DMD-based software-configurable spatially-offset Raman spectroscopy for spectral depth-profiling of optically turbid samples

Spectral depth-profiling of optically turbid samples is of high interest to a broad range of applications. We present a method for measuring spatially-offset Raman spectroscopy (SORS) over a range of length scales by incorporating a digital micro-mirror device (DMD) into a sample-conjugate plane in the detection optical path. The DMD can be arbitrarily programmed to collect/reject light at spatial positions in the 2D sample-conjugate plane, allowing spatially offset Raman measurements. We demonstrate several detection geometries, including annular and simultaneous multi-offset modalities, for both macro- and micro-SORS measurements, all on the same instrument. Compared to other SORS modalities, DMD-based SORS provides more flexibility with only minimal additional experimental complexity for subsurface Raman collection

Holographic optical trapping Raman micro-spectroscopy for non-invasive measurement and manipulation of live cells

We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems

Ex-vivo Raman spectroscopy mapping of lung tissue: label-free molecular characterisation of non-tumorous and cancerous tissues

Raman spectroscopy mapping was used to study ex vivo fresh lung tissues and compare to histology sections. The Raman mapping measurements revealed differences in the molecular composition of normal lung tissue, adenocarcinoma, and squamous cell carcinoma (SCC). Molecular heterogeneity of the tissue samples was well captured by the k-means clustering analysis of the Raman datasets, as confirmed by the correlation with the adjacent haematoxylin and eosin (H&E) stained tissue sections. The results indicate that the fluorescence background varies considerably even in samples that appear structurally uniform in the H&E images, both for normal and tumor tissue. The results show that characteristic Raman bands can be used to discriminate between tumorous and nontumorous lung tissues and between adenocarcinoma and SCC tissues. These results indicate the potential to develop Raman classifications models for lung tissues based on the Raman spectral differences at the microscopic level, which can be used for tissue diagnosis or treatment stratification

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

Label-free Raman hyperspectral imaging of single cells cultured on polymer substrates

While Raman hyper-spectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in-vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: 1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; 2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples we show that Raman hyperspectral datasets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering

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

In-situ fabrication of gold nanoparticle functionalized probes for tip-enhanced Raman spectroscopy by dielectrophoresis

We report the use of dielectrophoresis to fabricate in-situ probes for tip-enhanced Raman spectroscopy (TERS) based on Au nanoparticles. A typical conductive atomic force microscope (AFM) was used to functionalize iridium-coated conductive silicon probes with Au nanoparticles of 10-nm diameter. Suitable TERS probes can be rapidly produced (30 to 120 s) by applying a voltage of 10 Vpp at a frequency of 1 MHz. The technique has the advantage that the Au-based probes are ready for immediate use for TERS measurements, minimizing the risks of tip contamination and damage during handling. Scanning electron microscopy and energy dispersive x-ray spectroscopy were used to confirm the quality of the probes, and used samples of p-ATP monolayers on silver substrates were used to demonstrate experimentally TERS measurements

Co-localised Raman and force spectroscopy reveal the roles of hydrogen bonds and ?-? interactions in defining the mechanical properties of diphenylalanine nano- and micro-tubes

An integrated atomic force and polarized Raman microscope were used to measure the elastic properties of individual diphenylalanine (FF) nano- and micro-tubes and to obtain quantitative information regarding the inter-molecular interactions that define their mechanical properties. For individual tubes, co-localised force spectroscopy and Raman spectroscopy measurements allowed the calculation of the Young’s and shear moduli (2565 GPa and 0.2860.05 GPa, respectively) and the contribution of hydrogen bonding network to the Young’s modulus (!17.6 GPa). The p-p interactions between the phenyl rings, dominated by T-type arrangements, were estimated based on previously published X-ray data to only 0.20 GPa. These results provide experimental evidence obtained from individual FF tubes that the network of H-bonds dominates the elastic properties of the FF tubes

Time-gated Raman spectroscopy for biomedical application under ambient or strong background light conditions

Many biomedical applications require measurements of Raman spectra of tissue under ambient lighting conditions. However, the background light often swamps the weaker Raman signal. The use of time-gated (TG) Raman spectroscopy based on a single photon avalanche diode (SPAD) operating in time-correlated single photon counting and near-infrared laser excitation was investigated for acquisition of Raman spectra and spectral images of biological tissue. The results obtained using animal tissue samples (adipose tissue and muscle) show that the time gating modality enables measurement of Raman spectra under background light conditions of similar quality as conventional continuous wave Raman spectroscopy in the absence of background light. Optimal suppression of the background light was observed for time gate widths of 300–1000 ps. The results also showed that TG Raman spectroscopy was able to detect subtle spectral differences required for medical diagnostics, such as differences in Raman spectra of cancer and normal tissue. While the current instrument required scanning of the grating in order to obtain full Raman spectra, leading to impractical times for multi-wavenumber Raman mapping, imaging time could be drastically reduced by spectral multiplexing (compressed detection) using digital micromirror devices or by using SPAD arrays

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