Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

Bonnier, Franck; Knief, Peter; Lim, Basil; Meade, Aidan D.; Dorney, Jennifer; Bhattacharya, Kunal; Lyng, Fiona M.; Byrne, Hugh J.

doi:10.1039/c0an00539h

Cited by 59 publications

(84 citation statements)

References 48 publications

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“…The specific information contained in the Raman spectra can be related to changes to the physiology as a result of external stimuli [27][28][29][30]. The spatial resolution is of the order of 0.5-2 mm, providing access to the subcellular organisation of the cells at a molecular level [31][32][33][34][35]. In the case of FTIR spectroscopy, the spatial resolution is limited to some 5-10 mm in free space applications, although this can be reduced to 1-2 mm using high numerical aperture microscopic objectives, as in the case of Attenuated Total Reflection (ATR) FTIR microspectroscopy.…”

Section: Biophotonicsmentioning

confidence: 99%

“…Whereas dry samples are often required for FTIR analysis of tissue sections, Raman microspectroscopy is perfectly compatible with water immersion measurement which enhances the signal to background ratio and the collection of higher quality data [36,37]. The weak contribution from water offers the possibility to study cells in an aqueous environment and thus to keep them alive for the duration of the measurement [2,31,38], whereas the strong absorption from water means that such measurements in infrared can only be performed with high brightness sources such as synchrotrons [39].…”

Section: Biophotonicsmentioning

confidence: 99%

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Improved protocols for vibrational spectroscopic analysis of body fluids

Bonnier¹,

Petitjean

Baker

et al. 2013

Journal of Biophotonics

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The applications of vibrational spectroscopy to the examination of human blood serum are explored. Although FTIR spectra can be recorded in aqueous solutions at (gelatin) concentrations as low as 100 mg/L, the high-wavenumber region remains obscured by water absorption. Using Raman spectroscopy, high quality spectra of gelatine solutions as low as 10 mg/L can be achieved, also covering the high-wavenumber regions. In human serum, spectral profiles are weak and partially obscured by water features. Dried deposits are shown to be physically and chemically inhomogeneous resulting in reduced measurement reproducibility. Concentration of the serum using commercially available centrifugal filter devices results in an improvement in the spectral intensity and quality. Additionally, in Raman spectroscopy, reduced background and significantly enhanced signal collection is achievable by measurement in an inverted geometry. The improved protocols for spectroscopic measurement of human serum are applicable to a range of bodily fluids and should accelerate potential clinical applications

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Section: Biophotonicsmentioning

confidence: 99%

Section: Biophotonicsmentioning

confidence: 99%

Improved protocols for vibrational spectroscopic analysis of body fluids

Bonnier¹,

Petitjean

Baker

et al. 2013

Journal of Biophotonics

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show abstract

“…In principle, each principal component is a linear combination of the original variables for the simplified variables, therefore, var (PC1) ≥ var (PC2) ≥ (PCp), where var (PCi) represents the variance of PCi in the dataset. A scatter plot is generated from the data which shows groups of points representing variations within the dataset [25]. PCA was performed using Matlab (Mathworks, USA) following the different data analysis steps described in the previous section.…”

Section: Principal Component Analysismentioning

confidence: 99%

Assessment of an osteoblast-like cell line as a model for human primary osteoblasts using Raman spectroscopy

McManus¹,

Bonnier²,

Burke³

et al. 2012

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Raman spectroscopy is employed to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts, with emphasis on the ability of these cell types to replicate their tissue of origin. It was found that both cell types demonstrated early stage mineral deposition that followed significantly different growth patterns. Analysis of the growth pattern and spectral data from primary cells revealed increasing bone quality ratios and a high crystallinity, consistent with previous reports. Conversely the investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone in terms of its crystallinity and bone quality ratios. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently described them as non-osteoinductive when cultured in various conditions on a number of substrates. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analysis for the investigation of osteoblast-like U20S cells and human primary osteoblasts, specifically with focus on the osteoinductive ability of the osteoblast-like cell line and the comparative differences in relation to the primary osteoblasts.

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“…Furthermore, confocal operation is not available on many commercial spectrometers. Recent studies have demonstrated the benefits of using 3D collagen gels for Raman mapping of single live cells [39]. The substrate (collagen) contribution to the spectrum is shown to be negligible, reducing the requirement for substrate subtraction.…”

Section: Spectral Preprocessing: Raman Spectroscopymentioning

confidence: 99%

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells

et al. 2016

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Vibrational Spectroscopy, both infrared absorption and Raman spectroscopy, have attracted increasing attention for biomedical applications, from in vivo and ex vivo disease diagnostics and screening, to in vitro screening of therapeutics. There remain, however, many challenges related to the accuracy of analysis of physically and chemically inhomogeneous samples, across heterogeneous sample sets. Data preprocessing is required to deal with variations in instrumental responses and intrinsic spectral backgrounds and distortions in order to extract reliable spectral data. Data postprocessing is required to extract the most reliable information from the sample sets, based on often very subtle changes in spectra associated with the targeted pathology or biochemical process. This review presents the current understanding of the factors influencing the quality of spectra recorded and the pre-processing steps commonly employed to improve on spectral quality. It further explores some of the most common techniques which have emerged for classification and analysis of the spectral data for biomedical applications. The importance of sample presentation and measurement conditions to yield the highest quality spectra in the first place is emphasised, as is the potential of model simulated datasets to validate both pre-and post-processing protocols.

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Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

Cited by 59 publications

References 48 publications

Improved protocols for vibrational spectroscopic analysis of body fluids

Improved protocols for vibrational spectroscopic analysis of body fluids

Assessment of an osteoblast-like cell line as a model for human primary osteoblasts using Raman spectroscopy

Spectral pre and post processing for infrared and Raman spectroscopy of biological tissues and cells

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