead to collateral damage in adjacent tissues. We demonstrate photodisruptive surgery in animal skin tissue and single cells using 100-fs laser pulses. In mouse skin, we create surface incisions and subsurface cavities with much less collateral damage to the surrounding tissue than is produced with picosecond pulses. Using pulses with only a few nanojoules of energy obtained from an unamplified femtosecond oscillator, we destroy single mitochondria in live cells without affecting cell viability, providing insights into the structure of the mitochondrial network. An apparatus is constructed to perform subcellular surgery and multiphoton 3D laser scanning imaging simultaneously with a single laser and objective lens.