Multimodal virtual histology of rabbit vocal folds by nonlinear microscopy and nano computed tomography.

Human vocal folds (VFs) possess a unique anatomical structure and mechanical properties for human communication. However, VFs are prone to scarring as a consequence of overuse, injury, disease or surgery. Accumulation of scar tissue on VFs inhibits proper phonation and leads to partial or complete loss of voice, with significant consequences for the patient's quality of life. VF regeneration after scarring provides a significant challenge for tissue engineering therapies given the complexity of tissue microarchitecture. To establish an effective animal model for VF injury and scarring, new histological methods are required to visualize the wound repair process of the tissue in its three-dimensional native environment. In this work, we propose the use of a combination of nonlinear microscopy and nanotomography as contrast methods for virtual histology of rabbit VFs. We apply these methods to rabbit VF tissue to demonstrate their use as alternatives to conventional VF histology that may enable future clinical studies of this injury model.

Nonlinear microscopy of common histological stains reveals third harmonic generation harmonophores.

Since its invention over a hundred years ago, histological analysis using coloured dye staining remains the gold standard for histopathology. While these stains provide critical information for a variety of diagnostic purposes, they offer limited two-dimensional histological information. Extending classical histological analysis to three dimensions requires novel imaging approaches such as multiphoton microscopy. Multiphoton microscopy enables multimodal, three-dimensional imaging of histologically stained samples. Specifically, third harmonic generation (THG), a nonlinear optical process in which three incident photons are combined into one by the sample, allows high contrast imaging of tissues stained with absorbing dyes, which in turn act as harmonophores. While this technique has previously been applied to hematoxylin and eosin (H&E) tissue sections, we extend this approach to other commonly used histological stains to demonstrate further potential applications of the technique. We demonstrate THG imaging of both human skin and liver tissue stained with H&E, Verhoeff-Van Gieson (VVG) and Picrosirius Red stains. We find that these stains provide excellent contrast as THG harmonophores, enabling high resolution imaging of histological samples. THG imaging of the Verhoeff stain enables easy detection of elastic fibers while Picrosirius Red acts as an effective harmonophore for imaging collagen fibers of all sizes.

Malaria Detection by Third-Harmonic Generation Image Scanning Cytometry.

Despite global efforts aimed at its elimination, malaria is still a significant health concern in many countries across the world. The disease is caused by blood-borne parasites, Plasmodium species, and is transmitted by female Anopheles mosquitoes and presents with generic febrile symptoms that are challenging to diagnose clinically. To adequately tackle this issue, an effective detection method is required for screening potential malaria patients for infection. To this day, the gold standard for malaria detection remains basic light microscopy of Giemsa-stained patient blood smears to first enable detection and manual counting to determine the parasite density by a microscopist. While effective at detecting parasites, this method requires both significant time and skilled personnel. As an alternate approach, we propose a new malaria detection method that we call third-harmonic generation image scanning cytometry (THGISC) based on the combination of third-harmonic generation imaging, high-speed motorized scanning, and automated software processing. Third-harmonic generation (THG) is a nonlinear optical process in which the frequency of incident photons is tripled within the sample material. We have previously demonstrated that hemozoin, a metabolic byproduct of the malaria parasite, presents a significant THG signal. We now present a practical approach that uses the selectivity of this contrast mechanism to perform label-free image scanning cytometry of patient blood smears for automated malaria detection. In this work, we applied this technique to lab-cultured parasites and parasites in whole blood obtained from malaria patients. We also compared its effectiveness to parasite counts obtained by classical methods. The ability to easily and rapidly determine parasitemia by THG offers potential not only for the easy confirmation of malaria diagnoses following symptoms, but also the tracking of treatment progress in existing patients, potentially allowing physicians to adjust medication and dosage for each individual.

Low-cost multimodal light sheet microscopy for optically cleared tissues and living specimens.

Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology. However, the current major limitation of light sheet microscopy arises from optical aberrations, with the main drawback being the defocusing introduced by refractive index variations that accompany clearing techniques. Here, we propose an inexpensive and easy to build light sheet based instrumentation to overcome this limitation by optomechanically decoupling the sample scanning movement from the detection step. Our solution is relatively simple to implement and also provides increased modularity by using a swappable excitation arm. This expands the range of samples we can image on a single system, from high resolution for single cells at ? m spatial resolution, to tissues with mm spatial resolution. We demonstrate our approach, using the system to image iDISCO cleared embryos and sciatic nerves, and provide the full three-dimensional reconstruction of these objects in minutes.

Rapid prototyping of pneumatically actuated hydrocarbon gel valves for centrifugal microfluidic devices.

A novel, easy to prototype hydrocarbon gel-based active valve was developed for use in centrifugal microfluidic devices. The valve has been demonstrated to restrict flow by an additional 1000 revolutions per minute (RPM) when compared to a passive capillary valve of the same size located at the same radius. Opening of the valve is accomplished in a contactless manner using a stream of focused compressed air. The ease of fabrication, low cost and small dimensions of the gel valve offer the potential for integration of multiple valves of this type into multi-process centrifugal microfluidic systems.

Volumetric measurements by image segmentation on centrifugal microfluidic platforms in motion.

An image segmentation based method was developed to perform volumetric measurements of liquid aliquots in centrifugal microfluidic platforms in motion. The method was designed to be as automated as possible to allow its applicability to the large variety of available design features that tend to be included on such platforms. Experiments have indicated a relative standard deviation (RSD) of 0.3% for replicate measurements and 1% for same volume aliquots injected into different sized chambers. The versatility of the method in regards to chamber shape and size, liquid colour and platform rotational frequency was demonstrated. This flexibility should allow it to be used for a variety of applications including real time metering of volumes in platforms, quantitative monitoring of a design's performance in real time and could result in the elimination of metering chambers for some applications.

Automated liquid-liquid extraction by pneumatic recirculation on a centrifugal microfluidic platform.

In this technical note, a liquid-liquid extraction technique was performed using pneumatic liquid recirculation on a centrifugal microfluidic device. Non-contact pneumatic pumping enabled a multi-cycle liquid-liquid extraction process using aqueous iodine in a potassium iodide solution and hexadecane while requiring a minimal amount of space on the device. The extraction process was completely automated on the device following sample introduction and required only 50 s for each extraction cycle. The pumping rate achieved during liquid recirculation was 120 ± 10 µL/min. A recycling process such as the one demonstrated would be difficult to implement in a conventional centrifugal microfluidic system.