Ultrafast laser ablation offers unparalleled spatial and thermal confinement, making it a compelling candidate for high-precision spinal bone surgery. However, the inherently low ablation rates of ultrafast lasers and the challenges associated with probe miniaturization have significantly impeded their clinical translation. In this work, we present the design and prototyping of a new-generation, fiber-delivered ultrafast laser surgical scalpel optimized for both minimally invasive and open spinal procedures. The system integrates high-efficiency laser delivery via a Kagome hollow-core photonic crystal fiber, a piezoelectric fiber-scanning mechanism for compact beam steering, a high-demagnification miniaturized objective for enhanced ablation efficiency, and an opto-mechanical architecture enabling controlled depth-wise material removal and handheld operation. Prior efforts in parametric optimization informed the selection of pulse duration, spot size, and field-of-view to enable ablation rates exceeding 20 mm(3)/min under high-average-power operation. The optical design achieves a focused spot radius of 7.87 mu m with a working distance of 3.8 mm, suitable for deep spinal bone incisions. Preliminary handheld tests on bone and synthetic spine models demonstrate stable focal-plane ablation, ergonomic maneuverability, and access to confined anatomical regions. These results establish a scalable and clinically translatable platform for ultrafast laser-based spinal surgery and represent a significant step toward replacing conventional mechanical bone removal tools.

Two-Photon Line Excitation Array Detection (2p-LEAD) is a novel, high-speed imaging method designed to overcome the speed limitations of traditional multi-photon microscopy. While conventional point-scanning is restricted to sub-30 Hz frame rates, 2p-LEAD can achieve 4 kHz frame rates at a 250 mu m x 96 mu m field-of-view. By coupling galvanometric line scanning with a parallelized 32-channel photomultiplier tube (PMT) array, we have constructed one of the fastest two-photon microscopes. Our system maintains the critical balance of subcellular resolution, kHz temporal resolution, and signal-to-noise ratio (SNR) through several key features. Temporal focusing confines the point-spread function (PSF) axially to reduce out-of-focus background, while the 32-channel PMT array enables highly efficient, parallelized photon collection. Additionally, optimizations of the optical configuration, excitation conditions, and detection hardware drastically improve the SNR for a given laser power, thereby mitigating the risk of phototoxicity and photodamage during sensitive, long-duration in vivo experiments. We demonstrate this advancement in capability by imaging the mammalian brain in vivo, resolving highly dynamic neurovascular events. The system's combination of high spatial resolution, temporal resolution, and SNR enables the quantitative measurement of fast-flowing red blood cells through cortical capillaries without motion artifacts. This demonstrated performance establishes a robust platform for future upgrades that may help enable comprehensive 4D investigation of functional and hemodynamic dynamics throughout large volumes of the mouse brain.

Recent advancements in ultrafast laser ablation technology redefine surgical precision while minimizing thermal damage, offering a promising alternative to traditional methods. However, the slow material removal rates (MRRs) have hindered clinical adoption. Addressing this challenge, we present a compact fiber-based laser delivery system, exhibiting an 82-fold increase in MRR compared to previous femtosecond laser probes. The system leverages a hollow-core Kagome fiber to deliver 10 ps laser pulses with high transmission efficiency and minimal nonlinear effects, even at high peak powers. The system distributes ultrashort pulses utilizing a piezo-scanned Lissajous-based beam steering mechanism onto the target surface over a larger field-of-view (FOV), enabling the scope for easy scalability of the system to a miniaturized probe. Drawing insights from our prior work, the focusing optics were carefully selected to deliver fluence three times the ablation threshold. An optimal combination of FOV size, translation speed, and repetition rate was identified, enabling clean ablations (devoid of carbonization) even at maximum laser power. We achieved a maximum MRR of 10.7 mm3/min with 8.8 W of laser power at 333 kHz, validating our hypothesis that high MRR can be achieved by ablating at high average powers over a large FOV with fast scanning of a large spot size. Numerical simulations further suggest that MRR up to 30 mm3/min can be achieved through increased repetition rates, expanded FOVs, and high translation speeds, defining an optimal parameter space for future probe designs. To validate the clinical relevance of high MRR, experiments were conducted to create deep bone incisions over a 3×3 mm2 area within a clinically relevant timeframe. An ablation depth of ∼3 mm was achieved in ∼2 minutes without auxiliary cooling mechanisms. Scanning electron microscopy (SEM) of the deep incisions confirmed the preservation of healthy bone tissue, with clear evidence of canaliculi along the slopes and at the bottom surface of the ablated region. This study outlines a clear pathway toward developing a high-performance, miniaturized surgical probe with significant potential for spinal decompression surgery and other clinical applications, representing a transformative tool for future surgical precision.

High-content imaging (HCI) and analysis are the keys for advancing our understanding of the science behind organogenesis. To this end, culturing adult stem cell-derived organoids (ASOs) in a platform that also enables live imaging, staining, immobilization, and fast high-resolution imaging is crucial. However, existing platforms only partially satisfy these requirements. In this study, we present the OrganoidChip+, an all-in-one microfluidic device designed to integrate both culturing and HCI of ASOs all within one platform. We previously developed the OrganoidChip as a robust imaging tool. Now, the OrganoidChip+ incorporates several additional features for culturing organoids in addition to fluorescence staining and imaging without the need for sample transfer. The organoids grown within a culture chamber are stained and then transferred to immobilization chambers for blur-free, high-resolution imaging at predetermined locations. We cultured adult stem cell-derived intestinal organoids in the chip for 7 days and tracked growth rates of each organoid using intermittent brightfield images, followed by multiple image-based assays, including viability assay using widefield fluorescence imaging, a redox ratio assay using label-free, two-color, two-photon microscopy, and immunofluorescence assays using confocal microscopy. These assays serve as proof-of-concept to showcase the chip's capabilities in HCI of ASOs. Organoids cultured in the chip exhibited superior average growth rates over those in traditional Matrigel dome cultures, off-chip. Viability and redox ratio measurements of on-chip organoids were comparable or slightly better than their off-chip counterparts. Confocal imaging further confirmed that the OrganoidChip+ supports robust organoid culture while enabling detailed, high-resolution analysis. This all-in-one platform holds great potential for advancing ASO-based research, offering a scalable and cost-effective solution for HCI and analysis in organogenesis, drug screening, and disease modeling.

High-content imaging (HCI) and analysis are the keys for advancing our understanding of the science behind organogenesis. To this end, culturing adult stem cell-derived organoids (ASOs) in a platform that also enables live imaging, staining, immobilization, and fast high-resolution imaging is crucial. However, existing platforms only partially satisfy these requirements. In this study, we present the OrganoidChip+, an all-in-one microfluidic device designed to integrate both culturing and HCI of ASOs all within one platform. We previously developed the OrganoidChip as a robust imaging tool. Now, the OrganoidChip+ incorporates several additional features for culturing organoids in addition to fluorescence staining and imaging without the need for sample transfer. The organoids grown within a culture chamber are stained and then transferred to immobilization chambers for blur-free, high-resolution imaging at predetermined locations. We cultured adult stem cell-derived intestinal organoids in the chip for 7 days and tracked growth rates of each organoid using intermittent brightfield images, followed by multiple image-based assays, including viability assay using widefield fluorescence imaging, a redox ratio assay using label-free, two-color, two-photon microscopy, and immunofluorescence assays using confocal microscopy. These assays serve as proof-of-concept to showcase the chip’s capabilities in HCI of ASOs. Organoids cultured in the chip exhibited superior average growth rates over those in traditional Matrigel dome cultures, off-chip. Viability and redox ratio measurements of on-chip organoids were comparable or slightly better than their off-chip counterparts. Confocal imaging further confirmed that the OrganoidChip+ supports robust organoid culture while enabling detailed, high-resolution analysis. This all-in-one platform holds great potential for advancing ASO-based research, offering a scalable and cost-effective solution for HCI and analysis in organogenesis, drug screening, and disease modeling.

In this paper, we present a 2-photon imaging probe system featuring a novel fluorescence collection method with improved and reliable efficiency. The system aims to miniaturize the potential of 2-photon imaging in the metabolic and morphological characterization of cervical tissue at sub-micron resolution over large imaging depths into a flexible and clinically viable platform towards the early detection of cancers. Clinical implementation of such a probe system is challenging due to inherently low levels of autofluorescence, particularly when imaging deep in highly scattering tissues. For an efficient collection of fluorescence signals, our probe employs 12 0.5 NA collection fibers arranged around a miniaturized excitation objective. By bending and terminating a multitude of collection fibers at a specific angle, we increase collection area and directivity significantly. Positioning of these fibers allows the collection of fluorescence photons scattered away from their ballistic trajectory multiple times, which offers a system collection efficiency of 4%, which is 55% of what our bench-top microscope with 0.75 NA objective achieves. We demonstrate that the collection efficiency is largely maintained even at high scattering conditions and high imaging depths. Radial symmetry of arrangement maintains uniformity of collection efficiency across the whole FOV. Additionally, our probe can image at different tissue depths via axial actuation by a dc servo motor, allowing depth dependent tissue characterization. We designed our probe to perform imaging at 775 nm, targeting 2-photon autofluorescence from NAD(P)H and FAD molecules, which are often used in metabolic tissue characterization. An air core photonic bandgap fiber delivers laser pulses of 100 fs duration to the sample. A miniaturized objective designed with commercially available lenses of 3 mm diameter focuses the laser beam on tissue, attaining lateral and axial imaging resolutions of 0.66 µm and 4.65 µm, respectively. Characterization results verify that our probe achieves collection efficiency comparable to our optimized bench-top 2-photon imaging microscope, minimally affected by imaging depth and radial positioning. We validate autofluorescence imaging capability with excised porcine vocal fold tissue samples. Images with 120 µm FOV and 0.33 µm pixel sizes collected at 2 fps confirm that the 300 µm imaging depth was achieved.

We extend the resolution of our 2p autofluorescence imaging probe by incorporation of custom designed and fabricated optics. We will add an NA extender piece, inserted at excitation fiber tip to boost the beam divergence at the excitation optics input. The new custom designed miniaturized objective will achieve a resolution of 0.5 μm, without any changes to the diameter of the objective design. Custom design objective also will allow us to perform autofluorescence imaging at two colors. This will enable the optical redox ratio characterization of tissues, which is an important hallmark in diagnosis of cancers at early stages.

The major advancements in ultrafast laser ablation technology are revolutionizing surgical precision and minimizing thermal impact compared to traditional methods. However, the primary challenge hindering widespread clinical adoption has been the slow material removal rate (MRR). Towards this gap, a compact fiber-based laser delivery system has been developed, boasting an impressive 82-fold increase in MRR over the previous femtosecond laser surgical probes. This benchtop setup utilizes a hollow-core Kagome fiber (NA approximate to 0.02) coupled to a high-power Yb-doped fiber laser (lambda=1035 nm) to deliver laser pulses onto the sample. Employing a piezo-scanned Lissajous-based beam steering mechanism, the system achieves efficient distribution of ultrashort pulses onto the target surface. Remarkably, the system maintains a high transmission efficiency of 74% while operating at peak intensities, with no components exhibiting nonlinear behavior. For a FOV scan width of 550 mu m, the logarithmic relationship between the ablation depth and laser fluence was determined for two different translational velocities. The system achieved material removal rates of similar to 10.7 mm(3)/min for the maximum applied laser fluence of 9.3 J/cm(2), without initiating carbonization. Moreover, by fine-tuning laser parameters, the system can swiftly create clean-cut trenches of significant dimensions, 3 x 3 mm(2) size and similar to 1 mm deep, mimicking conventional surgical procedures such as spinal decompression within a minute, all without carbonization or tissue damage. This remarkable achievement underscores the reliability and potential of ultrashort-laser ablation techniques for a wide array of surgical interventions.

Tightly‐focused ultrafast laser pulses (pulse widths of 100 fs–10 ps) provide high peak intensities to produce a spatially confined tissue ablation effect. The creation of sub‐epithelial voids within scarred vocal folds (VFs) via ultrafast laser ablation may help to localize injectable biomaterials to treat VF scarring. Here, we demonstrate the feasibility of this technique in an animal model using a custom‐designed endolaryngeal laser surgery probe.

Accurate real-time measurement of the glucose concentration change is vital for both biomedical applications and fundamental studies. Wireless biosensors offer appealing solutions to sensitive, real-time, and non-invasive glucose measurements. Resonant type RF sensors operating on the principle of frequency shift with respect to glucose concentration change have the potential of realizing simple, miniaturized, reproducible, biocompatible, accurate, portable yet cheap solutions. Such sensors have a profound impact particularly on diabetic patients to monitor their condition and to test the efficiency of the drugs. Recently, split ring resonators opened new door of opportunities for developing the next generation of biosensors. This chapter concentrates on glucose sensors with a particular focus of radio frequency SRR resonant types. Theoretical background, operation as well as applications of metamaterial SRR biosensor devices covering a wide range of frequencies from VHF to THz are discussed.

Imaging modalities capable of detecting functional changes over small areas can increase sensitivity and specificity of early cancer detection. Label-free imaging of metabolic activity at cellular level resolution over full thickness of cervix epithelium is possible with 2p imaging. However, low probability of 2p excitation and scattering nature of tissues limit autofluorescence levels in 2p imaging. We present a 2p autofluorescence imaging endoscope system for detection of metabolic changes in cervix in a clinical setting, with an increased collection efficiency in scattering media. Collection of autofluorescence signals is done with a multitude of high NA fibers arranged around a miniaturized excitation objective. By cleaving the collection fibers at a specific angle, we increase the directivity of the collection and the collection efficiency per fiber. The endoscope performs imaging at 775 nm, which is capable of exciting NAD(P)H and FAD molecules. Laser pulses of 100 fs duration are delivered to the sample with an air core photonic bandgap fiber. Fiber is scanned in spiral pattern via a piezo actuator tube. Scanning at different tissue depths is possible with the axial actuation of the endoscope via a linear stepper motor. Benchtop tests indicate that the endoscope system has lateral and axial resolutions of 0.65 μm and 4.33 μm, respectively. Fluorescence images of pollen cores are presented to demonstrate the imaging quality of the endoscope system.

The functional meaning associated with neuronal activity in the mammalian brain and sensory systems remains to be fully understood. Exploring this area of neuroscience requires high-speed 3D imaging operating at >1 kHz volumetric scan rates with sub-cellular resolution, as neuronal signals propagate on sub-millisecond time scales. Additionally, since these studies must be performed in vivo, care must be taken to avoid invasive or damaging methods. Multi-photon imaging allows for non-invasive studies that deeply penetrate brain tissue, but has traditionally been limited to volumetric imaging between 10 to 100 Hz. We propose an improvement upon these systems with the novel imaging modality 2-photon Line Excitation and Array Detection (2p-LEAD) microscopy. 2p-LEAD is built on the main concept in our previous work where we developed single photon LEAD microscopy operating at 0.8 million FPS for 3D flow cytometry. In 2p-LEAD, we scan a 1035 nm excitation line of 2.4 μm x 220 μm (1/e² beam intensity diameter) at the focal plane. The resulting fluorescence is collected by a 16-channel linear PMT array. With a scanning mirror, we scan the line over a 140 μm x 160 μm FOV at 3,000 FPS, creating a frame of 16 x 320 pixels. Here we will present the design and imaging capabilities of our current 2p-LEAD instrument. This system lays the groundwork for higher speed imaging at 125 kHz frame rates with an acoustooptic deflector replacing the scanning mirror. When combined with vertical scanning, we will be able to volumetrically image at sub-millisecond time scales to allow for in vivo calcium imaging of the visual cortex.

A scanning fiber endoscope system utilizing an alternative collection method for improved collection efficiency is presented. Low probability of two-photon excitation and high tissue scattering make signal collection a challenging aspect of two-photon imaging. Our work demonstrates a collection strategy using a high NA optical fiber bundle utilized in addition to the excitation fiber. This scheme significantly increases collection area without requiring collection optics at distal end. Experimentally determined collection efficiency of the method will be compared against our table-top two-photon microscope setup. Various effects of increased collection area on efficiency will be discussed with simulation results.

Microfluidics is an innovative technological platform with a vast potential to streamline processes in biology, chemistry, and biomedical fields. Microfluidic integrated biosensors attract much attention due to extreme miniaturization, low sample consumption, and increased homogeneity in mixing conditions, leading to enhanced sensitivity. Nowadays, many researchers focus on inexpensive and flexible laser production of polymer-based microfluidic devices for sensing applications due to their ease of production and rapid processing benefits. In this article, we present some key factors for the simple and rapid production of microfluidic components of the microwave sensor by using the CO2 laser ablation technique. The technique does not require any cleanroom or complex laboratory setups and provides short fabrication times for prototyping. It is observed that, at high laser power (30 W) and low scan speed (125 cm s−1), both the channel depth and the surface roughness increase greatly as opposed to channel waviness. It is also demonstrated that heat treatment is a viable method to reduce the channel roughness with a trade of channel depth. In the second section, prepared channels are bonded onto the split ring resonators (SRRs) fabricated using polymethyl methacrylate as a substrate. Power reflection measurements from SRR are performed using a continuous flow system that injects 100 mM glucose solutions into the channels. Change of dielectric constant due to glucose loading generates a meaningful resonance frequency shift, showing a possible use scenario of the device as a biosensor.

Although molecular communication systems have been shown to bear great potential for many useful in-body applications, they require the intervention, action, or input of an out-of-body actor. From an Internet of Bio-Nano Things perspective, a successful overall network aims to bring together the two links belonging to the in-body and out-of-body networks for end-to-end communications. For most applications, the uplink from the in-body sensor is more significant since it provides the multi-scalar connection required to relay the information sensed and carried by the molecular communication system to a macro-scale smart terminal. This article proposes two different mechanisms to sense the output of the molecular communication system and transmit the information to an on-body reader. Each mechanism involves different genetically engineered bacteria and specific antenna designs. An experimental setup is provided to demonstrate each proposed concept. The results constitute a proof of concept to detect the in-body bacterial activity from the on-body reader.

This paper describes a microwave resonator incorporating microfluidic lab-on-chip sensor system capable of performing simultaneous differential measurement based sensing of liquid samples. The resonators are split-ring resonator shapes made of gold on glass substrates. Directly bonded on glass substrates are polydimethylsiloxane microchannels. Sensor system design incorporates a pair of identical resonators, one of which performs reference reading from the background. Tracking the difference of the responses of both resonators simultaneously, rather than a single one, is used to obtain a more linear and noise-free reading. The sensor system was produced with conventional fabrication techniques. It is compatible with low-cost, simple, easy to handle sensing applications. Results indicate that reliable differential measurement was possible owing to a well-matched pair of sensors with a response error as low as 0.1%. It was also demonstrated that differential measurement capability enables sensing with improved linearity. Measurements were performed with glucose solutions in the range of 3.2-16.1 mM, achieving a sensitivity of 0.16 MHz/mM.

High throughput microfluidic devices coupled with optical detection systems bring several advantages to study molecular communication (MC) by mimicking capillary vessels and arterioles. Motivated by this, we present an MC platform using fluorescence polystyrene (PS) beads as messenger molecules to transfer encoded information in microfluidic channels via flow induced diffusion. To this end, we couple multiple production and analysis techniques to construct and characterize our micro scale MC system. PS microbeads are introduced into microchannels via programmable syringe pumps serving as transmitters, while the received signal is recorded by inverted fluorescence microscope. Time lapsed images of microparticles are presented as they move across diffusion channels.