Enhanced characterization of breast cancer phenotypes using Raman micro-spectroscopy on stainless steel substrate.

Biochemical insights into varying breast cancer (BC) phenotypes can provide a fundamental understanding of BC pathogenesis, while identifying novel therapeutic targets. Raman spectroscopy (RS) can gauge these biochemical differences with high specificity. For routine RS, cells are traditionally seeded onto calcium fluoride (CaF2) substrates that are costly and fragile, limiting its widespread adoption. Stainless steel has been interrogated previously as a less expensive alternative to CaF2 substrates, while reporting increased Raman signal intensity than the latter. We sought to further investigate and compare the Raman signal quality measured from stainless steel versus CaF2 substrates by characterizing different BC phenotypes with altered human epidermal growth factor receptor 2 (HER2) expression. Raman spectra were obtained on stainless steel and CaF2 substrates for HER2 negative cells - MDA-MB-231, MDA-MB-468 and HER2 overexpressing cells - AU565, SKBr3. Upon analyzing signal-to-noise ratios (SNR), stainless steel provided a stronger Raman signal, improving SNR by 119% at 1450 cm-1 and 122% at 2925 cm-1 on average compared to the CaF2 substrate. Utilizing only 22% of laser power on sample relative to the CaF2 substrate, stainless steel still yielded improved spectral characterization over CaF2, achieving 96.0% versus 89.8% accuracy in BC phenotype discrimination and equivalent 100.0% accuracy in HER2 status classification. Spectral analysis further highlighted increased lipogenesis and altered metabolism in HER2 overexpressing cells, which was subsequently visualized with coherent anti-Stokes Raman scattering microscopy. Our findings demonstrate that stainless steel substrates deliver improved Raman signal and enhanced spectral characterization, underscoring its potential as a cost-effective alternative to CaF2 for non-invasively monitoring cellular biochemical dynamics in translational cancer research.

Recurrent activity within microcircuits of macaque dorsolateral prefrontal cortex tracks cognitive flexibility.

Human and non-human primate data clearly implicate the dorsolateral prefrontal cortex (dlPFC) as critical for advanced cognitive functions 1,2 . It is thought that intracortical synaptic architectures within dlPFC are the integral neurobiological substrate that gives rise to these processes, including working memory, inferential reasoning, and decision-making 3-7 . In the prevailing model, each cortical column makes up one fundamental processing unit composed of dense intrinsic connectivity, conceptualized as the 'canonical' cortical microcircuit 3,8 . Each cortical microcircuit receives sensory and cognitive information from a variety of sources which are represented by sustained activity within the microcircuit, referred to as persistent or recurrent activity 4,9 . Via recurrent connections within the microcircuit, activity can propagate for a variable length of time, thereby allowing temporary storage and computations to occur locally before ultimately passing a transformed representation to a downstream output 4,5,10 . Competing theories regarding how microcircuit activity is coordinated have proven difficult to reconcile in vivo where intercortical and intracortical computations cannot be fully dissociated 5,9,11,12 . Here, we interrogated the intrinsic features of isolated microcircuit networks using high-density calcium imaging of macaque dlPFC ex vivo . We found that spontaneous activity is intrinsically maintained by microcircuit architecture, persisting at a high rate in the absence of extrinsic connections. Further, using perisulcal stimulation to evoke persistent activity in deep layers, we found that activity propagates through stochastically assembled intracortical networks, creating predictable population-level events from largely non-overlapping ensembles. Microcircuit excitability covaried with individual cognitive performance, thus anchoring heuristic models of abstract cortical functions within quantifiable constraints imposed by the underlying synaptic architecture.

Visualizing the lipid dynamics role in infrared neural stimulation using stimulated Raman scattering.

Infrared neural stimulation (INS) uses pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, INS's mechanistic and biophysical underpinnings have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. We used hyperspectral stimulated Raman scattering microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. The findings suggest that lipid bilayer structural changes occur during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell stimulated Raman scattering spectra varied with stimulation energy and radiation exposure. The spectroscopic observations agree with high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)pyridinium hydroxide. The findings support the hypothesis that INS causes changes in the lipid membrane of neural cells by changing the lipid membrane packing order. This work highlights the potential of hyperspectral stimulated Raman scattering as a method to safely study biophysical and biochemical dynamics in live cells.

Identifying optimal parameters for infrared neural stimulation in the peripheral nervous system.

Significance: Infrared neural stimulation (INS) utilizes pulsed infrared light to selectively elicit neural activity without exogenous compounds. Despite its versatility in a broad range of biomedical applications, no comprehensive comparison of factors pertaining to the efficacy and safety of INS such as wavelength, radiant exposure, and optical spot size exists in the literature. Aim: Here, we evaluate these parameters using three of the wavelengths commonly used for INS, 1450 nm, 1875 nm, and 2120 nm. Approach: In an in vivo rat sciatic nerve preparation, the stimulation threshold and transition rate to 100% activation probability were used to compare the effects of each parameter. Results: The pulsed diode lasers at 1450 nm and 1875 nm had a consistently higher ( ∼ 1.0 J / cm 2 ) stimulation threshold than that of the Ho:YAG laser at 2120 nm ( ∼ 0.7 J / cm 2 ). In addition, the Ho:YAG produced a faster transition rate to 100% activation probability compared to the diode lasers. Our data suggest that the superior performance of the Ho:YAG is a result of the high-intensity microsecond spike at the onset of the pulse. Acute histological evaluation of diode irradiated nerves revealed a safe range of radiant exposures for stimulation. Conclusion: Together, our results identify measures to improve the safety, efficacy, and accessibility of INS technology for research and clinical applications.

Multi-modal nonlinear optical and thermal imaging platform for label-free characterization of biological tissue.

The ability to characterize the combined structural, functional, and thermal properties of biophysically dynamic samples is needed to address critical questions related to tissue structure, physiological dynamics, and disease progression. Towards this, we have developed an imaging platform that enables multiple nonlinear imaging modalities to be combined with thermal imaging on a common sample. Here we demonstrate label-free multimodal imaging of live cells, excised tissues, and live rodent brain models. While potential applications of this technology are wide-ranging, we expect it to be especially useful in addressing biomedical research questions aimed at the biomolecular and biophysical properties of tissue and their physiology.

Stimulation of water and calcium dynamics in astrocytes with pulsed infrared light.

Astrocytes are non-neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca2+ -mediated signaling. As they are tightly integrated into neural networks, label-free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live-cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca2+ and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP3 R, TRPA1, TRPV4, and Aquaporin-4 are all involved in shaping the dynamics of infrared pulse-evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy.

Beyond the H&E: Advanced Technologies for in situ Tissue Biomarker Imaging.

For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.