Magnetic Resonance Microscopy Contribution to Interpret High-Resolution Magic Angle Spinning Metabolomic Data of Human Tumor Tissue

HRMAS NMR is considered a valuable technique to obtain detailed metabolic profile of unprocessed tissues. To properly interpret the HRMAS metabolomic results, detailed information of the actual state of the sample inside the rotor is needed. MRM (Magnetic Resonance Microscopy) was applied for obtaining structural and spatially localized metabolic information of the samples inside the HRMAS rotors. The tissue was observed stuck to the rotor wall under the effect of HRMAS spinning. MRM spectroscopy showed a transference of metabolites from the tissue to the medium. The sample shape and the metabolite transfer after HRMAS indicated that tissue had undergone alterations and it can not be strictly considered as intact. This must be considered when HRMAS is used for metabolic tissue characterization, and it is expected to be highly dependent on the manipulation of the sample. The localized spectroscopic information of MRM reveals the biochemical compartmentalization on tissue samples hidden in the HRMAS spectrum

Urinary metabolic signatures detect recurrences in non-muscle invasive bladder cancer

Patients with non-muscle invasive bladder cancer (NMIBC) undergo lifelong monitoring based on repeated cystoscopy and urinary cytology due to the high recurrence rate of this tumor. Nevertheless, these techniques have some drawbacks, namely, low accuracy in detection of low-grade tumors, omission of pre-neoplastic lesions and carcinomas in situ (CIS), invasiveness, and high costs. This work aims to identify a urinary metabolomic signature of recurrence by proton Nuclear Magnetic Resonance (1H NMR) spectroscopy for the follow-up of NMIBC patients. To do this, changes in the urinary metabolome before and after transurethral resection (TUR) of tumors are analyzed and a Partial Least Square Discriminant Analysis (PLS-DA) model is developed. The usefulness of this discriminant model for the detection of tumor recurrences is assessed using a cohort of patients undergoing monitoring. The trajectories of the metabolomic profile in the follow-up period provide a negative predictive value of 92.7% in the sample classification. Pathway analyses show taurine, alanine, aspartate, glutamate, and phenylalanine perturbed metabolism associated with NMIBC. These results highlight the potential of 1H NMR metabolomics to detect bladder cancer (BC) recurrences through a non-invasive approach

Development of a three-dimensional cell culture system based on microfluidics for nuclear magnetic resonance and optical monitoring.

A new microfluidic cell culture device compatible with real-time nuclear magnetic resonance (NMR) is presented here. The intended application is the long-term monitoring of 3D cell cultures by several techniques. The system has been designed to fit inside commercially available NMR equipment to obtain maximum readout resolution when working with small samples. Moreover, the microfluidic device integrates a fibre-optic-based sensor to monitor parameters such as oxygen, pH, or temperature during NMR monitoring, and it also allows the use of optical microscopy techniques such as confocal fluorescence microscopy. This manuscript reports the initial trials culturing neurospheres inside the microchamber of this device and the preliminary images and spatially localised spectra obtained by NMR. The images show the presence of a necrotic area in the interior of the neurospheres, as is frequently observed in histological preparations; this phenomenon appears whenever the distance between the cells and fresh nutrients impairs the diffusion of oxygen. Moreover, the spectra acquired in a volume of 8 nl inside the neurosphereshow an accumulation of lactate and lipids, which are indicative of anoxic condi-tions. Additionally, a basis for general temperature control and monitoring and a graphical control software have been developed and are also described. The complete platform will allow biomedical assays of therapeutic agents to be performed in the early phases of therapeutic development. Thus, small quantities of drugs or advanced nanodevices may be studied long-term under simulated living conditions that mimic the flow and distribution of nutrient

Alzheimer Demenz in verschiedenen Wohn- und Betreuungsformen : Konsequenzen für die psychosoziale Arbeit

nicht vorhande

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies

[EN] Quantitative multinuclear high-resolution magic angle spinning (HRMAS) was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D 1 H and 31P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. 1 H–1 H homonuclear and 1 H–31P heteronuclear correlation experiments enabled the direct assessment of the 1 H–31P spin systems for signals that suffered from overlapping in the 1D 1 H spectra, and linked the information present in the 1D 1 H and 31P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the 31P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from 31P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH3)3 signals of phosphocholine and choline in 1 H spectra of the tissue in these tumour samples.The authors acknowledge the SCSIE-University of Valencia Microscopy Service for the histological preparations. They also acknowledge Martial Piotto (Bruker BioSpin, France) for providing the ERETIC synthetic signal. Furthermore, they acknowledge financial support from the Spanish Government project SAF2007-6547, the Generalitat Valenciana project GVACOMP2009-303, and the E.U.'s VI Framework Programme via the project "Web accessible MR decision support system for brain tumor diagnosis and prognosis, incorporating in vivo and ex vivo genomic and metabolomic data" (FP6-2002-LSH 503094). CIBER-BBN is an initiative funded by the VI National R&D&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.Esteve Moya, V.; Celda, B.; Martínez Bisbal, MC. (2012). Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies. Analytical and Bioanalytical Chemistry. 403:2611-2625. https://doi.org/10.1007/s00216-012-6001-zS26112625403Cheng LL, Chang IW, Louis DN, Gonzalez RG (1998) Cancer Res 58:1825–1832Opstad KS, Bell BA, Griffiths JR, Howe FA (2008) Magn Reson Med 60:1237–1242Sjobakk TE, Johansen R, Bathen TF, Sonnewald U, Juul R, Torp SH, Lundgren S, Gribbestad IS (2008) NMR Biomed 21:175–185Martinez-Bisbal MC, Marti-Bonmati L, Piquer J, Revert A, Ferrer P, Llacer JL, Piotto M, Assemat O, Celda B (2004) NMR Biomed 17:191–205Erb G, Elbayed K, Piotto M, Raya J, Neuville A, Mohr M, Maitrot D, Kehrli P, Namer IJ (2008) Magn Reson Med 59:959–965Wilson M, Davies NP, Brundler MA, McConville C, Grundy RG, Peet AC (2009) Mol Cancer 8:6Martinez-Bisbal MC, Monleon D, Assemat O, Piotto M, Piquer J, Llacer JL, Celda B (2009) NMR Biomed 22:199–206Martínez-Granados B, Monleón D, Martínez-Bisbal MC, Rodrigo JM, del Olmo J, Lluch P, Ferrández A, Martí-Bonmatí L, Celda B (2006) NMR Biomed 19:90–100Hubesch B, Sappey-Marinier D, Roth K, Meyerhoff DJ, Matson GB, Weiner MW (1990) Radiology 174:401–409Albers MJ, Krieger MD, Gonzalez-Gomez I, Gilles FH, McComb JG, Nelson MD Jr, Bluml S (2005) Magn Reson Med 53:22–29Wijnen JP, Scheenen TW, Klomp DW, Heerschap A (2010) NMR Biomed 23:968–976Podo F (1999) NMR Biomed 12:413–439Griffiths JR, Cady E, Edwards RH, McCready VR, Wilkie DR, Wiltshaw E (1983) Lancet 1:1435–1436Robitaille PL, Robitaille PA, Gordon Brown G, Brown GG (1991) J Magn Reson 92:73–84, 1969Griffiths JR (1991) Br J Cancer 64:425–427Payne GS, Troy H, Vaidya SJ, Griffiths JR, Leach MO, Chung YL (2006) NMR Biomed 19:593–598De Silva SS, Payne GS, Thomas V, Carter PG, Ind TE, deSouza NM (2009) NMR Biomed 22:191–198Wang Y, Cloarec O, Tang H, Lindon JC, Holmes E, Kochhar S, Nicholson JK (2008) Anal Chem 80:1058–1066Lehnhardt FG, Rohn G, Ernestus RI, Grune M, Hoehn M (2001) NMR Biomed 14:307–317Srivastava NK, Pradhan S, Gowda GA, Kumar R (2010) NMR Biomed 23:113–122Akoka S, Barantin L, Trierweiler M (1999) Anal Chem 71:2554–2557Albers MJ, Butler TN, Rahwa I, Bao N, Keshari KR, Swanson MG, Kurhanewicz J (2009) Magn Reson Med 61:525–532Ben Sellem D, Elbayed K, Neuville A, Moussallieh FM, Lang-Averous G, Piotto M, Bellocq JP, Namer IJ (2011) J Oncol 2011:174019Bourne R, Dzendrowskyj T, Mountford C (2003) NMR Biomed 16:96–101Martinez-Bisbal MC, Esteve V, Martinez-Granados B, Celda B (2011) J Biomed Biotechnol 2011:763684, Epub 2010 Sep 5Celda B, Montelione GT (1993) J Magn Reson B 101:189–193Esteve V, Celda B (2008) Magn Reson Mater Phys MAGMA 21:484–484Collins TJ (2007) Biotechniques 43:25–30Govindaraju V, Young K, Maudsley AA (2000) NMR Biomed 13:129–153Fan TW-M (1996) Prog Nucl Magn Reson Spectrosc 28:161–219Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley JL (2008) Nucleic Acids Res 36:D402–D408Kriat M, Vion-Dury J, Confort-Gouny S, Favre R, Viout P, Sciaky M, Sari H, Cozzone PJ (1993) J Lipid Res 34:1009–1019Subramanian A, Shankar Joshi B, Roy AD, Roy R, Gupta V, Dang RS (2008) NMR Biomed 21:272–288Daykin CA, Corcoran O, Hansen SH, Bjornsdottir I, Cornett C, Connor SC, Lindon JC, Nicholson JK (2001) Anal Chem 73:1084–1090Griffin JL, Lehtimaki KK, Valonen PK, Grohn OH, Kettunen MI, Yla-Herttuala S, Pitkanen A, Nicholson JK, Kauppinen RA (2003) Cancer Res 63:3195–3201Petroff OAC, Prichard JW (1995) In: Kraicer J, Dixon SJ (eds) Methods in neurosciences. 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Chronic erythropoietin treatment improves diet-induced glucose intolerance in rats

Erythropoietin (EPO) ameliorates glucose metabolism through mechanisms not fully understood. In this study, we investigated the effect of EPO on glucose metabolism and insulin signaling in skeletal muscle. A 2-week EPO treatment of rats fed with a high-fat diet (HFD) improved fasting glucose levels and glucose tolerance, without altering total body weight or retroperitoneal fat mass. Concomitantly, EPO partially rescued insulin-stimulated AKT activation, reduced markers of oxidative stress, and restored heat-shock protein 72 expression in soleus muscles from HFD-fed rats. Incubation of skeletal muscle cell cultures with EPO failed to induce AKT phosphorylation and had no effect on glucose uptake or glycogen synthesis. We found that the EPO receptor gene was expressed in myotubes, but was undetectable in soleus. Together, our results indicate that EPO treatment improves glucose tolerance but does not directly activate the phosphorylation of AKT in muscle cells. We propose that the reduced systemic inflammation or oxidative stress that we observed after treatment with EPO could contribute to the improvement of whole-body glucose metabolism.Corinne Caillaud, Mie Mechta, Heidi Ainge, Andreas N Madsen, Patricia Ruell, Emilie Mas, Catherine Bisbal, Jacques Mercier, Stephen Twigg, Trevor A Mori, David Simar and Romain Barrè

Integrative Metabolomic and Transcriptomic Analysis for the Study of Bladder Cancer

[EN] Metabolism reprogramming is considered a hallmark of cancer. The study of bladder cancer (BC) metabolism could be the key to developing new strategies for diagnosis and therapy. This work aimed to identify tissue and urinary metabolic signatures as biomarkers of BC and get further insight into BC tumor biology through the study of gene-metabolite networks and the integration of metabolomics and transcriptomics data. BC and control tissue samples (n = 44) from the same patients were analyzed by High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance and microarrays techniques. Besides, urinary profiling study (n = 35) was performed in the same patients to identify a metabolomic profile, linked with BC tissue hallmarks, as a potential non-invasive approach for BC diagnosis. The metabolic profile allowed for the classification of BC tissue samples with a sensitivity and specificity of 100%. The most discriminant metabolites for BC tissue samples reflected alterations in amino acids, glutathione, and taurine metabolic pathways. Transcriptomic data supported metabolomic results and revealed a predominant downregulation of metabolic genes belonging to phosphorylative oxidation, tricarboxylic acid cycle, and amino acid metabolism. The urinary profiling study showed a relation with taurine and other amino acids perturbed pathways observed in BC tissue samples, and classified BC from non-tumor urine samples with good sensitivities (91%) and specificities (77%). This urinary profile could be used as a non-invasive tool for BC diagnosis and follow-up.This research was funded by FEDER cofounded MINECO grant SAF2015-66015-R, MAT2015-64139-C4-1-R, MAT2015-64139-C4-3-R, ISCIII-RETICRD12/0036/0009, PIE 15/00076, CB/16/00228, CTQ2016-79561-P; and the PROMETEO II/2014/047 and PROMETEO 2018/24 projects.Loras, A.; Suárez-Cabrera, C.; Martínez-Bisbal, M.; Quintás, G.; Paramio, JM.; Martínez-Máñez, R.; Gil Grau, S.... (2019). Integrative Metabolomic and Transcriptomic Analysis for the Study of Bladder Cancer. Cancers. 11(5):1-19. https://doi.org/10.3390/cancers1105068611911

Quantitative determination of spring water quality parameters via electronic tongue

[EN] The use of a voltammetric electronic tongue for the quantitative analysis of quality parameters in spring water is proposed here. The electronic voltammetric tongue consisted of a set of four noble electrodes (iridium, rhodium, platinum, and gold) housed inside a stainless steel cylinder. These noble metals have a high durability and are not demanding for maintenance, features required for the development of future automated equipment. A pulse voltammetry study was conducted in 83 spring water samples to determine concentrations of nitrate (range: 6.9-115 mg/L), sulfate (32-472 mg/L), fluoride (0.08-0.26 mg/L), chloride (17-190 mg/L), and sodium (11-94 mg/L) as well as pH (7.3-7.8). These parameters were also determined by routine analytical methods in spring water samples. A partial least squares (PLS) analysis was run to obtain a model to predict these parameter. Orthogonal signal correction (OSC) was applied in the preprocessing step. Calibration (67%) and validation (33%) sets were selected randomly. The electronic tongue showed good predictive power to determine the concentrations of nitrate, sulfate, chloride, and sodium as well as pH and displayed a lower R-2 and slope in the validation set for fluoride. Nitrate and fluoride concentrations were estimated with errors lower than 15%, whereas chloride, sulfate, and sodium concentrations as well as pH were estimated with errors below 10%.The authors gratefully acknowledge the Ministerio de Economia y Competitividad and FEDER, providing funds amounting to (sic)664.525,75 for the project "Desarrollo de sistemas basados en lenguas electronicas para el control y monitorizacion del ciclo integral del agua" INNPACTO. IPT-2012-0069-310000. Moreover, the authors gratefully acknowledge the Ministerio de Economia y Competitividad and FEDER (projects MAT2015-64139-C4-1-R (MINECO/FEDER), MAT2015-64139-C4-3-R (MINECO/FEDER)), the Generalitat Valenciana (project PROMETEOII/2014/047), and CIBER-BBN (NANOPROBE project) for their financial support.Carbó-Mestre, N.; López-Carrero, J.; Garcia-Castillo, FJ.; Tormos, I.; Olivas, E.; Folch, E.; Alcañiz Fillol, M.... (2018). Quantitative determination of spring water quality parameters via electronic tongue. Sensors. 18(1):1-12. https://doi.org/10.3390/s18010040S112181Winquist, F., Olsson, J., & Eriksson, M. (2011). Multicomponent analysis of drinking water by a voltammetric electronic tongue. Analytica Chimica Acta, 683(2), 192-197. doi:10.1016/j.aca.2010.10.027Storey, M. V., van der Gaag, B., & Burns, B. P. (2011). Advances in on-line drinking water quality monitoring and early warning systems. Water Research, 45(2), 741-747. doi:10.1016/j.watres.2010.08.049Winquist, F. (2008). Voltammetric electronic tongues – basic principles and applications. Microchimica Acta, 163(1-2), 3-10. doi:10.1007/s00604-007-0929-2Garçon, L.-A., Genua, M., Hou, Y., Buhot, A., Calemczuk, R., Livache, T., … Hou, Y. (2017). A Versatile Electronic Tongue Based on Surface Plasmon Resonance Imaging and Cross-Reactive Sensor Arrays—A Mini-Review. Sensors, 17(5), 1046. doi:10.3390/s17051046Hou, Y., Genua, M., Tada Batista, D., Calemczuk, R., Buhot, A., Fornarelli, P., … Livache, T. (2012). Continuous Evolution Profiles for Electronic-Tongue-Based Analysis. Angewandte Chemie International Edition, 51(41), 10394-10398. doi:10.1002/anie.201205346Holmin, S., Spångeus, P., Krantz-Rülcker, C., & Winquist, F. (2001). Compression of electronic tongue data based on voltammetry — a comparative study. Sensors and Actuators B: Chemical, 76(1-3), 455-464. doi:10.1016/s0925-4005(01)00585-8Campos, I., Alcañiz, M., Aguado, D., Barat, R., Ferrer, J., Gil, L., … Vivancos, J.-L. (2012). A voltammetric electronic tongue as tool for water quality monitoring in wastewater treatment plants. Water Research, 46(8), 2605-2614. doi:10.1016/j.watres.2012.02.029Martı́nez-Máñez, R., Soto, J., Garcia-Breijo, E., Gil, L., Ibáñez, J., & Llobet, E. (2005). An «electronic tongue» design for the qualitative analysis of natural waters. Sensors and Actuators B: Chemical, 104(2), 302-307. doi:10.1016/j.snb.2004.05.022Garcia-Breijo, E., Atkinson, J., Gil-Sanchez, L., Masot, R., Ibañez, J., Garrigues, J., … Olguin, C. (2011). A comparison study of pattern recognition algorithms implemented on a microcontroller for use in an electronic tongue for monitoring drinking waters. Sensors and Actuators A: Physical, 172(2), 570-582. doi:10.1016/j.sna.2011.09.039Moreno, L., Merlos, A., Abramova, N., Jiménez, C., & Bratov, A. (2006). Multi-sensor array used as an «electronic tongue» for mineral water analysis. Sensors and Actuators B: Chemical, 116(1-2), 130-134. doi:10.1016/j.snb.2005.12.063Sipos, L., Kovács, Z., Sági-Kiss, V., Csiki, T., Kókai, Z., Fekete, A., & Héberger, K. (2012). Discrimination of mineral waters by electronic tongue, sensory evaluation and chemical analysis. Food Chemistry, 135(4), 2947-2953. doi:10.1016/j.foodchem.2012.06.021Braga, G. S., Paterno, L. G., & Fonseca, F. J. (2012). Performance of an electronic tongue during monitoring 2-methylisoborneol and geosmin in water samples. Sensors and Actuators B: Chemical, 171-172, 181-189. doi:10.1016/j.snb.2012.02.092Escobar, J. D., Alcaniz, M., Masot, R., Fuentes, A., Bataller, R., Soto, J., & Barat, J. M. (2013). Quantification of organic acids using voltammetric tongues. Food Chemistry, 138(2-3), 814-820. doi:10.1016/j.foodchem.2012.11.078Martínez-Bisbal, M. C., Loeff, E., Olivas, E., Carbó, N., García-Castillo, F. J., López-Carrero, J., … Soto, J. (2017). A Voltammetric Electronic Tongue for the Quantitative Analysis of Quality Parameters in Wastewater. Electroanalysis, 29(4), 1147-1153. doi:10.1002/elan.201600717Bonastre, A., Ors, R., Capella, J. V., Fabra, M. J., & Peris, M. (2005). In-line chemical analysis of wastewater: present and future trends. TrAC Trends in Analytical Chemistry, 24(2), 128-137. doi:10.1016/j.trac.2004.09.008Ivarsson, P., Johansson, M., Höjer, N.-E., Krantz-Rülcker, C., Winquist, F., & Lundström, I. (2005). Supervision of rinses in a washing machine by a voltammetric electronic tongue. Sensors and Actuators B: Chemical, 108(1-2), 851-857. doi:10.1016/j.snb.2004.12.088Garcia-Breijo, E., Peris, R. M., Pinatti, C. O., Fillol, M. A., Civera, J. I., & Prats, R. B. (2013). Low-Cost Electronic Tongue System and Its Application to Explosive Detection. IEEE Transactions on Instrumentation and Measurement, 62(2), 424-431. doi:10.1109/tim.2012.2215156Winquist, F., Wide, P., & Lundström, I. (1997). An electronic tongue based on voltammetry. Analytica Chimica Acta, 357(1-2), 21-31. doi:10.1016/s0003-2670(97)00498-4Campos, I., Sangrador, A., Bataller, R., Aguado, D., Barat, R., Soto, J., & Martínez-Máñez, R. (2014). Ammonium and Phosphate Quantification in Wastewater by Using a Voltammetric Electronic Tongue. Electroanalysis, 26(3), 588-595. doi:10.1002/elan.201300538Geladi, P., & Kowalski, B. R. (1986). Partial least-squares regression: a tutorial. Analytica Chimica Acta, 185, 1-17. doi:10.1016/0003-2670(86)80028-9Wold, S., Antti, H., Lindgren, F., & Öhman, J. (1998). Orthogonal signal correction of near-infrared spectra. Chemometrics and Intelligent Laboratory Systems, 44(1-2), 175-185. doi:10.1016/s0169-7439(98)00109-9Sjöblom, J., Svensson, O., Josefson, M., Kullberg, H., & Wold, S. (1998). An evaluation of orthogonal signal correction applied to calibration transfer of near infrared spectra. Chemometrics and Intelligent Laboratory Systems, 44(1-2), 229-244. doi:10.1016/s0169-7439(98)00112-9Rouhollahi, A., Rajabzadeh, R., & Ghasemi, J. (2006). Simultaneous determination of dopamine and ascorbic acid by linear sweep voltammetry along with chemometrics using a glassy carbon electrode. Microchimica Acta, 157(3-4), 139-147. doi:10.1007/s00604-006-0668-9Riahi, S., Ganjali, M., Moghaddam, A., Pourbasheer, E., & Norouzi, P. (2009). Development of a New Combined Chemometrics Method, Applied in the Simultaneous Voltammetric Determination of Cinnamic Acid and 3, 4-Dihydroxy Benzoic Acid. Current Analytical Chemistry, 5(1), 42-47. doi:10.2174/157341109787047925Palacios-Santander, J. M., Cubillana-Aguilera, L. M., Cocchi, M., Ulrici, A., Naranjo-Rodríguez, I., Seeber, R., & Hidalgo-Hidalgo de Cisneros, J. L. (2008). Multicomponent analysis in the wavelet domain of highly overlapped electrochemical signals: Resolution of quaternary mixtures of chlorophenols using a peg-modified Sonogel–Carbon electrode. Chemometrics and Intelligent Laboratory Systems, 91(2), 110-120. doi:10.1016/j.chemolab.2007.10.00

Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin,spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodicskeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronalextensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limitof conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costlyequipment and special analysis routines, which remain inaccessible to most research groups. Thisreport aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to revealthe MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiologylaboratories. We first explore means to accurately estimate the expansion factors of protein structureswithin cells. We then describe the protocol that produces an expanded specimen that can be examinedwith any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. Wevalidate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. Weconclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of theMPS using accessible reagents and conventional fluorescent microscopes.Fil: Martínez, Gaby F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Gazal, Nahir Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Quassollo Infanzon, Gonzalo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Szalai, Alan Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Del Cid Pellitero, Esther. No especifíca;Fil: Durcan, Thomas M.. No especifíca;Fil: Fon, Edward A.. No especifíca;Fil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Unsain, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin