Noninvasive Detection of Differential Water Content Inside Biological Samples Using Deep Raman Spectroscopy.

Here we conceptually demonstrate the capability of deep Raman spectroscopy to noninvasively monitor changes in the water content within biological tissues. Water was added by injection into an isolated tissue volume (a 20 mm diameter disk of 5 mm thickness) representing a 20% increase in the overall mass, which was equivalent to a 5% increase in the water/tissue content. The elevated water content was detected through a larger volume of tissue with a total thickness of approximately 12 mm and a spiked tissue segment located in its center using transmission Raman spectroscopy (TRS) by monitoring the change of the OH (∼3390 cm-1) Raman band area (3350-3550 cm-1 spectral region) after being normalized to the neighboring CH stretching band. The tissue sample was raster scanned with TRS to yield a spatial map of the water concentration within the sample encompassing the spiked tissue zone. The mapping revealed the presence and location of the spiked region. The results provide the first conceptual demonstration using a deep Raman-based architecture, which can be used noninvasively for the detection of an elevated water content deep within biological tissues. It is envisaged that this concept could play a role in rapid in vivo detection and localization of cancerous lesions (generally exhibiting a higher water content) beneath the tissue surface.

Enhanced deep detection of Raman scattered light by wavefront shaping.

Light scattering limits the penetration depth of non-invasive Raman spectroscopy in biological media. While safe levels of irradiation may be adequate to analyze superficial tissue, scattering of the pump beam reduces the Raman signal to undetectable levels deeper within the tissue. Here we demonstrate how wavefront shaping techniques can significantly increase the Raman signal at depth, while keeping the total irradiance constant, thus increasing the amount of Raman signal available for detection.

Exploring the effect of laser excitation wavelength on signal recovery with deep tissue transmission Raman spectroscopy.

The aim of this research was to find the optimal Raman excitation wavelength to attain the largest possible sensitivity in deep Raman spectroscopy of breast tissue. This involved careful consideration of factors such as tissue absorption, scattering, fluorescence and instrument response function. The study examined the tissue absorption profile combined with Raman scattering and detection sensitivity at seven different, laser excitation wavelengths in the near infrared region of the spectrum. Several key scenarios in regards to the sample position within the tissue were examined. The highest Raman band visibility over the background ratio in respect to biological tissue provides the necessary information for determining the optimum laser excitation wavelength for deep tissue analysis using transmission Raman spectroscopy, including detection of breast calcifications. For thick tissues with a mix of protein and fat, such as breast tissue, 790-810 nm is concluded to be the optimum excitation wavelength for deep Raman measurements.

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman spectroscopy.

Label-free imaging using Raman micro-spectroscopy (RMS) was used to characterize the spatio-temporal molecular changes of T. gondii tachyzoites and their host cell microenvironment. Raman spectral maps were recorded from isolated T. gondii tachyzoites and T. gondii-infected human retinal cells at 6 h, 24 h and 48 h post-infection. Principal component analysis (PCA) of the Raman spectra of paraformaldehyde-fixed infected cells indicated a significant increase in the amount of lipids and proteins in the T. gondii tachyzoites as the infection progresses within host cells. These results were confirmed by experiments carried out on live T. gondii-infected cells and were correlated with an increase in the concentration of proteins and lipids required for the replication of this intracellular pathogen. These findings demonstrate the potential of RMS to characterize time- and spatially-dependent molecular interactions between intracellular pathogens and the host cells. Such information may be useful for discovery of pharmacological targets or screening compounds with potential neuro-protective activity for eminent effects of changes in brain infection control practices.

Monitoring the mineralisation of bone nodules in vitro by space- and time-resolved Raman micro-spectroscopy.

Raman microscopy was used as a label-free method to study the mineralisation of bone nodules formed by mesenchymal stem cells cultured in osteogenic medium in vitro. Monitoring individual bone nodules over 28 days revealed temporal and spatial changes in the crystalline phase of the hydroxyapatite components of the nodules.

Cytoplasmic RNA in undifferentiated neural stem cells: a potential label-free Raman spectral marker for assessing the undifferentiated status.

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA.

The Effect of Food on the Single-Dose Bioavailability and Tolerability of the Highest Marketed Strength of Duloxetine.

Duloxetine is a combined serotonin and norepinephrine reuptake inhibitor indicated in adults for the treatment of major depressive disorder, diabetic peripheral neuropathic pain, and generalized anxiety disorder. The aim of these studies was to evaluate the effect of food on the pharmacokinetics and safety of duloxetine 60-mg gastroresistant hard capsules following single-dose administration. The data were obtained from 2 phase 1 bioequivalence studies, 1 in a fasting state and the other under fed conditions. Both studies have shown that, when administered as a single dose in the same prandial state, the test and reference duloxetine treatments were bioequivalent and exhibited similar safety profiles. The mean fed and fasting pharmacokinetic parameters and drug-related adverse events from the 2 studies were compared in order to assess the effect of food on the duloxetine bioavailability and respectively, tolerability. Administration of duloxetine in fed conditions increased peak plasma concentration by up to 30% and delayed mean time to peak concentration by an average of 1.15 hours while having an insignificant effect on extent of absorption (area under the plasma concentration-time curve in fed state within ±6% as compared with fasting conditions). Even though peak plasma levels were substantially higher in the fed state, there was no negative impact on the drug's safety profile. Actually, administration with food resulted in a lower average number of adverse events per single dose exposure. The negligible variation in overall systemic exposure suggests that efficacy remains unchanged irrespective of administration conditions; however, a better tolerability of the 60-mg dose is expected when the drug is taken with food.

High sensitivity non-invasive detection of calcifications deep inside biological tissue using Transmission Raman Spectroscopy.

The aim of this research was to develop a novel approach to probe non-invasively the composition of inorganic chemicals buried deep in large volume biological samples. The method is based on advanced Transmission Raman Spectroscopy (TRS) permitting chemical specific detection within a large sampling volume. The approach could be beneficial to chemical identification of the breast calcifications detected during mammographic X-ray procedures. The chemical composition of a breast calcification reflects the pathology of the surrounding tissue, malignant or benign and potentially the grade of malignancy. However, this information is not available from mammography, leading to excisional biopsy and histopathological assessment for a definitive diagnosis. Here we present, for the first time, a design of a new high performance deep Raman instrument and demonstrate its capability to detect type II calcifications (calcium hydroxyapatite) at clinically relevant concentrations and depths of around 40 mm in phantom tissue. This is around double the penetration depth achieved with our previous instrument design and around two orders of magnitude higher than that possible when using conventional Raman spectroscopy.

Sensitivity of Transmission Raman Spectroscopy Signals to Temperature of Biological Tissues.

Optical properties of biological tissues can be influenced by their temperature, thus affecting light transport inside the sample. This could potentially be exploited to deliver more photons inside large biological samples, when compared with experiments at room temperature, overcoming some of difficulties due to highly scattering nature of the tissue. Here we report a change in light transmitted inside biological tissue with temperature elevation from 20 to 40 °C, indicating a considerable enhancement of photons collected by the detector in transmission geometry. The measurement of Raman signals in porcine tissue samples, as large as 40 mm in thickness, indicates a considerable increase in signal ranging from 1.3 to 2 fold, subject to biological variability. The enhancements observed are ascribed to phase transitions of lipids in biological samples. This indicates that: 1) experiments performed on tissue at room temperature can lead to an underestimation of signals that would be obtained at depth in the body in vivo and 2) that experiments at room temperature could be modified to increase detection limits by elevating the temperature of the material of interest.

Applications of Raman micro-spectroscopy to stem cell technology: label-free molecular discrimination and monitoring cell differentiation.

Stem cell therapy is widely acknowledged as a key medical technology of the 21st century which may provide treatments for many currently incurable diseases. These cells have an enormous potential for cell replacement therapies to cure diseases such as Parkinson's disease, diabetes and cardiovascular disorders, as well as in tissue engineering as a reliable cell source for providing grafts to replace and repair diseased tissues. Nevertheless, the progress in this field has been difficult in part because of lack of techniques that can measure non-invasively the molecular properties of cells. Such repeated measurements can be used to evaluate the culture conditions during differentiation, cell quality and phenotype heterogeneity of stem cell progeny. Raman spectroscopy is an optical technique based on inelastic scattering of laser photons by molecular vibrations of cellular molecules and can be used to provide chemical fingerprints of cells or organelles without fixation, lysis or use of labels and other contrast enhancing chemicals. Because differentiated cells are specialized to perform specific functions, these cells produce specific biochemicals that can be detected by Raman micro-spectroscopy. This mini-review paper describes applications of Raman micro-scpectroscopy to measure moleculare properties of stem cells during differentiation in-vitro. The paper focuses on time- and spatially-resolved Raman spectral measurements that allow repeated investigation of live stem cells in-vitro.

Development of Raman microspectroscopy for automated detection and imaging of basal cell carcinoma.

We investigate the potential of Raman microspectroscopy (RMS) for automated evaluation of excised skin tissue during Mohs micrographic surgery (MMS). The main aim is to develop an automated method for imaging and diagnosis of basal cell carcinoma (BCC) regions. Selected Raman bands responsible for the largest spectral differences between BCC and normal skin regions and linear discriminant analysis (LDA) are used to build a multivariate supervised classification model. The model is based on 329 Raman spectra measured on skin tissue obtained from 20 patients. BCC is discriminated from healthy tissue with 90+/-9% sensitivity and 85+/-9% specificity in a 70% to 30% split cross-validation algorithm. This multivariate model is then applied on tissue sections from new patients to image tumor regions. The RMS images show excellent correlation with the gold standard of histopathology sections, BCC being detected in all positive sections. We demonstrate the potential of RMS as an automated objective method for tumor evaluation during MMS. The replacement of current histopathology during MMS by a "generalization" of the proposed technique may improve the feasibility and efficacy of MMS, leading to a wider use according to clinical need.