Raman spectroscopy and hyperspectral imaging ,
Osteoporosis and osteoarthritis ,
Lung tissue spectroscopy ,
Tissue and protein characterization ,
Infrared imaging and flow cell fabrication ,
Pharmaceutical polymorphs and screening
Publications (9)
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Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.
Bone is a highly specialized connective tissue comprised of cross-linked collagen fibers interspersed with apatitic
mineral crystallites of various sizes, shapes, orientation, and composition. However, the nucleation, growth, and
propagation of mineral crystallite into the collagenous matrix are not clearly understood. By using a research grade
inverted microscope fitted with a line-shaped 830 nm laser and spectrograph, we show that the Raman scatter from
mineralizing cell cultures in an incubation chamber can be collected and monitored directly through the bottom of the
well-plates over a period of 24 hours. In our studies, murine-derived MC3T3 cells were incubated at 37°C in the
presence of 5% CO2 and 85% humidity. Results show a gradual shift in the phosphate ν1 apatitic band center (955-957 cm-1) during the first hour of mineralization. The phosphate ν1 apatitic band width also narrowed during this time. To quantify the amount of crystal growth in vivo, we used a calibration curve derived from X-ray powder diffraction and Raman studies performed on a series of synthetic carbonated apatites and deproteinated mouse femoral specimens.
Mineralization in neonatal mouse calvarial culture was observed along the lambdoid suture. Deposition proceeded in a
stepwise fashion over the course of ~30 h.
As part of our ongoing assessment of bone tissue composition and structure, we report the first experimental protocols of
a prospective study to investigate the potential of using Raman spectroscopy to diagnose and predict skeletal fragility in
postmenopausal osteoporosis patients. This multi-center study will assess several potential spectroscopic and X-ray
based diagnostic techniques. One hundred and twenty participants will be enrolled in this five year study and the
investigators will be blinded to information concerning patient history and status. Iliac crest bone biopsy specimens are
provided with no identifying information except a patient study number. Our team will use micro-computed tomography
(micro-CT) to identify regions of interest in both cortical and cancellous bone from specimens delivered to us. Raman
mapping will be performed using a line-focused 785 nm laser in order to obtain local and averaged values on several
spectroscopic metrics of bone quality. These metrics include carbonate/phosphate and phosphate/matrix ratios. Results
from an initial set of biopsies will be presented. Protocols for obtaining measurements are discussed, with emphasis on
the challenges presented by the use of fixed and polymer embedded specimens. These protocols are illustrated will data
from a biopsy specimen.
For many years, viscosity has been the primary method used by researchers in rheumatology to assess the physiochemical properties of synovial fluid in both normal and osteoarthritic patients. However, progress has been limited by the lack of methods that provide multiple layers of information, use small sample volumes, and are rapid. Raman spectroscopy was used to assess the biochemical composition of synovial fluid collected from 40 patients with clinical evidence of knee osteoarthritis (OA) at the time of elective surgical treatment. Severity of knee osteoarthritis was assessed by a radiologist using Kellgren/Lawrence (K/L) scores from knee joint x rays, while light microscopy and Raman spectroscopy were used to examine synovial fluid (SF) aspirates (2 to 10 µL), deposited on fused silica slides. We show that Raman bands used to describe protein secondary structure and content can be used to detect changes in synovial fluid from osteoarthritic patients. Several Raman band intensity ratios increased significantly in spectra collected from synovial fluid in patients with radiological evidence of moderate-to-severe osteoarthritis damage. These ratios can be used to provide a "yes/no" damage assessment. These studies provide evidence that Raman spectroscopy would be a suitable candidate in the evaluation of joint damage in knee osteoarthritis patients.
Astronauts exposed to spaceflight conditions can lose 1-2% of their bone mineral density per month from the weight-bearing portions of the skeletal system. Low bone mineral density, termed osteopenia, is the result of decreased bone formation and/or increased bone resorption. In this study, Raman spectroscopy is used to examine if the physicochemical composition of murine femurs is altered in response to simulated spaceflight conditions (hindlimb suspension). Female C57BL/6J mice, aged 53 days, were divided into ground control and simulated spaceflight groups for a period of 12 days, modeling the experiment profile of mice flown on Space Shuttle flight STS-108. After the study, the mice were sacrificed and femur specimens harvested. Mid-diaphysis sections were probed using near-infrared Raman microscopy. Spectra were collected at various anatomical sites (anterior, lateral, medial, and posterior quadrants) and/or cortical locations (periosteal, midosteal, and endosteal). Chemometric recovery of spectra was employed to reduce signal contributions from the epoxy embedding agent. Mean values for mineralization, carbonation, crystallinity, and other parameters associated with the matrix were estimated. Correlations between mineralization and carbonation were observed, despite the small absolute changes between the two groups. We present more detailed analysis of this data and comment on the prospects for Raman spectroscopic evaluation of bone quality in hindlimb suspended (HLS) specimens.
We describe the use of Raman spectroscopy to investigate synovial fluid drops deposited onto fused silica microscope slides. This spectral information can be used to identify chemical changes in synovial fluid associated with osteoarthritis (OA) damage to knee joints. The chemical composition of synovial fluid is predominately proteins (enzymes, cytokines, or collagen fragments), glycosaminoglycans, and a mixture of minor components such as inorganic phosphate crystals. During osteoarthritis, the chemical, viscoelastic and biological properties of synovial fluid are altered. A pilot study was conducted to determine if Raman spectra of synovial fluid correlated with radiological scoring of knee joint damage. After informed consent, synovial fluid was drawn and x-rays were collected from the knee joints of 40 patients. Raman spectra and microscope images were obtained from the dried synovial fluid drops using a Raman microprobe and indicate a coarse separation of synovial fluid components. Individual protein signatures could not be identified; Raman spectra were useful as a general marker of overall protein content and secondary structure. Band intensity ratios used to describe protein and glycosaminoglycan structure were used in synovial fluid spectra. Band intensity ratios of Raman spectra indicate that there is less ordered protein secondary structure in synovial fluid from the damage group. Combination of drop deposition with Raman spectroscopy is a powerful approach to examining synovial fluid for the purposes of assessing osteoarthritis damage.
Human synovial fluid droplets were investigated using drop deposition in combination with Raman spectroscopy.
Following informed consent, synovial fluid was obtained from forty human patients with various severities of knee pain
and/or osteoarthritis at the time of knee arthroscopy or total joint replacement. Synovial fluid was aspirated from the
knee joint of each patient and stored at -80°C until examination by near-infrared Raman spectroscopy. Synovial fluid
aspirates from the knee joint of each patient were deposited onto a clean fused silica microscope slide and the droplet
dried under ambient laboratory conditions. Each droplet was illuminated by a line-focused or a ring-focused 785 nm
laser. As the droplet dries, biofluid components segregated based on solubility differences and a deposit that is spatially
heterogeneous was made. Spectra taken from the droplet edges and center were dominated by protein bands and showed
the presence of at least two protein moieties in the droplet. Band area and band height ratios (1410 cm-1/1450 cm-1)
showed the greatest change between specimens from patients with mild/early osteoarthritis compared to those with
severe/late stage osteoarthritis. The greatest differences were found in the center of the droplet, which contains more
soluble protein components than the edges.
A novel application of surface-enhanced Raman spectroscopy (SERS) for in-vitro osteoarthritis (OA) biomarker detection is described. Hyaluronic acid (HA) is a potential OA biomarker and synovial fluid levels of HA have been correlated with progression of joint space narrowing. However, current immunoassay and chromatographic methods that identify HA in synovial fluid are cumbersome and often require sophisticated instrumentation. Raman spectroscopy may be an alternative to these analytical methods, providing rapid identification of HA using characteristic Raman bands. Yet, previous reports of un-enhanced Raman spectroscopy for hyaluronic acid are in aqueous solutions exceeding 1000X in-vivo concentrations because HA is a weakly scattering polysaccharide. Surface-enhanced Raman spectroscopy can improve detection limits by 100-1000 times and we present, to our best knowledge, the first surface-enhanced Raman spectra of hyaluronic acid. Moreover, the recent commercial availability of stable SERS gold substrates has enabled rapid SERS detection of this biomarker at concentrations diluted by more than an order of magnitude, compared to previous literature reports. Preliminary results of easily and rapidly observing hyaluronic acid at low concentrations in aqueous solutions supported further studies in synthetic models of biofluids, such as artificial synovial fluid, that contain HA at low concentrations. These complex fluids contain proteins that compete for the SERS-active sites on the substrate, and the resulting spectra are dominated by protein Raman bands. We apply a simple and validated protein precipitation protocol to artificial synovial fluid prior to deposition onto the SERS substrate. We find that HA is easily detected in these fluids after protein removal treatment.
Background fluorescence can often complicate the use of Raman microspectroscopy in the study of musculoskeletal tissues. Such fluorescence interferences are undesirable as the Raman spectra of matrix and mineral phases can be used to differentiate between normal and pathological or microdamaged bone. Photobleaching with the excitation laser provides a non-invasive method for reducing background fluorescence, enabling 532 nm Raman hyperspectral imaging
of bone tissue. The signal acquisition time for a 400 point Raman line image is reduced to 1-4 seconds using electronmultiplying
CCD (EMCCD) detector, enabling acquisition of Raman images in less than 10 minutes. Rapid photobleaching depends upon multiple scattering effects in the tissue specimen and is applicable to some, but not all experimental situations.
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