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1.
Breast Cancer Res Treat ; 207(1): 223-232, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38769222

RESUMEN

BACKGROUND AND OBJECTIVES: Sentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer and normal axilla on imaging. Positive SLNs on histological examination can lead to a subsequent surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery. METHODS: A microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. During AF-Raman measurements, AF imaging determined optimal sampling locations for Raman spectroscopy measurements. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81 lymph nodes comprising 58 fixed and 23 fresh specimens. The sensitivity and specificity of AF-Raman were calculated using post-operative histology as a standard of reference. RESULTS: The independent test set contained 66 negative lymph nodes and 15 positive lymph nodes according to the reference standard, collected from 78 patients. For this set of specimens, the area under the receiver operating characteristic (ROC) curve for the AF-Raman technique was 0.93 [0.83-0.98]. AF-Raman was then operated in a regime that maximised detection specificity, producing a 94% detection accuracy: 80% sensitivity and 97% specificity. The main confounders for SLN metastasis were areas rich in histiocytes clusters, for which only few Raman spectra had been included in the training dataset. DISCUSSION: This preliminary study indicates that with further development and extension of the training dataset by inclusion of additional Raman spectra of histiocytes clusters and capsule, the AF-Raman may become a promising technique for intra-operative assessment of SLNs. Intra-operative detection of positive biopsies could avoid second surgery for axillary clearance.


Asunto(s)
Neoplasias de la Mama , Biopsia del Ganglio Linfático Centinela , Ganglio Linfático Centinela , Espectrometría Raman , Humanos , Neoplasias de la Mama/cirugía , Neoplasias de la Mama/patología , Femenino , Espectrometría Raman/métodos , Ganglio Linfático Centinela/patología , Ganglio Linfático Centinela/cirugía , Biopsia del Ganglio Linfático Centinela/métodos , Persona de Mediana Edad , Metástasis Linfática/patología , Anciano , Curva ROC , Sensibilidad y Especificidad , Adulto , Imagen Óptica/métodos
2.
Br J Dermatol ; 191(3): 428-436, 2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-38736216

RESUMEN

BACKGROUND: Autofluorescence (AF)-Raman microspectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh, surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 min. OBJECTIVES: To determine the accuracy of the AF-Raman instrument in detecting incomplete BCC excisions during Mohs micrographic surgery (MMS), using histology as the reference standard. METHODS: Skin layers from 130 patients undergoing MMS at the Nottingham University Hospitals NHS Trust (September 2022-July 2023) were investigated with the AF-Raman instrument. The layers were measured when fresh, immediately after excision. The AF-Raman results and the intraoperative assessment by Mohs surgeons were compared with a postoperative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, and positive and negative predictive values were calculated. The study was registered with ClinicalTrials.gov (NCT03482622). RESULTS: AF-Raman analysis was successfully completed for 125 of 130 layers and, on average, covered 91% of the specimen surface area, with the lowest surface area covered being 87% for the eyelid and the highest being 94% for forehead specimens. The AF-Raman instrument identified positive margins in 24 of 36 BCC-positive cases [67% sensitivity, 95% confidence interval (CI) 49-82] and negative margins in 65 of 89 BCC-negative cases (73% specificity, 95% CI 63-82). Only one of 12 false-negative cases was caused by misclassification by the AF-Raman algorithm. The other 11 false-negatives cases were a result of no valid Raman signal being recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and the cassette window. The intraoperative diagnosis by Mohs surgeons identified positive margins in 31 of 36 BCC-positive cases (86% sensitivity, 95% CI 70-95) and negative margins in 79 of 89 BCC-negative cases (89% specificity, 95% CI 81-95). CONCLUSIONS: The AF-Raman instrument has the potential to provide intraoperative microscopic assessment of surgical margins in BCC surgery. Further improvements are required for tissue processing, to ensure complete coverage of the surgical specimens.


Basal cell carcinoma (BCC) is one of the most common human cancers, occurring mostly on the face and neck. Most BCCs are treated by cutting them out under local anaesthetic. This is routinely done in a hospital by a dermatologist or plastic surgeon. Surgery aims to remove all the cancer leaving the smallest scar possible, but it is often difficult to know how much normal skin to remove. Results from the laboratory often take 1 to 2 weeks to show if the cancer is clear. A technique called 'Mohs' (micrographic surgery) is recommended for these 'high-risk' BCCs. Mohs surgery removes thin layers of skin and investigates them under a microscope while the patient is still in the hospital. This is repeated until all the layers are clear of cancer. Because of the patchy availability of Mohs surgery, many patients with high-risk BCCs are treated by traditional methods that may not be as good as Mohs. We have developed an instrument that scans layers of skin and can quickly detect BCC. The instrument allows surgeons to check each removed skin layer for cancer cells to decide if more layers need to be removed. In this study, the instrument was tested on skin tissue layers from 130 patients who had Mohs surgery at the Nottingham Treatment Centre. The results showed that the instrument can measure skin layers in approximately 30 minutes and identify BCC with a similar accuracy to a Mohs surgeon, but only when the skin layers are prepared properly. With future improvements, the technology might be used to guide Mohs surgery or help surgeons in centres that do not have access to Mohs surgery.


Asunto(s)
Carcinoma Basocelular , Márgenes de Escisión , Cirugía de Mohs , Neoplasias Cutáneas , Femenino , Humanos , Masculino , Carcinoma Basocelular/cirugía , Carcinoma Basocelular/patología , Carcinoma Basocelular/diagnóstico , Neoplasia Residual/patología , Imagen Óptica/métodos , Imagen Óptica/normas , Sensibilidad y Especificidad , Piel/patología , Neoplasias Cutáneas/cirugía , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/diagnóstico , Espectrometría Raman/métodos
3.
J Surg Res ; 288: 10-20, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-36940563

RESUMEN

INTRODUCTION: Identifying colorectal liver metastases (CRLM) during liver resection could assist in achieving clear surgical margins, which is an important prognostic variable for both disease-free and overall survival. The aim of this study was to investigate the effect of auto-fluorescence (AF) and Raman spectroscopy for ex vivo label-free discrimination of CRLMs from normal liver tissue. Secondary aims include exploring options for multimodal AF-Raman integration with respect to diagnosis accuracy and imaging speed on human liver tissue and CRLM. METHODS: Liver samples were obtained from patients undergoing liver surgery for CRLM who provided informed consent (15 patients were recruited). AF and Raman spectroscopy was performed on CRLM and normal liver tissue samples and then compared to histology. RESULTS: AF emission spectra demonstrated that the 671 nm and 775/785 nm excitation wavelengths provided the highest contrast, as normal liver tissue elicited on average around eight-fold higher AF intensity compared to CRLM. The use of the 785 nm wavelength had the advantage of enabling Raman spectroscopy measurements from CRLM regions, allowing discrimination of CRLM from regions of normal liver tissue eliciting unusual low AF intensity, preventing misclassification. Proof-of-concept experiments using small pieces of CRLM samples covered by large normal liver tissue demonstrated the feasibility of a dual-modality AF-Raman for detection of positive margins within few minutes. CONCLUSIONS: AF imaging and Raman spectroscopy can discriminate CRLM from normal liver tissue in an ex vivo setting. These results suggest the potential for developing integrated multimodal AF-Raman imaging techniques for intraoperative assessment of surgical margins.


Asunto(s)
Neoplasias Colorrectales , Neoplasias Hepáticas , Humanos , Espectrometría Raman , Márgenes de Escisión , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/cirugía , Hepatectomía
4.
Analyst ; 146(12): 3799-3809, 2021 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-34042924

RESUMEN

One of the main challenges in cancer surgery is to ensure the complete excision of the tumour while sparing as much healthy tissue as possible. Histopathology, the gold-standard technique used to assess the surgical margins on the excised tissue, is often impractical for intra-operative use because of the time-consuming tissue cryo-sectioning and staining, and availability of histopathologists to assess stained tissue sections. Raman micro-spectroscopy is a powerful technique that can detect microscopic residual tumours on ex vivo tissue samples with accuracy, based entirely on intrinsic chemical differences. However, raster-scanning Raman micro-spectroscopy is a slow imaging technique that typically requires long data acquisition times wich are impractical for intra-operative use. Selective-sampling Raman imaging overcomes these limitations by using information regarding the spatial properties of the tissue to reduce the number of Raman spectra. This paper reviews the latest advances in selective-sampling Raman techniques and applications, mainly based on multimodal optical imaging. We also highlight the latest results of clinical integration of a prototype device for non-melanoma skin cancer. These promising results indicate the potential impact of Raman spectroscopy for providing fast and objective assessment of surgical margins, helping surgeons ensure the complete removal of tumour cells while sparing as much healthy tissue as possible.


Asunto(s)
Márgenes de Escisión , Espectrometría Raman , Diagnóstico por Imagen , Humanos , Neoplasia Residual
5.
Biophys J ; 117(9): 1589-1598, 2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31587827

RESUMEN

Cellular plasma membrane deformability and stability is important in a range of biological processes. Changes in local curvature of the membrane affect the lateral movement of lipids, affecting the biophysical properties of the membrane. An integrated holographic optical tweezers and Raman microscope was used to investigate the effect of curvature gradients induced by optically stretching individual giant unilamellar vesicles (GUVs) on lipid packing and lateral segregation of cholesterol in the bilayer. The spatially resolved Raman analysis enabled detection of induced phase separation and changes in lipid ordering in individual GUVs. Using deuterated cholesterol, the changes in lipid ordering and phase separation were linked to lateral sorting of cholesterol in the stretched GUVs. Stretching the GUVs in the range of elongation factors 1-1.3 led to an overall decrease in cholesterol concentration at the edges compared to the center of stretched GUVs. The Raman spectroscopy results were consistent with a model of the bilayer accounting for cholesterol sorting in both bilayer leaflets, with a compositional asymmetry of 0.63 ± 0.04 in favor of the outer leaflet. The results demonstrate the potential of the integrated holographic optical tweezers-Raman technique to induce deformations to individual lipid vesicles and to simultaneously provide quantitative and spatially resolved molecular information. Future studies can extend to include more realistic models of cell membranes and potentially live cells.


Asunto(s)
Membrana Dobles de Lípidos/química , Espectrometría Raman , Liposomas Unilamelares/química , Colesterol/química , Pinzas Ópticas
6.
Breast Cancer Res ; 20(1): 69, 2018 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-29986750

RESUMEN

BACKGROUND: In over 20% of breast conserving operations, postoperative pathological assessment of the excised tissue reveals positive margins, requiring additional surgery. Current techniques for intra-operative assessment of tumor margins are insufficient in accuracy or resolution to reliably detect small tumors. There is a distinct need for a fast technique to accurately identify tumors smaller than 1 mm2 in large tissue surfaces within 30 min. METHODS: Multi-modal spectral histopathology (MSH), a multimodal imaging technique combining tissue auto-fluorescence and Raman spectroscopy was used to detect microscopic residual tumor at the surface of the excised breast tissue. New algorithms were developed to optimally utilize auto-fluorescence images to guide Raman measurements and achieve the required detection accuracy over large tissue surfaces (up to 4 × 6.5 cm2). Algorithms were trained on 91 breast tissue samples from 65 patients. RESULTS: Independent tests on 121 samples from 107 patients - including 51 fresh, whole excision specimens - detected breast carcinoma on the tissue surface with 95% sensitivity and 82% specificity. One surface of each uncut excision specimen was measured in 12-24 min. The combination of high spatial-resolution auto-fluorescence with specific diagnosis by Raman spectroscopy allows reliable detection even for invasive carcinoma or ductal carcinoma in situ smaller than 1 mm2. CONCLUSIONS: This study provides evidence that this multimodal approach could provide an objective tool for intra-operative assessment of breast conserving surgery margins, reducing the risk for unnecessary second operations.


Asunto(s)
Neoplasias de la Mama/cirugía , Carcinoma Ductal de Mama/cirugía , Carcinoma Intraductal no Infiltrante/cirugía , Mastectomía Segmentaria , Adulto , Mama/fisiopatología , Mama/cirugía , Neoplasias de la Mama/fisiopatología , Carcinoma Ductal de Mama/fisiopatología , Carcinoma Intraductal no Infiltrante/fisiopatología , Femenino , Humanos , Márgenes de Escisión , Persona de Mediana Edad , Neoplasia Residual/fisiopatología , Neoplasia Residual/cirugía , Espectrometría Raman
7.
Opt Express ; 26(19): 25211-25225, 2018 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-30469626

RESUMEN

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.


Asunto(s)
Bacterias/citología , Células Dendríticas/citología , Holografía/instrumentación , Pinzas Ópticas , Espectrometría Raman/instrumentación , Linfocitos T/citología , Diseño de Equipo , Luz
8.
Opt Lett ; 43(23): 5733-5736, 2018 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-30499980

RESUMEN

We present a time-gated Raman micro-spectroscopy technique suitable for fast Raman mapping of samples eliciting large laser-induced fluorescence backgrounds. To achieve the required time resolution for effective fluorescence rejection, a picosecond pulsed laser and a single-photon avalanche diode were used. A module consisting of a spectrometer, digital micromirror device, and two prisms was used for high-resolution spectral filtering and multiplexing, which is required for a high chemical specificity and short integration times. With this instrument, we demonstrated time-gated Raman imaging of highly fluorescent samples, achieving acquisition times as short as 3 min for 40×40 pixel resolution images.

9.
Anal Chem ; 89(1): 847-853, 2017 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-27983789

RESUMEN

We investigated the feasibility of using spatially offset Raman spectroscopy (SORS) for nondestructive characterization of bone tissue engineering scaffolds. The deep regions of these scaffolds, or scaffolds implanted subcutaneously in live animals, are typically difficult to measure by confocal Raman spectroscopy techniques because of the limited depth penetration of light caused by the high level of light scattering. Layered samples consisting of bioactive glass foams (IEIC16), three-dimensional (3D)-printed biodegradable poly(lactic-co-glycolic acid) scaffolds (PLGA), and hydroxyapatite powder (HA) were used to mimic nondestructive detection of biomineralization for intact real-size 3D tissue engineering constructs. SORS spectra were measured with a new SORS instrument using a digital micromirror device (DMD) to allow software selection of the spatial offsets. The results show that HA can be reliably detected at depths of 0-2.3 mm, which corresponds to the maximum accessible spatial offset of the current instrument. The intensity ratio of Raman bands associated with the scaffolds and HA with the spatial offset depended on the depth at which HA was located. Furthermore, we show the feasibility for in vivo monitoring mineralization of scaffold implanted subcutaneously by demonstrating the ability to measure transcutaneously Raman signals of the scaffolds and HA (fresh chicken skin used as a top layer). The ability to measure spectral depth profiles at high speed (5 s acquisition time) and the ease of implementation make SORS a promising approach for noninvasive characterization of cell/tissue development in vitro, and for long-term in vivo monitoring the mineralization in 3D scaffolds subcutaneously implanted in small animals.


Asunto(s)
Huesos/química , Durapatita/análisis , Ingeniería de Tejidos , Materiales Biocompatibles/análisis , Ácido Láctico/química , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Espectrometría Raman
10.
Opt Express ; 24(12): 12701-12, 2016 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-27410290

RESUMEN

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.

11.
Faraday Discuss ; 187: 199-212, 2016 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-27023675

RESUMEN

Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences.


Asunto(s)
Microscopía de Fuerza Atómica , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Espectrometría Raman , Animales , Análisis de los Mínimos Cuadrados , Ratones , Células-Madre Neurales/química , Análisis de Componente Principal , Proteínas/análisis , ARN/análisis
13.
Proc Natl Acad Sci U S A ; 110(38): 15189-94, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-24003124

RESUMEN

Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1-2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.


Asunto(s)
Técnicas de Diagnóstico Quirúrgico , Microscopía/métodos , Neoplasias/diagnóstico , Imagen Óptica/métodos , Espectrometría Raman/métodos , Técnicas Histológicas/métodos , Humanos , Neoplasias/patología
14.
Analyst ; 140(3): 756-64, 2015 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-25422831

RESUMEN

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.


Asunto(s)
Lípidos/análisis , Epitelio Pigmentado Ocular/metabolismo , Proteínas Protozoarias/metabolismo , Espectrometría Raman/métodos , Toxoplasma/fisiología , Toxoplasmosis/metabolismo , Células Cultivadas , Fluorescencia , Interacciones Huésped-Parásitos , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen Molecular , Epitelio Pigmentado Ocular/parasitología , Epitelio Pigmentado Ocular/patología , Análisis de Componente Principal , Toxoplasmosis/parasitología , Toxoplasmosis/patología
15.
Biochim Biophys Acta ; 1830(6): 3517-24, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23403134

RESUMEN

BACKGROUND: Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation. METHODS: EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals. RESULTS: The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm(-1), 1083cm(-1), 937cm(-1), 858cm(-1), 577cm(-1) and 482cm(-1). The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs. CONCLUSIONS: The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation. GENERAL SIGNIFICANCE: As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias , Miocitos Cardíacos , Espectrometría Raman/métodos , Actinina/biosíntesis , Antígenos de Diferenciación/biosíntesis , Técnicas de Cultivo de Célula , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo
16.
Analyst ; 139(1): 55-8, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24153382

RESUMEN

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.


Asunto(s)
Densidad Ósea/fisiología , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/fisiología , Espectrometría Raman/métodos , Tamaño de la Célula , Células Cultivadas , Humanos , Factores de Tiempo
17.
Appl Spectrosc ; 77(3): 246-260, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36320126

RESUMEN

Quantitative analysis of drug delivery with in biological systems is an integral challenge in drug development. Analytical techniques are important for assessing both drug target delivery, target action, and drug toxicology. Using mimetic tissue models, we have investigated the efficacy of Raman spectroscopy in quantitative detection of alkyne group and deuterated drugs in rat brain and rat liver tissue models. Lasers with 671 nm and 785 nm wavelengths were assessed for their feasibility in this application due to opposing relative benefits and disadvantages. Thin tissue sections have been tested as a practical means of reducing autofluorescent background by minimizing out-of-focus tissue and therefore maximizing photobleaching rates. Alkyne-tagged drugs were quantitatively measured at 18 ± 5 µg/g drug/tissue mass ratio in rat brain and at 34 ± 6 µg/g in rat liver. Quantification calibration curves were generated for a range of concentrations from 0-500 µg/g. These results show the potential of Raman spectroscopy as a diffraction-limited spatially resolved imaging technique for assessing drug delivery in tissue applications.


Asunto(s)
Hígado , Espectrometría Raman , Preparaciones Farmacéuticas , Espectrometría Raman/métodos , Encéfalo , Alquinos
18.
Biomed Opt Express ; 14(12): 6592-6606, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-38420302

RESUMEN

Diffuse Raman spectroscopy (DRS) allows subsurface molecular analysis of optically turbid samples. Numerical modeling of light propagation was used as a method for improving the design of an DRS instrument to maximize the signal to noise ratio (SNR) while ensuring safe laser exposure parameters required for in-vivo measurements. Experimental validation of the model was performed on both phantom samples and disks implanted postmortem to mimic the typical response to foreign bodies (formation of a fibrotic capsule around an implant). A reduction of laser exposure of over 1500-fold was achieved over previous studies whilst maintaining the same Raman collection rates and reaching the safe power density of 3 mW/mm2. The validation of this approach in a subcutaneous implant in a mouse cadaver showed a further improvement of 1.5-fold SNR, with a thickness limit of detection for the fibrotic layer of 23 µm, under the same acquisition times. In the animal body, a thickness limit of detection of 16 µm was achieved. These results demonstrate the feasibility of numerical model-based optimization for DRS, and that the technique can be improved sufficiently to be used for in-vivo measurement of collagenous capsule formation as a result of the foreign body response in murine models.

19.
Anal Chem ; 84(7): 3155-62, 2012 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-22436054

RESUMEN

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.


Asunto(s)
Diferenciación Celular , Citoplasma/metabolismo , Células-Madre Neurales/citología , ARN/metabolismo , Espectrometría Raman/métodos , Animales , Supervivencia Celular , Análisis Discriminante , Ratones , Análisis Multivariante , Neuroglía/citología , Fenotipo , Análisis de Componente Principal
20.
Opt Lett ; 37(12): 2256-8, 2012 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-22739873

RESUMEN

We report a new approach in tip-enhanced Raman spectroscopy (TERS) in which TERS-active tips with enhancement factors of ∼10(-5)× can be rapidly (1-3 min) produced in situ by laser-induced synthesis of silver nanoparticles at the tip apex. The technique minimizes the risks of tip contamination and damage during handling and provides in situ feedback control, which allows the prediction of the tip performance. We show that TERS tips produced by this technique enable the measurement of spatially resolved TERS spectra of self-assembled peptide nanotubes with a spatial resolution of ∼20 nm.


Asunto(s)
Rayos Láser , Nanopartículas del Metal/química , Nanotecnología/métodos , Nanotubos de Péptidos/química , Plata/química , Espectrometría Raman/métodos , Dipéptidos , Microscopía de Fuerza Atómica , Fenilalanina/análogos & derivados , Fenilalanina/química
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