Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy.

Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type-specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

Membrane Tension Modifies Redox Loading and Release in Single Liposome Electroanalysis.

Here, we present a study of how liposomes are loaded and release their contents during their electrochemical detection. We loaded 200 nm liposomes with a redox mediator, ferrocyanide, and used amperometry to detect their collision on a carbon-fiber microelectrode (CFE). We found that we could control the favorability of their electroporation process and the amount of ferrocyanide released by modifying the osmolarity of the buffer in which the liposomes were suspended. Interestingly, we observed that the quantity of the released ferrocyanide varied significantly with buffer osmolarity in a nonmonotonic fashion. Using stimulated Raman scattering (SRS), we confirmed that this behavior was partly explained by fluctuations in the intravesicular redox concentration in response to osmotic pressure. To our surprise, the redox concentration obtained from SRS was much greater than that obtained from amperometry, implying that liposomes may release only a fraction of their contents during electroporation. Consistent with this hypothesis, we observed barrages of electrochemical signals that far exceeded the frequency predicted by Poisson statistics, suggesting that single liposomes can collide with the CFE and electroporate multiple times. With this study, we have resolved some outstanding questions surrounding electrochemical detection of liposomes while extending observations from giant unilamellar vesicles to 200 nm liposomes with high temporal resolution and sensitivity.

Real-Time Microscale Temperature Imaging by Stimulated Raman Scattering.

Microscale thermometry of aqueous solutions is essential to understand the dynamics of local heat generation and dissipation in chemical and biological systems. A wide variety of fluorescent probes have been developed to map temperature changes with submicrometer resolution, but they often suffer from the uncertainty associated with microenvironment-dependent fluorescent properties. In this work, we develop a label-free ratiometric stimulated Raman scattering (SRS) microscopy technique to quantify microscale temperature by monitoring the O-H Raman stretching modes of water. By tracking the ratio changes of the hydrogen-bonding O-H band and the isosbestic band, we can directly quantify the temperature of water-based environments in real time without exogenous contrast agents. We demonstrate real-time measurement of localized intracellular and extracellular temperature changes due to laser absorption. This high-speed nonlinear optical imaging technique has the potential for in situ microscale imaging of thermogenesis in both chemical and biological systems.

Quantitative chemical imaging of breast calcifications in association with neoplastic processes.

Calcifications play an essential role in early breast cancer detection and diagnosis. However, information regarding the chemical composition of calcifications identified on mammography and histology is limited. Detailed spectroscopy reveals an association between the chemical composition of calcifications and breast cancer, warranting the development of novel analytical tools to better define calcification types. Previous investigations average calcification composition across broad tissue sections with no spatially resolved information or provide qualitative visualization, which prevents a robust linking of specific spatially resolved changes in calcification chemistry with the pathologic process. Method: To visualize breast calcification chemical composition at high spatial resolution, we apply hyperspectral stimulated Raman scattering (SRS) microscopy to study breast calcifications associated with a spectrum of breast changes ranging from benign to neoplastic processes, including atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. The carbonate content of individual breast calcifications is quantified using a simple ratiometric analysis. Results: Our findings reveal that intra-sample calcification carbonate content is closely associated with local pathological processes. Single calcification analysis supports previous studies demonstrating decreasing average carbonate level with increasing malignant potential. Sensitivity and specificity reach >85% when carbonate content level is used as the single differentiator in separating benign from neoplastic processes. However, the average carbonate content is limiting when trying to separate specific diagnostic categories, such as fibroadenoma and invasive ductal carcinoma. Second harmonic generation (SHG) data can provide critical information to bridge this gap. Conclusion: SRS, combined with SHG, can be a valuable tool in better understanding calcifications in carcinogenesis, diagnosis, and possible prognosis. This study not only reveals previously unknown large variations of breast microcalcifications in association with local malignancy but also corroborates the clinical value of linking microcalcification chemistry to breast malignancy. More importantly, it represents an important step in the development of a label-free imaging strategy for breast cancer diagnosis with tremendous potential to address major challenges in diagnostic discordance in pathology.

Fate of Barium Sulfate Nanoparticles Deposited in the Lungs of Rats.

We have shown that barium [from BaSO4 nanoparticles (NPs)] was cleared from the lungs faster than other poorly soluble NPs and translocated mostly to bone. We now studied barium biokinetics in rats during Study 1: two-year inhalation exposure to 50 mg/m3 BaSO4 NP aerosols, and Study 2: single intratracheal (IT) instillation of increasing doses of BaSO4 NPs or BaCl2. Study 1 showed that lung barium content measured by inductively coupled plasma mass spectrometry increased during 360 days of BaSO4 NP aerosol exposures. An equilibrium was established from that time until 2 years. Barium concentrations in BaSO4-exposed animals were in the order (lungs > lymph nodes > hard bone > bone marrow > liver). In Study 2, there was an increase in lung barium post-IT instillation of BaSO4 NPs while barium from BaCl2 was mostly cleared by day 28. Transmission electron microscopy showed intact BaSO4 NPs in alveolar macrophages and type II epithelial cells, and in tracheobronchial lymph nodes. Using stimulated Raman scattering microscopy, specific BaSO4 Raman spectra were detected in BaSO4 NP-instilled lungs and not in other organs. Thus, we posit that barium from BaSO4 NPs translocates from the lungs mainly after dissolution. Barium ions are then incorporated mostly into the bone and other organs.

Detecting and Quantifying Microscale Chemical Reactions in Pharmaceutical Tablets by Stimulated Raman Scattering Microscopy.

It has been estimated that approximately 50% of all marketed drug molecules are manufactured and administered in the form of salts, often with the goal of improving solubility, dissolution rate, and efficacy of the drug. However, salt disproportionation during processing or storage is a common adverse effect in these formulations. Due to the heterogeneous nature of solid drug formulations, it is essential to characterize the drug substances noninvasively at micrometer resolution to understand the molecular mechanism of salt disproportionation. However, there is a lack of such capability with current characterization methods. In this study, we demonstrate that stimulated Raman scattering (SRS) microscopy can be used to provide sensitive and quantitative chemical imaging of the salt disproportionation reaction of pioglitazone hydrochloride (PIO-HCl) at a very low drug loading (1% w/w). Our findings illuminate a water mediated pathway of drug disproportionation and highlight the importance of noninvasive chemical imaging in a mechanistic study of solid-state chemical reactions.

Broadband hyperspectral stimulated Raman scattering microscopy with a parabolic fiber amplifier source.

Hyperspectral stimulated Raman scattering (hsSRS) microscopy has recently emerged as a powerful non-destructive technique for the label-free chemical imaging of biological samples. In most hsSRS imaging experiments, the SRS spectral range is limited by the total bandwidth of the excitation laser to ~300 cm-1 and a spectral resolution of ~25 cm-1. Here we present a novel approach for broadband hsSRS microscopy based on parabolic fiber amplification to provide linearly chirped broadened Stokes pulses. This novel hsSRS instrument provides >600 cm-1 spectral coverage and ~10 cm-1 spectral resolution. We further demonstrated broadband hsSRS imaging of the entire Raman fingerprint region for resolving the distribution of major biomolecules in fixed cells. Moreover, we applied broadband hsSRS in imaging amyloid plaques in human brain tissue with Alzheimer's disease.

Label-free pathology by spectrally sliced femtosecond stimulated Raman scattering (SRS) microscopy.

Optical "virtual biopsy" is an attractive way to improve disease diagnosis and surgical guidance. Many optical microscopy techniques have been developed to provide diagnostic information without the need for tissue sectioning or staining. Among these techniques, label-free chemical imaging is the most desirable. Recently, it has been shown that narrowband, picosecond stimulated Raman scattering (SRS) can achieve comparable morphological contrast to hematoxylin and eosin staining (H&E staining), the 'gold standard' of pathology. However, to translate the technique from the bench to the bedside, optimal laser sources and parameters have yet to be identified. Here we describe an improvement to the narrowband SRS microscopy techniques for label-free tissue imaging. Through spectral slicing of broadband, femtosecond pulses, we are able to maintain the same protein/lipid contrast as narrowband SRS while achieving a higher signal-to-noise ratio (SNR). Our method draws upon the benefits of femtosecond pulses (e.g. higher peak power) while preserving those of picosecond pulses (e.g. adequate spectral resolution). We demonstrate this achievement through protein/lipid signal and contrast quantification of mouse brain tissue as a function of bandwidth, and comparison with numerical simulations. Further method validation is provided through imaging of additional mouse tissues: liver, kidney, and skin.

Label-Free Chemical Imaging of Latent Fingerprints with Stimulated Raman Scattering Microscopy.

Fingerprints have long been the gold standard for personal identification in forensic investigations. Methods for cultivating and enhancing the visualization of latent fingerprints (LFPs) are continuously evolving. One important challenge is to identify suspicious chemicals present in fingerprint residues, which requires chemical imaging capability. Recently, vibrational spectroscopy has shown that LFP analysis through tape-lift, Raman mapping, and multivariate data analysis presents a useful tool for forensic investigation. However, there are still major difficulties in terms of acquisition speed, poor spatial resolution, and lack of sensitivity. This paper demonstrates the feasibility of stimulated Raman scattering microscopy to quickly and easily extract LFP patterns from different substrates. Contrary to what has been reported, no obvious fingerprint degradation or lipid diffusion is observed with either glass or stainless steel substrate. Importantly, we demonstrate that trace exogenous chemicals can be detected in fingerprints. We further demonstrate an improvement in directly acquiring a LFP pattern lifted from tape by spectrally removing signals from tape.

[Gestational diabetes. Diagnosis in early stages of the pregnancy]. / Diabetes gestacional. Diagnóstico en etapas tempranas del embarazo.

OBJECTIVE: To determine whether women with early diagnosis of gestational diabetes have an increase of the rate of gestational hypertensive disease, metabolic imbalance, higher insulin dosage requirement, perinatal morbidity and perinatal death than those in whom diabetes developed after 24th week. METHODS: All pregnant women with gestational diabetes were studied. Maternal age, pregestational body mass indexes, total weight gain during pregnancy, gestational age at diagnosis, and the timing of delivery were recorded. Two groups were studied: women with gestational diabetes diagnosed before 24 gestation weeks and those in which diabetes was diagnosed at 24 weeks or later. Total insulin dosage, rate of gestational hypertension, perinatal morbidity, perinatal death, and percentages of women with good metabolic control were analysed. RESULTS: In the whole group 28% were diagnosed before 24 weeks of gestation. Women that were diagnosed earlier had grater pregestational body mass indexes (30 +/- 6 kg/m2 vs 27 +/- 6 kg/m2) and total weight gain during pregnancy was lower during over all gestation (8 +/- 5 kg vs 10 +/- 6 kg). Insulin requirements were higher in women diagnosed earlier and there were no differences in the rates of gestational hypertension, and perinatal death. Perinatal morbidity was significantly higher in patients who were diagnosed later CONCLUSIONS: Early diagnosed and treatment may result in decreased perinatal morbidity. Women with early diagnosed diabetes required higher insulin dose, but they did not represent a high risk subgroup.

Fractal radar scattering from soil.

A general technique is developed to retrieve the fractal dimension of self-similar soils through microwave (radar) scattering. The technique is based on a mathematical model relating the fractal dimensions of the georadargram to that of the scattering structure. Clear and different fractal signatures have been observed over four geosystems (soils and sediments) compared in this work.