Glass Microneedles Research Articles

A modified motor-clutch model reveals that neuronal growth cones respond faster to soft substrates.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

Membrane backtracking at the maximum capacity of nondigestible antigen phagocytosis in macrophages

The zipper model has been dominantly used to describe the driving mechanism of the engulfment process and its specific identification of antigens during phagocytosis in macrophages. However, the abilities and limitations of the zipper model, capturing the process as an irreversible reaction, have not been examined yet under the critical conditions of engulfment capacity. Here, we demonstrated the phagocytic behavior of macrophages after reaching the maximum engulfment capacity by tracking the progression of their membrane extension during engulfment using IgG-coated nondigestible polystyrene beads and glass microneedles. The results showed that, after macrophages reached their maximum engulfment capacity, they induced membrane backtracking (the reverse phenomenon of engulfment) in both polystyrene beads and glass microneedles, regardless of the difference in the shape of these antigens. We evaluated the correlation of engulfment in simultaneous stimulations of two IgG-coated microneedles and found that each microneedle was regurgitated by the macrophage independently of the advancement or backtracking of membranes on the other microneedle. Moreover, assessing the total engulfment capacity determined by the maximum amount the macrophage was capable of engulfing when imposing each antigen geometry showed that the capacity increased as the attached antigen areas increased. These results indicate that the mechanism of engulfment should imply the following: 1) macrophages have a backtracking function to recover their phagocytic activity after reaching maximal engulfment limit, 2) both phagocytosis and backtracking are local phenomena of the macrophage membrane that operates independently, and 3) the maximum engulfment capacity is determined not only by mere local cell membrane capacity but also by the whole-cell volume increase during simultaneous phagocytosis of multiple antigens by the single macrophages. Thus, the phagocytic activity may entail a hidden backtracking function, adding to the conventionally known irreversible zipper-like ligand-receptor binding mechanism during membrane progression to recover the macrophages that are saturated from engulfing targets beyond their capacity.

Solid and hollow metallic glass microneedles for transdermal drug-delivery

Metallic microneedles are attractive for painless transdermal drug-delivery. However, fabrication techniques for metal microneedles are often complex and multi-step. In this study, a scalable manufacturing of metallic microneedle arrays is presented using thermoplastic drawing of metallic glasses. Microneedles with tunable lengths and tips are produced by controlling the rheology and fracture of metallic glass. The same drawing process can generate solid and hollow microneedles simply by varying the thickness of metallic glass. The mechanism of thickness dependent transition from solid to hollow profiles is described by the viscous buckling of metallic liquid. In vitro skin insertion tests demonstrate that both solid and hollow metallic glass microneedles can pierce porcine skin and deliver model drugs.

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Printing of small molecular medicines from the vapor phase

There is growing need to develop efficient methods for early-stage drug discovery, continuous manufacturing of drug delivery vehicles, and ultra-precise dosing of high potency drugs. Here we demonstrate the use of solvent-free organic vapor jet printing to deposit nanostructured films of small molecular pharmaceutical ingredients, including caffeine, paracetamol, ibuprofen, tamoxifen, BAY 11-7082 and fluorescein, with accuracy on the scale of micrograms per square centimeter, onto glass, Tegaderm, Listerine tabs, and stainless steel microneedles. The printed films exhibit similar crystallographic order and chemistry as the original powders; controlled, order-of-magnitude enhancements of dissolution rate are observed relative to powder-form particles. In vitro treatment of breast and ovarian cancer cell cultures in aqueous media by tamoxifen and BAY 11-7082 films shows similar behavior to drugs pre-dissolved in dimethyl sulfoxide. The demonstrated precise printing of medicines as films, without the use of solvents, can accelerate drug screening and enable continuous manufacturing, while enhancing dosage accuracy.

Mechanical properties of spindle poles are symmetrically balanced

The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties.

Substrate Deformation Predicts Neuronal Growth Cone Advance

Although pulling forces have been observed in axonal growth for several decades, their underlying mechanisms, absolute magnitudes, and exact roles are not well understood. In this study, using two different experimental approaches, we quantified retrograde traction force in Aplysia californica neuronal growth cones as they develop over time in response to a new adhesion substrate. In the first approach, we developed a novel method, to our knowledge, for measuring traction forces using an atomic force microscope (AFM) with a cantilever that was modified with an Aplysia cell adhesion molecule (apCAM)-coated microbead. In the second approach, we used force-calibrated glass microneedles coated with apCAM ligands to guide growth cone advance. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. Both approaches showed that Aplysia growth cones can develop traction forces in the 100–102 nN range during adhesion-mediated advance. Moreover, our results suggest that the level of traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism previously observed in other cell types. Interestingly, the absolute level of traction force did not correlate with growth cone advance toward the adhesion site, but the amount of microneedle deflection did. In cases of adhesion-mediated growth cone advance, the mean needle deflection was 1.05 ± 0.07 μm. By contrast, the mean deflection was significantly lower (0.48 ± 0.06 μm) when the growth cones did not advance. Our data support a hypothesis that adhesion complexes, which can undergo micron-scale elastic deformation, regulate the coupling between the retrogradely flowing actin cytoskeleton and apCAM substrates, stimulating growth cone advance if sufficiently abundant.

The Level of Substrate Deformation and not Traction Force Regulates Adhesion-Mediated Neuronal Growth Cone Advance

Although pulling forces have been observed in axonal growth for several decades, their exact roles are not fully understood. Here, we quantified retrograde traction forces in neuronal growth cones as they develop over time in response to an adhesion substrate using two different experimental approaches. In the first approach, we used force-calibrated glass microneedles coated with ligands for the Aplysia cell adhesion molecule apCAM to guide the advance of Aplysia Californica growth cones. The traction force exerted by the growth cone was measured by monitoring the microneedle deflection using an optical microscope. In the second approach, we developed a novel method for measuring traction forces using an atomic force microscope (AFM) with a static cantilever that contained a ligand-coated microbead and was monitored in real time. Both approaches showed that Aplysia growth cones can develop maximum traction forces up to 100 nN, which is an order of magnitude higher than previously reported for other experimental methods or growth cones. Moreover, our results suggest that the traction force is directly correlated to the stiffness of the microneedle, which is consistent with a reinforcement mechanism supported by other research. Contrary to our expectation, the level of force does not predict whether the growth cone will advance towards the adhesion site or not, but the level of microneedle deflection does. In cases of adhesion-mediated growth cone advance, the mean deflection was 1.07 ± 0.09 μm. By contrast, the mean deflection was significantly lower (0.49 ± 0.04 μm) when the growth cones did not advance in response to the adhesion substrate. In summary, our results provide novel insights into significance of substrate deformation as opposed to traction force for the regulation of adhesion-mediated directional growth cone advance.

Symmetrical Shape of the Meiotic Spindle is Dynamically Balanced

Meiotic spindle assembly is critical for achieving accurate chromosome segregation. The spindle mainly consists of microtubules and molecular motors. Recent studies have suggested that bipolar spindle formation requires the force balance sustained by molecular motors and polymerization-depolymerization dynamics of microtubules. However, it is not well understood whether and how the two pole structures are symmetrically balanced in a spindle. In this study, we quantitatively measured the mechanical stiffness, the microtubule density, and the response to the deformation of spindle poles by micromanipulation techniques and 3D analysis. To deform the spindle self-assembled in Xenopus egg extract, we inserted two glass micro-needles into a pole region at one side and widened it perpendicularly to the pole-to-pole axis. We found that the stiffness and the microtubule density in the manipulated side of pole region reduced upon widening. Unexpectedly, the reduction was also observed after a while in the unmanipulated side, which resulted in the formation of a symmetrical defocused barrel-like shape. On the other hand, it has been reported that inhibition of the dynein function by the addition of dynein-dynactin inhibitor causes the defocusing of pole regions, such that the barrel-shaped structure is formed. We compressed one side of the barrel-shaped spindle using a pair of glass micro-needles and found that the stiffness and the microtubule density in the compressed region increased. These changes also occurred in the unmanipulated side, which was accompanied by the bipolar spindle formation. Our results suggest that symmetrical shape of the spindle is dynamically balanced for proper cell division.

Contractile Properties of Half-Sarcomeres Mechanically Isolated from Skeletal Muscle Myofibrils

The goal of this study was to develop a system to experiment with half-sarcomeres mechanically isolated from skeletal muscle. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (10°C) bath. Half-sarcomeres were isolated using two pre-calibrated glass microneedles; the first pierced externally adjacent to the Z-line, and the second internally, adjacent to the M-line. The force produced during activation of the half-sarcomere was measured by tracking the displacement of the microneedles. The half-sarcomere length (HSL) was obtained by interpolating the displacement of the needles from the initial to the final distances measured from the Z-line to the center of the sarcomere. Half-sarcomeres (n=12) produced a stress of 24.0 ±3.7 nN/um2 at HSL between 1.0 and 1.4µm. The result was comparable to that observed in isolated sarcomeres (25.5 ±3.1 nN/um2, at SL between 1.8 and 3.0µm). Preliminary trials in which we imposed stretches (ranging from 12 to 33% of the initial HSL/SL, at a speeds of 1.35-3.15 μm·s−1·HSL−1) (n = 6 half-sarcomeres; n = 13 single sarcomeres) during activation show an increase in force during and after stretch, which was long lasting (i.e. residual force enhancement). Despite a high variability, half-sarcomeres showed a tendency to produce a smaller level of residual force enhancement when compared to single sarcomeres. Our results show that half-sarcomeres generate levels of isometric force that are similar to those observed in sarcomeres and myofibrils. However, half-sarcomeres produced a lower level of stretch-induced force enhancement when compared to sarcomeres.

PDMS embedded microneedles as a controlled release system for the eye

To demonstrate intraocular drug delivery using a novel device fabricated by embedding hollow glass microneedles within a soft and flexible poly (dimethylsiloxane) (PDMS) substrate for ease of device insertion into the eye. Hollow glass microneedles (5 µm ID tips), fabricated using standard glass drawing techniques, were assembled into a photolithographically micropatterned PDMS substrate. The microneedles were fluidically coupled to a drug reservoir through a 300 µm microchannel to test for in vitro release of 6-aminoquinolone (144 Da) and Rose Bengal (1044 Da). Intravitreal delivery in ex vivo bovine eyes was also studied. The microneedles penetrated UV-crosslinked collagen and excised bovine sclera without breaking or delaminating from the PDMS matrix. A total of 45 ng of 6-aminoquinolone and 16 µg of Rose Bengal was released into buffered saline over a 20-min infusion at a delivery rate of 50 µL/min. Microinjection of Rose Bengal for 8 h into ex vivo bovine vitreous resulted in a total mass accumulation of 0.0202 mg into both phases of the vitreous humor and to the uveal face of the sclera without clogging of the internal needle microchannel. PDMS-embedded microneedles offer an integrated method of drug targeting to the intraocular tissues using a less invasive and less painful approach when compared with macroscale hypodermic needles. The release rates from the microneedles were controllable on demand using a syringe pump and were independent of the properties of the drugs tested. The device demonstrated a new hybrid approach of coupling rigid microneedles strong enough to penetrate the tough, fibrous sclera with a soft and pliable PDMS substrate that could conform to the contours of the eye.

Altered Cross-Bridge Relaxation Kinetics in Guinea Pig Cardiac Hypertrophy

Cardiac hypertrophy is associated with Diastolic Heart Failure (DHF), a syndrome in which systolic function is preserved, but cardiac filling dynamics are depressed. The molecular mechanisms underlying DHF and the potential role of altered cross-bridge cycling in this syndrome are poorly understood. Accordingly, we induced chronic pressure overload by surgically banding the thoracic ascending aorta in female Dunkin Hartley Guinea pigs weighing 400g, for 12-16 weeks. Guinea pigs were chosen to avoid the confounding effects of myosin isoform switch that occurs in other small rodent models. Left ventricular (LV) samples were frozen in liquid N2. Aortic banding resulted in (+31%) LV hypertrophy (LV/BW ratio) and reduced diastolic cardiac function, but normal systolic function. Single myofibrils were prepared by mechanical dissociation and subsequently attached between two glass micro-needles that were positioned on the stage of an inverted phase-contrast microscope. While the maximum calcium saturated force development was depressed (−18%), the time required for force relaxation was increased (+8%) in parallel to a significant decrease in the rate of relaxation (−25%) in DHF myofibrils; Myosin Heavy Chain (MHC) isoform distribution was unaltered. We conclude that slower cross-bridge relaxation kinetics contribute to diastolic dysfunction in cardiac hypertrophy.

Parameters that Regulate Size and Shape of the Vertebrate Meiotic Spindle

During meiosis, the spindle equally segregates replicated genomes into two daughter cells. Spindles are mainly composed of microtubules (MTs) and molecular motors. Various studies have revealed key regulators of the spindle structure, such as kinesin-5 and depolymerizing kinesins, which produce force for the sliding of MTs or regulating the polymerization/depolymerization dynamics of MTs. Forces generated by the spindle are known to play an essential role in the checkpoint of the metaphase-anaphase transition. At metaphase, the size of the spindle is maintained almost constant despite the dynamic nature of the spindle MTs. This indicates that the forces generated by molecular motors and MTs are well balanced along the pole-to-pole axis; however, how this is achieved remains enigmatic. In this study, using a pair of glass microneedles, we developed quantitative micromanipulation techniques to deform the spindle directly, such as pushing, stretching, cutting, and fusing spindles, and 3D analysis of the spindle. We found that the meiotic spindles assembled in the Xenopus egg extracts have the ability to spontaneously reorganize, acquiring stable size and shape, after physical perturbations. Surprisingly, the density and the amount of MTs within the spindle were also regulated during the spontaneous reorganization. These results indicate that force generators, such as the molecular motors and MTs, dynamically regulate the spindle structure. We also found that the stable spindle length is fully determined by 4 three-dimensional parameters, indicating that the fluctuations of the length of the stable spindle are characterized by the variability of these 4 parameters.

Low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications

Transdermal drug delivery is limited by the barrier properties of the outer skin layer. Microneedles (MNs) effectively circumvent the skin barrier to offer this route as a potential alternative to oral and parenteral delivery of therapeutics. Biodegradable microneedles offer particular advantages however processing commonly requires elevated temperatures that may adversely affect heat-labile molecules and macromolecules. In this study, solid amorphous sugar glasses containing low residual quantities of water were created by dehydration of trehalose and sucrose sugar combination solutions. Biodegradable sugar glass MNs were fabricated following optimisation of a simple and novel low temperature vacuum deposition micromoulding methodology. These had absolute morphological fidelity to silicon master structures and demonstrated sufficient structural rigidity to efficiently penetrate excised human breast skin. Sugar glass MNs incorporating a marker compound dissolved rapidly and completely in situ releasing dye into deeper skin layers. The biological activity of a model macromolecule was partially retained over extended storage following incorporation into sugar glass. This is the first demonstration that MNs created from amorphous sugar glasses can be used for incorporating and delivering molecules, and potentially biologically active macromolecules, via the transdermal route.

Probing the Force-Balancing Mechanism of the Meiotic Spindle in Xenopus Egg Extracts

During cell division, the meiotic spindle equally segregates replicated genomes into two daughter cells. Errors in this process cause birth defect and cancer.Spindles are mainly composed of microtubules (MTs) and molecular motors. Various studies have revealed key regulators, such as Kinesin-5 (plus-end directed kinesin tetramer), depolymerizing kinesins and microtubule-associated proteins (MAPs). These exert forces for sliding MTs or regulating MT dynamics in the spindle, so that the spindle maintains a rugby ball-like structure at metaphase. These forces are also known to generate a poleward flux of spindle MTs. At metaphase, size and shape of the spindle are maintained constant in spite of the dynamic nature of spindle MTs. This indicates forces exerted by molecular motors and MTs are well balanced in the spindle.In this study, we developed micromanipulation techniques for changing spindle shape to disrupt steady state force balance of the spindle without any changes in molecular components of the spindle. Spindles spontaneously assembled in Xenopus egg extracts were stretched along their pole-to-pole axis using two glass micro-needles. When the spindles were briefly stretched, they recovered their original size and shape after a while. In contrast, when spindles were kept stretched, they gradually recovered their original shape with the increase in the spindle width, resulting in the enlargement in size. This result indicates that the meiotic spindle has an ability to adjust its size and shape to the externally applied force. Our findings provide new insights into the force-balancing mechanism of the spindle.

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

The goal of this study was to develop a system to experiment with sarcomeres mechanically isolated from skeletal muscles. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (22 degrees C or 15 degrees C) experimental chamber, and sarcomeres were isolated using precalibrated glass microneedles that were pierced externally, adjacent to the Z-lines. The force produced during activation was measured by tracking the displacement of the microneedles, and the sarcomere and half-sarcomere changes were measured by continuously tracking the Z-lines and A-bands position during the experiments. Sarcomeres produced a stress (force/cross-sectional area) of 112.75 +/- 4.96 nN/microm(2) (15 degrees C) and 128.47 +/- 5.58 nN/microm(2) (22 degrees C) at lengths between 2.0 microm and 2.4 microm. The descending limb was fitted with linear regression for length between 2.4 microm and 3.5 microm, which provided an abscissa extrapolating to 3.87 microm. The force-length relation was remarkably similar to a theoretical curve based on the degree of filament overlap. During sarcomere activation, we tracked the distance between the center of the A-band and the Z-lines. At lengths below 1.6 microm, movements of A-band were not detected. A-band movements increased with length to achieve a maximum displacement of 59.40 +/- 10.1 nm from the center at 2.0 microm-2.4 microm. A-band displacement decreased linearly in sarcomere lengths between 2.6 microm and 3.6 microm. A technique for monitoring force and length in single sarcomeres isolated from myofibrils represents a reliable technique to evaluate contractile mechanisms at the most basic, intact level of muscle organization, opening the possibility to clarify long-standing issues in the field of muscle contraction.

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.

Glass microneedles for force measurements: a finite-element analysis model

Changes in developed force (0.1-3.0 microN) observed during contraction of single myofibrils in response to rapidly changing calcium concentrations can be measured using glass microneedles. These microneedles are calibrated for stiffness and deflect on response to developed myofibril force. The precision and accuracy of kinetic measurements are highly dependent on the structural and mechanical characteristics of the microneedles, which are generally assumed to have a linear force-deflection relationship. We present a finite-element analysis (FEA) model used to simulate the effects of measurable geometry on stiffness as a function of applied force and validate our model with actual measured needle properties. In addition, we developed a simple heuristic constitutive equation that best describes the stiffness of our range of microneedles used and define limits of geometry parameters within which our predictions hold true. Our model also maps a relation between the geometry parameters and natural frequencies in air, enabling optimum parametric combinations for microneedle fabrication that would reflect more reliable force measurement in fluids and physiological environments. We propose a use for this model to aid in the design of microneedles to improve calibration time, reproducibility, and precision for measuring myofibrillar, cellular, and supramolecular kinetic forces.

AFM picking-up manipulation of the metaphase chromosome fragment by using the tweezers-type probe

We have studied the development of a new procedure based on atomic force microscopy (AFM) for the analysis of metaphase chromosome. The aim of this study was to obtain detailed information about the specific locations of genes on the metaphase chromosome. In this research, we performed the manipulation of the metaphase chromosome by using novel AFM probes to obtain chromosome fragments of a smaller size than the ones obtained using the conventional methods, such as glass microneedles. We could pick up the fragment of the metaphase chromosome dissected by the knife-edged probe by using our tweezers-type probe.

Precise Microinjection into Skin Using Hollow Microneedles

Hollow needles of micron dimensions have previously been fabricated and envisioned for use with transdermal patches or infusion pumps to achieve painless delivery of drugs to the skin for local and systemic effects without the need for hypodermic needles. However, little work has been carried out to identify methods to effectively use hollow microneedles for drug delivery. To address this need, we inserted hollow, glass microneedles into hairless rat skin in vivo and human cadaver skin in vitro and then imaged infusion of dye molecules, insulin, polymer microparticles, and cells into the skin by brightfield and fluorescence microscopy. The depth of needle penetration into skin was controlled by inserting needles with a rotary drilling device, which enabled localized injection within the epidermis or dermis with +/-60 microm resolution. Although small quantities of fluid could be injected after needle insertion into skin, partial retraction of the needle by withdrawing back 100-300 microm or vibrating the microneedle array dramatically increased infusion flow rate. We conclude that hollow microneedles can be used for precise microinjection into skin, especially when a single needle is inserted by rotary drilling and then retracted part way before infusion or a microneedle array is inserted by mechanical vibration.