Name: applying hyperspectral reflectance imaging to investigate the palettes and the techniques of painters
Uploaded: 2021-06-18T13:00:00
Description: Watch this Scientific Journal Video about Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters at JoVE.com

This research was funded by Regione Lombardia in the framework of the Project MOBARTECH: una piattaforma mobile tecnologica, interattiva e partecipata per lo studio, la conservazione e la valorizzazione di beni storico-artistici - Call Accordi per la Ricerca e l&#39;Innovazione.
The authors are grateful to the staff at Museo del Novecento for the support during the&#160;in situ&#160;experimental sessions and to the Associazione Pellizza da Volpedo for the access to Studio Museo.

1. Set the spatial resolution of the hypercubes
<ol>
	<li>Perform a preliminary inspection of the painted surface (Figure 1) supported by art experts to identify the main features of the painting.
	<ol>
		<li>Recognize the pictorial techniques employed by the artist to create the painting.</li>
		<li>Identify the different brush strokes of paint on the canvas.</li>
		<li>Estimate, qualitatively, the characteristics of the brush strokes with particular attention to their size.</li>
	</ol>
	...

<ol>
	<li>Picollo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+Optics+Reflectance+Spectroscopy:+a+non-destructive+technique+for+the+analysis+of+works+of+art.">Fiber Optics Reflectance Spectroscopy: a non-destructive technique for the analysis of works of art.</a> Optical Sensors and Microsystems. , 259-265 (2002).</li><li>Bacci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-destructive+spectroscopic+investigations+on+paintings+using+optical+fibers.">Non-destructive spectroscopic investigations on paintings using optical fibers.</a> MRS Online Proceedings Library Archive. 267, (1992).</li><li>Liang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+multispectral+and+hyperspectral+imaging+for+archaeology+and+art+conservation.">Advances in multispectral and hyperspectral imaging for archaeology and art conservation.</a> Applied Physics A. 106, 309-323 (2012).</li><li>Bullock, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectance+spectrophotometry+for+measurement+of+colour+change.">Reflectance spectrophotometry for measurement of colour change.</a> National Gallery Technical Bulletin. 2, 49-55 (1978).</li><li>Saunders, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colour+change+measurement+by+digital+image+processing.">Colour change measurement by digital image processing.</a> National Gallery Technical Bulletin. , 66-77 (1988).</li><li>Appolonia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+use+of+FORS,+XRF+and+Raman+spectroscopy+in+the+study+of+mural+paintings+in+the+Aosta+Valley+(Italy).">Combined use of FORS, XRF and Raman spectroscopy in the study of mural paintings in the Aosta Valley (Italy).</a> Analytical and Bioanalytical Chemistry. 395, 2005-2013 (2009).</li><li>Pouyet, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+Pablo+Picasso's+La+Mis&#233;reuse+accroupie+using+X-ray+fluorescence+imaging+and+reflectance+spectroscopies+combined+with+micro-analyses+of+samples.">New insights into Pablo Picasso's La Mis&#233;reuse accroupie using X-ray fluorescence imaging and reflectance spectroscopies combined with micro-analyses of samples.</a> SN Applied Sciences. 2, 1-6 (2020).</li><li>Garofano, I., Perez-Rodriguez, J. L., Robador, M. D., Duran, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+combination+of+non-invasive+UV-Visible-FORS,+XRD+and+XRF+techniques+to+study+Roman+wall+paintings+from+Seville,+Spain.">An innovative combination of non-invasive UV-Visible-FORS, XRD and XRF techniques to study Roman wall paintings from Seville, Spain.</a> Journal of Cultural Heritage. 22, 1028-1039 (2016).</li><li>Dupuis, G., Elias, M., Simonot, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigment+identification+by+fiber-optics+diffuse+reflectance+spectroscopy.">Pigment identification by fiber-optics diffuse reflectance spectroscopy.</a> Applied Spectroscopy. 56, 1329-1336 (2002).</li><li>Bacci, M., Picollo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-destructive+spectroscopic+detection+of+cobalt+(II)+in+paintings+and+glass.">Non-destructive spectroscopic detection of cobalt (II) in paintings and glass.</a> Studies in Conservation. 41, 136-144 (1996).</li><li>Cosentino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FORS+spectral+database+of+historical+pigments+in+different+binders.">FORS spectral database of historical pigments in different binders.</a> E-Conservation Journal. , 54-65 (2014).</li><li>Leona, M., Winter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+optics+reflectance+spectroscopy:+a+unique+tool+for+the+investigation+of+Japanese+paintings.">Fiber optics reflectance spectroscopy: a unique tool for the investigation of Japanese paintings.</a> Studies in Conservation. 46, 153-162 (2001).</li><li>Cheilakou, E., Troullinos, M., Koui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pigments+on+Byzantine+wall+paintings+from+Crete+(14th+century+AD)+using+non-invasive+Fiber+Optics+Diffuse+Reflectance+Spectroscopy+(FORS).">Identification of pigments on Byzantine wall paintings from Crete (14th century AD) using non-invasive Fiber Optics Diffuse Reflectance Spectroscopy (FORS).</a> Journal of Archaeological Science. 41, 541-555 (2014).</li><li>Kubik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+imaging:+a+new+technique+for+the+non-invasive+study+of+artworks.">Hyperspectral imaging: a new technique for the non-invasive study of artworks.</a> Physical Techniques in the Study of Art, Archaeology and Cultural. 2, 199-259 (2007).</li><li>Fischer, C., Kakoulli, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+and+hyperspectral+imaging+technologies+in+conservation:+current+research+and+potential+applications.">Multispectral and hyperspectral imaging technologies in conservation: current research and potential applications.</a> Studies in Conservation. 51, 3-16 (2006).</li><li>Daniel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+imaging+applied+to+the+analysis+of+Goya+paintings+in+the+Museu+of+Zaragoza+(Spain).">Hyperspectral imaging applied to the analysis of Goya paintings in the Museu of Zaragoza (Spain).</a> Microchemical Journal. 126, 113-120 (2016).</li><li>Baronti, S., Casini, A., Lotti, F., Porcinai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principal+component+analysis+of+visible+and+near-infrared+multispectral+images+of+works+of+art.">Principal component analysis of visible and near-infrared multispectral images of works of art.</a> Chemometrics and Intelligent Laboratory Systems. 39, 103-114 (1997).</li><li>Mansfield, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+spectroscopic+reflectance+imaging:+supervised+vs.+unsupervised+analysis+using+an+art+conservation+application.">Near infrared spectroscopic reflectance imaging: supervised vs. unsupervised analysis using an art conservation application.</a> Vibrational Spectroscopy. 19, 33-45 (1999).</li><li>Clodius, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+and+Hyperspectral+Image+Processing,+Part+1:+Initial+Processing.">Multispectral and Hyperspectral Image Processing, Part 1: Initial Processing.</a> Encyclopedia of Optical Engineering: Las-Pho. 2, 1390 (2003).</li><li>Kruse, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectral+image+processing+system+(SIPS)-interactive+visualization+and+analysis+of+imaging+spectrometer+data.">The spectral image processing system (SIPS)-interactive visualization and analysis of imaging spectrometer data.</a> AIP Conference Proceedings. , (1993).</li><li>Yang, C., Everitt, J. H., Bradford, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yield+estimation+from+hyperspectral+imagery+using+spectral+angle+mapper+(SAM).">Yield estimation from hyperspectral imagery using spectral angle mapper (SAM).</a> Transactions of the ASABE. 51, 729-737 (2008).</li><li>Delaney, J. K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+X-ray+fluorescence+and+diffuse+visible-to-near-infrared+reflectance+scanner+for+standoff+elemental+and+molecular+spectroscopic+imaging+of+paints+and+works+on+paper.">Integrated X-ray fluorescence and diffuse visible-to-near-infrared reflectance scanner for standoff elemental and molecular spectroscopic imaging of paints and works on paper.</a> Heritage Science. 6, 1-12 (2018).</li><li>Richards, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Remote sensing digital image analysis. 3, (1999).</li><li>Mobaraki, N., Amigo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HYPER-Tools.+A+graphical+user-friendly+interface+for+hyperspectral+image+analysis.">HYPER-Tools. A graphical user-friendly interface for hyperspectral image analysis.</a> Chemometrics and Intelligent Laboratory Systems. 172, 174-187 (2018).</li><li>Zhu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+hyperspectral+imaging+system+for+rapid+detection+of+microplastics+down+to+100+&#181;m.">Optimization of a hyperspectral imaging system for rapid detection of microplastics down to 100 &#181;m.</a> MethodsX. 8, 101175 (2021).</li><li>Behmann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specim+IQ:+evaluation+of+a+new,+miniaturized+handheld+hyperspectral+camera+and+its+application+for+plant+phenotyping+and+disease+detection.">Specim IQ: evaluation of a new, miniaturized handheld hyperspectral camera and its application for plant phenotyping and disease detection.</a> Sensors. 18, 441 (2018).</li><li>Spectral and XYZ Color Functions. MATLAB Central File Exchange Available from: <a target="_blank" href="https://www.mathworks.com/matlabcentral/fileexchange/7021-spectral-and-xyz-color-functions">https://www.mathworks.com/matlabcentral/fileexchange/7021-spectral-and-xyz-color-functions</a> (2021)</li><li>Chen, C. -. Y., Klette, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+stitching-Comparisons+and+new+techniques.">Image stitching-Comparisons and new techniques.</a> International Conference on Computer Analysis of Images and Patterns. , (1999).</li><li>read_envihdr. MATLAB Central File Exchange Available from: <a target="_blank" href="https://www.mathworks.com/matlabcentral/fileexchange/38500-read_envihdr">https://www.mathworks.com/matlabcentral/fileexchange/38500-read_envihdr</a> (2021)</li><li>Jolliffe, I. T., Cadima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principal+component+analysis:+a+review+and+recent+developments.">Principal component analysis: a review and recent developments.</a> Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences. 374, 20150202 (2016).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9, 671-675 (2012).</li><li>Rinnan, &. #. 1. 9. 7. ;., Van Den Berg, F., Engelsen, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+most+common+pre-processing+techniques+for+near-infrared+spectra.">Review of the most common pre-processing techniques for near-infrared spectra.</a> Trends in Analytical Chemistry. 28, 1201-1222 (2009).</li></ol>

Hyperspectral reflectance imaging datasets are large reservoirs of information; therefore, the development of robust and, possibly, automated protocols to analyze the data is a key turn to exploit their potential15,17. The proposed algorithm answers this need in the field of cultural heritage with particular attention to the characterization of the pigments of paintings. Based on SAM20,21, the algorithm s...

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are based on the detection of the light reflected by surfaces once illuminated by a light source, typically a tungsten-halogen lamp. The output of the acquisition system is constituted by spectra where the reflectance is monitored as a function of the wavelength in a range that depends on the characteristics of the employed experimental setup1,2,3. Introduced during the last four decades4,5, RS and FORS are typically used in combination with X-ray fluorescence and other spectroscopies to describe the materials and the techniques used by artists to realize their masterpieces6,7,8,9. The study of the reflectance spectra is usually performed by comparing the data from the sample with a group of reference spectra selected by the user in personal or public databases. Once the reference spectra that comply with the realization period of the sample and with the&#160;modus operandi&#160;of the artist have been identified, the user recognizes the main features of the reflectance spectra (i.e., transition, absorption, and reflection bands1,2,10,11) and then, with the help of other techniques6,7,8 they distinguish&#160;the pigments that have been used in the paintings. Finally they discusses the slight differences that there exist between the references and the experimental spectra7,9.
In most cases, the experimental datasets are composed of a few spectra, collected from areas chosen by art experts and assumed to be significant for the characterization of the painting6, 12,13. Despite the skills and the experience of the user, a few spectra cannot fully exhaust the characteristics of the whole painted surface. Moreover, the result of the analysis will always be strongly dependent on the expertise of the performer. In this scenario, Hyperspectral Reflectance Imaging (HRI3,14,15) could be a useful resource. Instead of a few isolated spectra, the experimental setups return the reflectance properties of extended portions or even of the whole artefact under investigation16. The two main advantages with respect to the acquisition of the isolated spectra are evident. On one hand, the availability of the spatial distribution of the reflectance properties allows the identification of areas that hide interesting features, even though they may not seem peculiar17. On the other hand, the hypercubes guarantee a number of spectra high enough to enable the statistical analysis of the data. These facts support the comprehension of the distribution of pigments within the painted surface18,19.
With HRI, the comparison of the experimental data with the references could be hard to handle15. A typical detector returns hypercubes of at least 256 x 256 spectra. This would require the user to evaluate more than 65,000 reflectance spectra against each reference, a task almost impossible to be carried out manually in a reasonable time. Therefore, the request for robust and, possibly, automated protocols to manage and analyze HRI datasets is more than justified15,17. The proposed method answers this need by handling the whole analytic procedure with the minimum involvement and the maximum flexibility.
An algorithm comprising a set of home-made codes (Table of Materials) reads, manages, and organizes the files returned by the experimental setup. It allows the fine selection of the portions of the Fields of View (FOVs, one field of view is the area of the painting monitored by a single hypercube) to be studied and performs the analysis of the data based on the Spectral Angle Mapper (SAM) method20,21 and on the manipulation of the original spectra. SAM returns false color gray-scale images called similarity maps. The values of the pixels of these maps correspond to the spectral angles that are the angles between the spectra stored in the hypercubes&#160;and the so-called End Members (EMs, a group of reference spectra that should describe the features of the surface monitored by the hypercubes)22. In the case of RS applied to paintings, the EMs are the reflectance spectra of pigments that should match the palette of the Master. They are chosen based on the available information about the artist, the realization period of the painting, and the expertise of the user. Therefore, the output of the SAM is a set of maps that describes the spatial distributions of these pigments over the painting surface and that supports the user to infer the materials used by the artist and their organization in the artefact. The algorithm offers the possibility of employing all kind of references independently from their origin. The references can be specific spectra selected within the hypercubes, come from databases, be acquired by a different instrument on a different surface (such as samples of pigments or the palette of the artist, for instance), or be obtained employing any kind of reflectance spectroscopy, FORS included.
SAM has been preferred among the available classification methods because it has been demonstrated to be effective for characterizing pigments (refer to the book by Richard23&#160;to have an overview of the main available classification methods).&#160;Instead, the idea of developing a home-made protocol rather than adopting one of the many tools freely available on the net24,25 relies on a practical consideration. Despite the effectiveness and scientific foundation of the existing GUIs and software, a single tool hardly satisfies all the needs of the user. There could be an Input/Output (I/O) issue because a tool does not manage the file containing the raw data. There could be an issue regarding the analysis of the data because another tool does not provide the desired approach. There could be a limitation in the handling of the data because the simultaneous analysis of multiple datasets is not supported. In any case, a perfect tool does not exist. Each method must be adjusted to the data or vice versa. Therefore, the development of a home-made protocol has been preferred.
The presented approach offers neither a complete set of analytical methods (see, for comparison, the tool proposed by Mobaraki and Amigo24) nor an easy-to-manage user-interface (see, for comparison, the software employed by Zhu and co-workers25), but, in exchange, it focuses on a still underrated aspect of hyperspectral data analysis: the opportunity to manipulate the detected spectra. The power of the approach is illustrated by applying it to the painting Quarto Stato by Giuseppe Pellizza da Volpedo (Figure 1), an iconic oil on canvas held in the Museo del Novecento in Milan, Italy. Note that, since the approach requires running home-made codes, the developer arbitrarily chose the names of the codes and both the input and output variables used in the description of the protocol. The names of the variables can be changed by the user but they must be provided as follow: the input and out variables must be written respectively within brackets and eventually separated by comma and within square brackets and eventually separated by a white space.&#160;On the contrary the names of the codes cannot be altered.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ImageJ/Fiji</td>
			<td>Specim (Oulo, Finlad)</td>
			<td>N/A</td>
			<td>Portable reflectance hyperspectral camera used to acquire the hypercubes</td>
		</tr>
		<tr>
			<td>MATLAB 2019b</td>
			<td>StellarNet Inc (Tampa, Florida, USA)</td>
			<td>N/A</td>
			<td>Portable reflectance spectrometer used to acquire independent reflectance spectra</td>
		</tr>
		<tr>
			<td>Specim IQ Hyperspectral Camera</td>
			<td>National Institutes of Health (Bethesda, Maryland, USA)</td>
			<td>N/A</td>
			<td>&#160;Open source Java image processing program</td>
		</tr>
		<tr>
			<td>StellarNet BLUE-wave Miniature Spectrometer</td>
			<td>&#160;MathWorks (Natick, Massachusset, USA)</td>
			<td>N/A</td>
			<td>Program Language and numerical computing environment</td>
		</tr>
	</tbody>
</table>

applying hyperspectral reflectance imaging to investigate the palettes and the techniques of painters

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with particular attention to paintings. Most modern museums put at the disposal of their research groups portable equipment that, together with the intrinsic non-invasiveness of RS and FORS, makes possible the in situ collection of reflectance spectra from the surface of artefacts. The comparison, performed by experts in pigments and painting materials, of the experimental data with databases of reference spectra drives the characterization of the palettes and of the techniques used by the artists. However, this approach requires specific skills and it is time consuming especially if the number of the spectra to be investigated becomes large as is the case of Hyperspectral Reflectance Imaging (HRI) datasets. The HRI experimental setups are multi-dimensional cameras that associate the spectral information, given by the reflectance spectra, with the spatial localization of the spectra over the painted surface. The resulting datasets are 3D-cubes (called hypercubes or data-cubes) where the first two dimensions locate the spectrum over the painting and the third is the spectrum itself (i.e., the reflectance of that point of the painted surface versus the wavelength in the operative range of the detector). The capability of the detector to simultaneously collect a great number of spectra (typically much more than 10,000 for each hypercube) makes the HRI datasets large reservoirs of information and justifies the need for the development of robust and, possibly, automated protocols to analyze the data. After the description of the procedure designed for the data acquisition, we present an analysis method that systematically exploits the potential of the hypercubes. Based on Spectral Angle Mapper (SAM) and on the manipulation of the collected spectra, the algorithm handles and analyzes thousands of spectra while at the same time it supports&#160;the user to unveil the features of the samples under investigation. The power of the approach is illustrated by applying it to Quarto Stato, the iconic masterpiece by Giuseppe Pellizza da Volpedo, held in the Museo del Novecento in Milan (Italy).

The proposed protocol offers a set of interesting features for the management and the analysis of HRI data. The I/O (step 3.1) of the raw data is always the first problem that must be solved before applying any analysis method and it can become a critical issue when dealing with large amounts of data. In the present case, the only task regarding the raw data is to store the experimental results into a dedicated folder and select it by browsing the hard disk when running the reading code (step 3.1.1). Thereafter, the crop...

Watch this Scientific Journal Video about Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters at JoVE.com

Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters

Hyperspectral Reflectance Imaging hypercubes include remarkable information into a large amount of data. Therefore, the request for automated protocols to manage and study the datasets is widely justified. The combination of Spectral Angle Mapper, data manipulation, and a user-adjustable analysis method constitutes a key-turn for exploring the experimental results.

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with ...

This protocol offers new opportunities involving the analysis of paintings of faces. It supports the users step by step along the whole data analysis process. This protocol has two main advantages.The users can tailor the analysis according to their preferences, and for the first time, spectrum manipulation has been introduced to analyze the hyperspectral data. Hyperspectral reflectance imaging is successfully used to study skin disease or tumor diagnosis. Even if a protocol was born in the counter heritage field it can be applied to clinical health dataset as well.With the support of art experts, perform a preliminary inspection of the painted surface to identify the main features of the painting. Make a note of the pictorial techniques used by the artist, the different brush strokes of paint on the canvas, and qualitatively estimate the characteristics of the brush strokes with particular attention to their size. Create ad hoc samples where the brush strokes show characteristics similar to those applied by the artist by mimicking the pictorial technique used by the artist.Acquire the hyperspectral data and check whether the spatial resolution of the hypercubes can distinguish the different brushstrokes on the RGB images of the painted surface. Run PointSel, the isolated measuring point selection code to manually select some reference spectra on the surfaces of the test samples. Type the command line including the semicolon in the terminal window and press enter to run the code.Select the measurement points by clicking the interactive window that one by one shows the two dimensional RGB images of the fields of view. Run SAM_Standard, the standard SAM maps evaluation code to extract the SAM maps using the whole spectra. Type the command line, including semicolon in the terminal window, then press enter to run the code.SAM maps are saved as PNG images in the current work folder. Check whether the obtain similarity maps display the details of the brush strokes used to realize the test samples. If not, restart the process by readjusting the distance between the test sample surface and the acquisition equipment.According to the evaluation obtained by the test samples set the distance between the surface of the painting under investigation and the acquisition equipment. Perform IO of the hyperspectral data by organizing, reading and managing the hypercubes. Run the HS FileLister code to store the list of the files containing the hypercubes and the related information into two variables at the disposal of the algorithm.Run the HS_Crop PNG code to select the portion of each FOV to be used in the analysis of the data. Next, run the PointSel code and click within the displayed interactive window to identify the reference spectra as isolated measuring points over the surface of the monitored areas. Type the command line including the semicolon in the terminal window and press enter to run ReticularSel, the Reticular selection code.This automatically selects the reference spectra as a regular reticulum of measuring points superimposed to the surface of the monitored areas. This selection method makes the analysis very time consuming as the number of references is large. Type the command line including the semicolon in the terminal window and press enter to run SaveImPoint.This saves a location at the selected measurement point superimposed to the pictures of the fields of view. Run Spectra_Importer, the external references importer code to create a variable containing references from data sets and databases independent from the hypercubes acquired on quarto stato. Note that the spectra have different size with respect to those obtained with the hyperspectral camera.Run the SAM complete code to evaluate the similarity maps. Feed the code with the desired pre-processing option entering zero or one in the dialogue box. Zero to require the spectra normalization only or one to require that after the normalization the spectra are derived one time.enter this sequence of numbers corresponding to the desired columns of the references matrix into the dialogue box by typing the numbers separated by a white space. Press Enter to continue. Set the method to zero for no manipulation of the data.One to require manual selection of the wavelength ranges of the spectra to be considered before starting the analysis or two to require the algorithm to order the data based on a specific criterion and before the evaluation of the SAM maps. To select the end members for the SAM analysis, the algorithm either retrieves the references spectra among the hypercubes by manually selecting some isolated measuring points or automatically samples the painting surface, providing a particular selection of measuring points within one or more FOVs. The algorithm can also compare the hypercubes with external spectra like the ones obtained by a portable FORS miniature spectrometer.When the pre-processed references appear on an interactive window, one or more wavelength intervals to be analyzed can be manually selected. In automatic selection, the algorithm computes a maximum variance within the desired references and orders spectra according to this criterion. If the maximum variance corresponds to the nth wavelength the content of the nth component of each pre-processed spectrum will be moved to the first position of a rearranged hypervector and so on.Following the automatic manipulation, the algorithm applies a floating threshold to the variance values and evaluates the SAM maps at the increasing threshold which results in a total of two n plus one sets of maps where n is a number of values assumed by the threshold. The obtained similarity maps provide new insights into the details of the mapped area. They can help to compare the samples and the references.The possibility to customize the analysis and to exploit any spectrum as reference, expands the user horizons but simultaneously asks the user for a careful evaluation of their choices This approach allows the use of spectrum manipulation as an analysis tool, therefore computer vision and statistical studies can help deepen the knowledge about the possibility of this matter.

applying-hyperspectral-reflectance-imaging-to-investigate-palettes

Research

JoVE Journal

Chemistry

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based molecular single crystals. As representative example, we chose a single crystal of the heterodinuclear Ln-based complex [TbEu(bpm)(tfaa)6] (bpm=2,2&#8217;-bipyrimidine, tfaa&#8211; =1,1,1-trifluoroacetylacetonate) exhibiting bright visible emission under UV excitation. HSI is an emerging technique that combines 2-dimensional spatial imaging of a luminescent structure with spectral information from each pixel of the obtained image. Specifically, HSI on single crystals of the [Tb-Eu] complex provided local spectral information unveiling variation of the luminescence intensity at different points along the studied crystals. These changes were attributed to the optical anisotropy present in the crystal, which results from the different molecular packing of Ln3+ ions in each one of the directions of the crystal structure. The HSI herein described is an example of the suitability of such technique for spectro-spatial investigations of molecular materials. Yet, importantly, this protocol can be easily extended for other types of luminescent materials (such as micron-sized molecular crystals, inorganic microparticles, nanoparticles in biological tissues, or labelled cells, among others), opening many possibilities for deeper investigation of structure-property relationships. Ultimately, such investigations will provide knowledge to be leveraged into the engineering of advanced materials for a wide range of applications from bioimaging to technological applications, such as waveguides or optoelectronic devices.

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...