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Conventional methods for intraoperative histopathologic diagnosis are labour- and time-intensive, and may delay decision-making during brain-tumour surgery. Stimulated Raman scattering (SRS) microscopy, a label-free optical process, has been shown to rapidly detect brain-tumour infiltration in fresh, unprocessed human tissues. Here, we demonstrate the first application of SRS microscopy in the operating room using a portable fibre-laser-based microscope and unprocessed specimens from 101 neurosurgical patients. We also introduce an image-processing method—stimulated Raman histology (SRH)—that leverages SRS images to create virtual haematoxylin-and-eosin-stained slides, revealing essential diagnostic features. In a simulation of intraoperative pathologic consultation in 30 patients, we found a remarkable concordance of SRH and conventional histology for predicting diagnosis (Cohen’s kappa, κ  > 0.89), with accuracy exceeding 92%. We also built and validated a multilayer perceptron based on quantified SRH image attributes that predicts brain-tumour subtype with 90% accuracy. Our findings provide insight into how SRH can now be used to improve the surgical care of brain-tumour patients. By taking advantage of stimulated Raman spectroscopy and fibre-laser technology, virtual histology images can be obtained in real time in the operating room, with diagnostic quality comparable with that achieved via conventional histopathology.

We have implemented an unbiased cell morphology-based screen to identify small-molecule modulators of cellular processes using the Cytometrix (TM) automated imaging and analysis system. This assay format provides unbiased analysis of morphological effects induced by small molecules by capturing phenotypic readouts of most known classes of pharmacological agents and has the potential to read out pathways for which little is known. Four human-cancer cell lines and one noncancerous primary cell type were treated with 107 small molecules comprising four different protein kinase-inhibitor scaffolds. Cellular phenotypes induced by each compound were quantified by multivariate statistical analysis of the morphology, staining intensity, and spatial attributes of the cellular nuclei, microtubules, and Golgi compartments. Principal component analysis was used to identify inhibitors of cellular components not targeted by known protein kinase inhibitors. Here we focus on a hydroxyl-substituted analog (hydroxy-PP) of the known Src-family kinase inhibitor PP2 because it induced cell-specific morphological features distinct from all known kinase inhibitors in the collection. We used affinity purification to identify a target of hydroxy-PP, carbonyl reductase 1 (CBR1), a short-chain dehydrogenase-reductase. We solved the X-ray crystal structure of the CBR1/hydroxy-PP complex to 1.24 A resolution. Structure-based design of more potent and selective CBR1 inhibitors provided probes for analyzing the biological function of CBR1 in A549 cells. These studies revealed a previously unknown function for CBR1 in serum-withdrawal-induced apoptosis. Further studies indicate CBR1 inhibitors may enhance the effectiveness of anticancer anthracyclines. Morphology-based screening of diverse cancer cell types has provided a method for discovering potent new small-molecule probes for cell biological studies and anticancer drug candidates.

▪ Abstract  Recent developments in optical studies of single molecules at room temperature are reviewed, with an emphasis on the underlying principles and the potential of single-molecule experiments. Examples of single-molecule studies are given, including photophysics and photochemistry pertinent to single-molecule measurements, spectral fluctuations, Raman spectroscopy, diffusional motions, conformational dynamics, fluorescence resonant energy transfer, exciton dynamics, and enzymatic turnovers. These studies illustrate the information obtainable with the single-molecule approach that is hidden in ensemble-averaged measurements.

The near-field optical interaction between a sharp probe and a sample of interest can be exploited to image, spectroscopically probe, or modify surfaces at a resolution (down to ∼12 nm) inaccessible by traditional far-field techniques. Many of the attractive features of conventional optics are retained, including noninvasiveness, reliability, and low cost. In addition, most optical contrast mechanisms can be extended to the near-field regime, resulting in a technique of considerable versatility. This versatility is demonstrated by several examples, such as the imaging of nanometric-scale features in mammalian tissue sections and the creation of ultrasmall, magneto-optic domains having implications for high-density data storage. Although the technique may find uses in many diverse fields, two of the most exciting possibilities are localized optical spectroscopy of semiconductors and the fluorescence imaging of living cells.

Photoswitch compounds such as DENAQ confer light-sensitivity on endogenous neuronal ion channels, enabling photocontrol of neuronal activity without genetic manipulation. DENAQ treatment restores both retinal light responses and visual behaviors in rodent models of Retinitis pigmentosa. However, retinal photosensitization requires a high dose of DENAQ and disappears within several days after treatment. Here we report that BENAQ, an improved photoswitch, is 20-fold more potent than DENAQ and persists in restoring visual responses to the retina for almost 1 month after a single intraocular injection. Studies on mice and rabbits show that BENAQ is non-toxic at concentrations 10-fold higher than required to impart light-sensitivity. These favorable properties make BENAQ a potential drug candidate for vision restoration in patients with degenerative blinding diseases.

The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas. The clinical detection of fluorescent protoporphyrin IX during glioma-resection surgery can be improved via a microscope that pairs stimulated Raman histology and two-photon excitation fluorescence microscopy.

Recent developments in optical studies of single molecules at room temperature are reviewed, with an emphasis on the underlying principles and the potential of single-molecule...

Photoinduced electronic energy and charge transfer processes in condensed media are two areas of research amenable to study with ultrafast lasers. The majority of the experimental work presented here concerns electronic energy transfer from carotenoids to (bacterio)chlorophyll in photosynthetic systems. Specifically, femtosecond transient absorption experiments on light-harvesting complexes of a purple photosynthetic bacterium, Rhodobacter sphaeroides revealed carotenoid to bacteriochlorophyll energy transfer times for two spectrally distinct bacteriochlorophylls, called B850 and B800, of 0.2 and 0.34 ps, respectively. The transfer time from B800 to B850 is approximately 2.5 ps. In addition, a picosecond decay component of the excited B850 population was discovered which is attributed to excitation annihilation within single units of the light-harvesting complex. In experiments on thylakoid membrane preparations from two algae, a carotenoid to chlorophyll a transfer time of 0.24 ps was found in Nannochloropsis sp., and, in Phaeodactylum tricornutum, the transfer was determined to be biexponential with time constants of 0.5 and 2.0 ps. Direct evidence for transfer from the carotenoid first excited singlet state (the 'A' state) is provided by the two-photon excitation spectrum for chlorophyll $\\alpha$ emission of P. tricornutum thylakoids. Energy transfer coupling involving the carotenoid A state is analyzed. The general conclusions are that the coupling is likely best described in terms of an intermolecular exchange interaction, but that binding-site induced mixing of the carotenoid A and B states leading to significant Coulombic coupling cannot be excluded. An investigation of the dynamics of tris(2,2$\\sp\\prime$-bipyridine)ruthenium(II) in its metal-to-ligand-charge-transfer states by femtosecond transient absorption spectroscopy is also reported. It was found that the transient difference spectrum at 0.3 ps is indistinguishable from that observed in nanosecond experiments where the spectrum is that of a localized state. That the excited states are localized due to a spontaneous symmetry breaking interaction between the complex and the medium is suggested. Also, as the transient spectrum does not evolve beyond the rise time of the apparatus, it is proposed that the intersystem crossing has occurred in less than 0.3 ps. A semi-quantitative argument is given to support this proposition. Finally, a calculation on vibronic activity in resonant third harmonic generation is presented.

Objective Overconsumption of processed foods has led to an increase in chronic diet-related diseases such obesity and type 2 diabetes. Although diets high in fresh fruits and vegetables are linked with healthier outcomes, the specific mechanisms for these relationships are poorly understood. Experiments examining plant phytochemical production and breeding programs, or separately on the health effects of nutritional supplements have yielded results that are sparse, siloed, and difficult to integrate between the domains of human health and agriculture. To connect plant products to health outcomes through their molecular mechanism an integrated computational resource is necessary. Results We created the Aliment to Bodily Condition Knowledgebase (ABCkb) to connect plants to human health by creating a stepwise path from plant → plant product → human gene → pathways → indication. ABCkb integrates 11 curated sources as well as relationships mined from Medline abstracts by loading into a graph database which is deployed via a Docker container. This new resource, provided in a queryable container with a user-friendly interface connects plant products with human health outcomes for generating nutritive hypotheses. All scripts used are available on github ( https://github.com/atrautm1/ABCkb ) along with basic directions for building the knowledgebase and a browsable interface is available ( https://abckb.charlotte.edu ).

Forty-five adult Asian and Middle Eastern immigrants living in Oklahoma City at the time of the 1995 bombing were surveyed 114 to 2 years later as part of a disaster mental health outreach program. Demographic variables, physical and interpersonal exposure, initial physiologic and emotional responses to the bombing, and posttraumatic stress symptoms associated with this disaster and with earlier trauma were measured. Most participants had experienced prior trauma in their homeland. PTSD symptomatology from prior trauma was most predictive of initial physiologic and emotional response and of later bomb-related PTSD symptoms. Bomb-related PTSD symptoms increased with current age and were inversely related to age at the time of prior trauma. Results underscore the importance of providing long-term disaster assistance to immigrants with prior trauma.