Christopher J. Rowlands

Diagnosis of tumors during tissue-conserving surgery with integrated autofluorescence and Raman scattering microscopy.

Significance Histopathology is the standard method for diagnosis of cancer. However, this method requires time-consuming procedures for sectioning and staining of tissues, making histopathology impractical for use during surgery for most cancer types. We report a unique method based on two optical spectroscopy techniques—autofluorescence imaging and Raman scattering—that can accurately measure molecular differences between tumor cells and healthy tissue and allows diagnosis of tumors faster than histopathology, without requiring tissue sectioning or staining. Our study demonstrates the potential of this technique for diagnosis of tissues during cancer surgery, providing a quick and objective way to determine whether the tissue layers removed by the surgeon are clear of tumor. Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1–2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.

Next-generation in vivo optical imaging with short-wave infrared quantum dots

For in vivo imaging, the short-wavelength infrared region (SWIR; 1000–2000 nm) provides several advantages over the visible and near-infrared regions: general lack of autofluorescence, low light absorption by blood and tissue, and reduced scattering. However, the lack of versatile and functional SWIR emitters has prevented the general adoption of SWIR imaging by the biomedical research community. Here, we introduce a class of high-quality SWIR-emissive indium-arsenide-based quantum dots (QDs) that are readily modifiable for various functional imaging applications, and that exhibit narrow and size-tunable emission and a dramatically higher emission quantum yield than previously described SWIR probes. To demonstrate the unprecedented combination of deep penetration, high spatial resolution, multicolor imaging and fast-acquisition-speed afforded by the SWIR QDs, we quantified, in mice, the metabolic turnover rates of lipoproteins in several organs simultaneously and in real time as well as heartbeat and breathing rates in awake and unrestrained animals, and generated detailed three-dimensional quantitative flow maps of the mouse brain vasculature.

Microfluidic device for the formation of optically excitable, three-dimensional, compartmentalized motor units

Microfluidics and optogenetics enable the formation of light-excitable motor units in a compartmentalized and 3D environment. Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord–limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.

Rapid acquisition of Raman spectral maps through minimal sampling: applications in tissue imaging

A method is presented for acquiring high-spatial-resolution spectral maps, in particular for Raman micro-spectroscopy (RMS), by selectively sampling the spatial features of interest and interpolating the results. This method achieves up to 30 times reduction in the sampling time compared to raster-scanning, the resulting images have excellent correlation with conventional histopathological staining, and are achieved with sufficient spectral signal-to-noise ratio to identify individual tissue structures. The benefits of this selective sampling method are not limited to tissue imaging however; it is expected that the method may be applied to other techniques which employ point-by-point mapping of large substrates.

Application of multiphoton microscopy in dermatological studies: A mini-review

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

High-throughput nonlinear optical microscopy.

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.

Nanostructures fabricated in chalcogenide glass for use as surface-enhanced Raman scattering substrates

We describe chalcogenide glass (ChG)-based nanostructures for use as substrates for surface-enhanced Raman scattering (SERS). Such substrates were fabricated by exploiting the photosensitivity of ChG. This allows convenient control of the shape, size, and spacing of the nanostructures. The substrates were used to investigate the sample-concentration and excitation-power dependences of SERS from Rhodamine 6G molecules. A sensitivity of 1 muM was achieved at low excitation irradiance, and a semilinear concentration dependence was found for concentrations below 100 muM, demonstrating the potential of these ChG-based SERS substrates for high-sensitivity quantitative analysis.

Quantification of labile heme in live malaria parasites using a genetically encoded biosensor

Significance Malaria parasites degrade substantial quantities of hemoglobin to release heme within a specialized digestive vacuole. Most of this heme is sequestered in an inert crystal. However, the concentration of bioavailable, labile heme in the parasite’s cytosol was unknown. We developed a biosensor to provide the first quantitative insights into labile heme concentrations in malaria parasites. We find that ∼1.6 µM labile cytosolic heme is maintained, including during a period coincident with intense hemoglobin degradation. The heme-binding antimalarial drug, chloroquine, which interferes with heme crystallization, specifically induces an increase in labile heme. The ability to quantify labile heme in malaria parasites opens opportunities for better understanding heme homeostasis, signaling, and metabolism, and its association with antimalarial potency. Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.

Label-free molecular analysis of live Neospora caninum tachyzoites in host cells by selective scanning Raman micro-spectroscopy.

A selective scanning method was used to measure spatially resolved Raman spectra of live Neospora caninum tachyzoites colonizing human brain microvascular-endothelial cells. The technique allowed the detection of nucleic acids, lipids and proteins linked to the parasites and their cellular micro-environment at ∼10× shorter acquisition time compared to raster scanning.

Wide-field three-photon excitation in biological samples

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two- and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.