Mapping the brain’s fiber network is crucial for understanding its function and malfunction, but resolving nerve trajectories over large fields of view is challenging. Here, we show that computational scattered light imaging (ComSLI) can map fiber networks in histology independent of sample preparation, also in formalin-fixed paraffin-embedded (FFPE) tissues including whole human brain sections. We showcase this method in new and archived, animal and human brain sections, for different sample preparations (in paraffin, deparaffinized, various stains, unstained fresh-frozen). We convert microscopic orientations to microstructure-informed fiber orientation distributions (μFODs). Adapting tractography tools from diffusion magnetic resonance imaging (dMRI), we trace axonal trajectories revealing white and gray matter connectivity. These allow us to identify altered microstructure or deficient tracts in demyelinating or neurodegenerating pathology, and to show key advantages over dMRI, polarization microscopy, and structure tensor analysis. Finally, we map fibers in non-brain tissues, including muscle, bone, and blood vessels, unveiling the tissue’s function. Our cost-effective, versatile approach enables micron-resolution studies of intricate fiber networks across tissues, species, diseases, and sample preparations, offering new dimensions to neuroscientific and biomedical research.

Silver oxide (AgO) thin films were prepared by using a cylindrical direct current reactive magnetron sputtering system at 10⁻⁵ torr initial pressure on BK7 glass substrate. Samples deposited for 3, 5 and 7 minutes. Surface characterization of AgO thin films in the nanometer scale can be accurately determined using the atomic force microscopy (AFM) and X-ray diffraction (XRD). The average roughness (R_avg), maximum peak to valley height (R_t) and root mean square (R_rms) roughness are used to analyze the surface morphology of AgO films. The linear optical absorption data were measured in the visible-near infrared spectral regions and the nonlinear refractive index (n₂) of thin films is evaluated by the moiré deflectometery technique. The investigation indicates that, increase in AgO thickness leads to reduction in nonlinear refractive index.

Near-infrared (NIR) fluorescence imaging using exogenous fluorescent agents provides whole-field images in real-time to assist the surgeon in the excision of a tumor. Although the method has high sensitivity, the specificity can sometimes be lower than expected. Raman spectroscopy can detect tumors with high specificity. Therefore, a combination of both techniques can be advantageous. A complication that must be addressed is that the NIR spectral region is favored by both techniques for (in vivo) tissue analysis. When fluorescence and Raman emissions spectrally overlap, it becomes challenging or impossible to detect the Raman signal. In this paper, by avoiding this overlap, we describe a Raman spectroscopy setup capable of recording high-quality Raman spectra from tissue containing NIR exogenous fluorescent agents. We identify an optimal wavelength interval (900-915 nm) for Raman excitation, which avoids both excitation of fluorescent dyes and Raman signal self-absorption by the tissue. In this way, Raman spectroscopy can be combined with the currently most-used NIR fluorescent dyes. This combined novel setup could pave the way for clinical trials benefiting from both fluorescence imaging and Raman spectroscopy to avoid positive margins in cancer surgery.

Minimally-invasive laser surgeries could benefit from a fiber-optic laser-induced breakdown spectroscopy (FO-LIBS) setup for real-time tissue characterization. In FO-LIBS, the sample receives limited light irradiance due to the fiber's low damage threshold and diminished laser beam quality. Therefore, the plasma created with FO-LIBS is less luminant than that of free-space LIBS. Furthermore, only a small portion of plasma emission can be collected, as the lens's size at the fiber tip is restricted to fit inside the narrow channel of an endoscope. A high optical throughput Echelle spectrometer was developed to compensate for low-intensity light collection with FO-LIBS. The Echelle spectrometer was tested for tissue differentiation when combined with a flexible fiber bundle delivery setup and a small lens at the bundle's tip. The customized FO-LIBS setup, coupled with multivariate data analysis, successfully differentiated bone from surrounding soft tissue (muscle, fat, and bone marrow) with 100% cross-validated (CV) sensitivity and specificity. The CV sensitivity and specificity for differentiation between all tissues were 90.2% and 96.7%, respectively. The results demonstrate, to the best of our knowledge, the first flexible FO-LIBS system, which may provide a further step towards the development of a smart endoscopic laser scalpel.

A clear margin is an important prognostic factor for most solid tumours treated by surgery. Intraoperative fluorescence imaging using exogenous tumour-specific fluorescent agents has shown particular benefit in improving complete resection of tumour tissue. However, signal processing for fluorescence imaging is complex, and fluorescence signal intensity does not always perfectly correlate with tumour location. Raman spectroscopy has the capacity to accurately differentiate between malignant and healthy tissue based on their molecular composition. In Raman spectroscopy, specificity is uniquely high, but signal intensity is weak and Raman measurements are mainly performed in a point-wise manner on microscopic tissue volumes, making whole-field assessment temporally unfeasible. In this review, we describe the state-of-the-art of both optical techniques, paying special attention to the combined intraoperative application of fluorescence imaging and Raman spectroscopy in current clinical research. We demonstrate how these techniques are complementary and address the technical challenges that have traditionally led them to be considered mutually exclusive for clinical implementation. Finally, we present a novel strategy that exploits the optimal characteristics of both modalities to facilitate resection with clear surgical margins.

To obtain efficient laser ablation in bone, dehydration, early carbonization and carbonization need to be avoided. Achieving this can only be provided by using an automated control of the ablation laser and irrigation system. As a preliminary study, we demonstrated a laser-induced breakdown spectroscopy based early carbonization detection system by analyzing carbonized bone tissues. Carbonization of bone samples was generated in a controlled way, by applying different number of Er:YAG pulses (0–25) at different locations on bone sample. To detect number of applied pulses, leading to the detection of carbonization level, we used a feed-forward Artificial Neural Network (ANN) with multi-layer perceptron structure. The results of the ANN were compared with the actual label, and R-squared of 0.85, 0.88, 0.86, 0.83, and 0.84 (0.85 on average) were achieved.

Preparation of biological samples for further processing or analysis is generally performed manually by means of standard mechanical tools such as scalpels or biopsy punches. While this approach is uncomplicated and swift, it entails constraints such as low, operator-dependent cutting accuracy and reproducibility. Tissue segments surrounding the cut may further suffer mechanical and thermal damage due to shear forces and friction between tool and sample. These hindrances affect procedures both in the laboratory environment as well as within clinical settings. A system has been developed leveraging robotic positioning and laser light for precise, controlled, and contactless tissue ablation, and providing a concise and intuitive graphical user interface. Additionally, sterility of the process is demonstrated, a paramount element for clinical application. The proposed process does not require sterilization of the robotic components or the lasers, easing a prospective integration into existing workflows. In the context of this work, mainly cartilage repair surgery is targeted. The proposed system allows for highly accurate and reproducible shaping of the cartilage lesion area as well as its corresponding engineered cartilage graft, possibly leading to better and faster integration at the defect site. Promising results could be obtained in a first test series with human cartilage samples, validating the functionality of the preparation system and the feasibility of the sterility concept.

Echelle spectrometers offer high-resolution (ca. one Angstrom or higher) and wide range (ca. half a micron or wider), simultaneously, without having a mechanical moving part. However, they require a complicated optical set-up as compared to conventional one-dimensional-dispersion/diffraction spectrometers, e.g., Czerny-Turner. Therefore, the simulation of the Echellogram is required before building the spectrometer. This paper aims to develop an application for visualizing the Echellogram using MATLAB App Designer followed by simulation of the optical aberrations by Zemax OpticStudio to optimize the spectrometer parameters. The developed application provides an interactive graphical interface with a user-friendly dashboard to control and monitor the required parameters. This spectrometer is being used in laser-induced breakdown spectroscopy (LIBS) system application lied of robot-guided laser ablation of biological tissues.

Minimally invasive laser surgeries that require the use of a flexible endoscope (flexiscope) could benefit from high-energy nanosecond laser pulses delivered through fibers for real-time tissue characterization and phenotyping. The damage threshold of the fiber's glass material limits the maximum amount of deliverable peak power. To transmit high-energy pulses without damaging the fiber material, large-diameter fibers are typically used, leading to a limited bending radius. Moreover, in a large-core fiber, self-focusing can damage the fiber even if the tip remains intact. In this work, we tested a fused-end fiber bundle combined with a beam shaper capable of delivering more than 20 MW (>100 mJ/5 ns). The fiber bundle was tested over more than eight hours of operation, with different bending radiuses down to 15 mm. The results demonstrate, to the best of our knowledge, the highest peak power delivered through a flexible fiber, for a frequency-doubled Q-switched Nd:YAG laser.

The preparation of small-sized biological samples is traditionally performed manually utilizing mechanical tools such as scalpels. The main drawbacks of such methods are a lack of accuracy and repeatability of the resulting cuts and damage to the surrounding tissue due to the high interaction forces and the accompanying mechanical stresses. One way to circumvent these issues is to substitute the mechanical tools for laser light. When used in conjunction with a high-accuracy positioning system, such a preparation procedure enables repeatable cutting of arbitrary geometries while largely preserving the integrity of the surrounding tissue. In this paper, a system leveraging the potential of laser-based ablation for bio-sample preparation is proposed. It integrates and synchronizes all key components with extensive safety features and an intuitive user interface, allowing novice operators to perform sample preparations easily. As a first application, the proposed system has been utilized to create microdamages in mouse tail tendon fascicles. Promising results could be obtained, but careful tuning of the laser parameters and further optimization of the mechanical setup is still required to attain the high repeatability striven for.

A novel real-time and non-destructive method for differentiating soft from hard tissue in laser osteotomy has been introduced and tested in a closed-loop fashion. Two laser beams were combined: a low energy frequency-doubled nanosecond Nd:YAG for detecting the type of tissue, and a high energy microsecond Er:YAG for ablating bone. The working principle is based on adjusting the energy of the Nd:YAG laser until it is low enough to create a microplasma in the hard tissue only (different energies are required to create plasma in different tissue types). Analyzing the light emitted from the generated microplasma enables real-time feedback to a shutter that prevents the Er:YAG laser from ablating the soft tissue.

During the past half-century, laser osteotomy has been studied for a broad range of lasers, which almost covers the entire range of available laser systems in the market, from early unsuccessful experiments with CW lasers to newly developed ultrashort pulse lasers. Although a large variety of laser parameters including wavelength, pulse energy, pulse duration, and repetition rate have been investigated to find an optimum laser system as an alternative osteotomy tool, there is not a universal agreement on a specific type of laser to replace conventional mechanical saws. The only universal agreement is on the speed of cutting (ablation rate) which went to longpulse Er:YAG and CO2 lasers. Microsecond pulse Er:YAG and CO2 lasers perform osteotomy by inducing efficient photothermal effect to the bone with the help of high absorption peak of water in the bone. However, having a speedy cut is not the only effective parameter to pave the way for transferring lasers to the operating room. Other parameters including cutting with the lowest thermal damage, ability for deep cutting, and compatibility with integrating sensors are among the other determinant parameters. Moreover, being able to be delivered through the fiber optic and as a consequence fit inside the endoscope channel could extend their application from the open surgery to minimally invasive ones. This chapter besides proving the necessary information on the physics behind the laser-bone interaction provides a short review on the history of bone surgery with laser and state-of-the-art studies in this field.

A cost-effective method employing a smartphone sensor to measure the laser-induced sound pressure level (SPL) has been introduced for lens-to-sample distance (LTSD) adjustment in micro laser-induced breakdown spectroscopy (µLIBS).

In the present work, a hyperspectral imaging system (imaging spectrometer) using a commercial webcam has been designed and developed. This system was able to capture two-dimensional spectra (in emission, transmission and reflection modes) directly from the scene in the desired wavelengths. Imaging of the object is done directly by linear sweep (pushbroom method). To do so, the spectrometer is equipped with a suitable collecting lens and a linear travel stage. A 1920 x 1080 pixel CMOS webcam was used as a detector. The spectrometer has been calibrated by the reference spectral lines of standard lamps. The spectral resolution of this system was about 2nm and its spatial resolution was about 1 mm for a 10 cm long object. The hardware solution is based on data acquisition working on the USB platform and controlled by a LabVIEW program. In this system, the initial output was a three-dimensional matrix in which two dimensions of the matrix were related to the spatial information of the object and the third dimension was the spectrum of any point of the object. Finally, the images in different wavelengths were created by reforming the data of the matrix. The free spectral range (FSR) of the system was 400 to 1100 nm. The system was successfully tested for some applications, such as plasma diagnosis as well as applications in food and agriculture sciences.

Although laserosteotomes have become generally accepted devices in surgical applications, they still suffer from a lack of information about the type of tissue currently being ablated; as a result, critical structures of the body under or near the focal spot of the laser beam are prone to inadvertent ablation. The lack of information about the properties of the ablated tissue can be solved by connecting the laserosteotome to an optical detection setup which can differentiate various types of tissues, especially bone from connective soft tissues. This study examines the applicability of laser-induced breakdown spectroscopy (LIBS) as a potential technique to differentiate bone from surrounding soft tissue (fat and muscle). In this experiment, fresh porcine femur bone, muscle, and fat were used as hard and soft tissue samples. The beam of a nanosecond frequency-doubled Nd:YAG laser was used to ablate the tissue samples and generate the plasma. The plasma light emitted from the ablated spot, which corresponds to the recombination spectra of ionized atoms and molecules, was gathered with a collection optic (including a reflective light collector and a fiber optic) and sent to an Echelle spectrometer for resolving the atomic composition of the ablated sample. Afterwards, Discriminant Function Analysis (DFA) based on the ratio of the intensity of selected peak pairs was performed to classify three sample groups (bone, muscle, and fat). Lastly, the sensitivity, specificity, and accuracy of the proposed method were calculated. Sensitivity and specificity of 100 % and 99 % were achieved, respectively, to differentiate bone from surrounding soft tissue.