Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Becker, Klaus; Jährling, Nina; Saghafi, Saiedeh; Weiler, Reto; Dodt, Hans‐Ulrich

doi:10.1371/journal.pone.0033916

Cited by 266 publications

(223 citation statements)

References 14 publications

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“…In our hands, even when storing the cell samples for one week in RNAlater, the relative amount of detectable GFP-positive cells was unchanged. This does not hold for other fixation reagents which led to a decrease in the fraction of GFP-positive cells over time, such as EtOH (time-and concentration-dependent quenching in accordance to previous findings (Becker et al, 2012)) or stop solution (time-dependent quenching; likely due to the phenol contained in the mixture).…”

Section: Transcriptome Fixationsupporting

confidence: 72%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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Section: Transcriptome Fixationsupporting

confidence: 72%

Dual RNA-seq of pathogen and host

2012

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“…In comparison with CM or twophoton microscopy, UM can rapidly create optical sectioning of macroscopic samples, since the objectives with small focal power and small numerical aperture can provide a large field of view. However, the use of UM is completely dependent upon the optical transparency of biological objects, so that the improvement of optical clearing efficiency, including that due to the development of new OCAs, remains to be an urgent problem [86,87,[258][259][260][261][262]. In Ref.…”

Section: Fluorescence Imagingmentioning

confidence: 99%

Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy

Genina

Bashkatov

Sinichkin

et al. 2015

JBPE

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Abstract.A review of specific features and methods of optical clearing and related interaction of light with tissues is presented. Physical and molecular mechanisms of immersion, compression, and photodynamic/photothermal optical clearing of some fibrous and cellular tissues are discussed. The possibility of efficient control of the tissue optical properties, particularly, the reduction of light scattering in tissues is demonstrated, which facilitates the increased efficiency of various optical visualisation methods (optical biopsy) used in medical purposes. © 2015 Samara State Aerospace University (SSAU).Keywords: tissue, optical clearing, optical diagnostics, imaging. 1, 404-413 (1997). 5. C. L. Smithpeter, A. K. Dunn, A. J. Welch, and R. Richards-Kortum, "Penetration depth limits of in vivo confocal reflectance imaging," Appl. Opt. 37, 2749Opt. 37, -2754Opt. 37, (1998. 6. V. V. Tuchin, L. V. Wang, and D. A. Zimnyakov, Optical Polarization in Biomedical Applications, SpringerVerlag, New York, NY, USA (2006). 7. R. Drezek, A. Dunn, and R. Richards-Kortum, "Light scattering from cells: finite-difference time-domain simulations and goniometric measurements," Appl. Opt. 38(16), 3651-3661 (1999). 8. K. Sokolov, R. Drezek, K. Gossagee, and R. Richards-Kortum, "Reflectance spectroscopy with polarized light:is it sensitive to cellular and nuclear morphology," Opt. Express 5, 302-317 (1999). 9. D. W. Leonard, and K. M. Meek, "Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma," Biophysical J. 72, 1382-1387 (1997). 10. A. G. Borovoi, E. I. Naats, and U. G. Oppel, "Scattering of light by a red blood cell," J. Biomed. Opt. 3, 364-372 (1998). 11. A. N. Yaroslavsky, A. V. Priezzhev, J. Rodriguez, I. V. Yaroslavsky, and H. Battarbee, "Optics of blood,"Chap. 2 in Handbook of Optical Biomedical Diagnostics, V. V. Tuchin, Ed., pp. 169-216, PM107 SPIE Press, Bellingham, WA, USA (2002). 12. G. Mazarevica, T. Freivalds, and A. Jurka, "Properties of erythrocyte light refraction in diabetic patients," J.Biomed. Opt. 7, 244-247 (2002). 13. M. Friebel, and M. Meinke, "Model function to calculate the refractive index of native hemoglobin in the wavelength range of 250-1100 nm dependent on concentration," Appl. Opt. 45(12), 2838-2842 (2006). Appl. Opt. 35(19), 3413-3420 (1996). 34. D. Zhu, J. Wang, Z. Zhi, X. Wen, and Q. Luo, "Imaging dermal blood flow through the intact rat skin with an optical clearing method," J. Biomed. Opt. 15, 026008 (2010 241. K. König, G. Flemming, and R. Hibst, "Laser-induced autofluorescence spectroscopy of dental caries lesion," Cell. Mol. Biol. 44, 1293-1300. 242. E. G. Borisova, T. T. Uzunov, and L. A. Avramov, "Early differentiation between caries and tooth demineralization using laser-induced autofluorescence spectroscopy," Lasers Surg. Med. 34, 249-253 (2004 -20(12), 1512-1516 (1984). 245. K. Konig, H. Schneckenburger, and R. Hibst, "Time-gated in vivo autofluorescence imaging of dental caries,"Cell. Mol. Biol. 45, 233-239 (1999). 246. E. Borisova, P. Tr...

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“…Various research groups have developed new tissue clearing protocols to be able to image their tissue of interest for different purposes. These include organic solvent [2][3][4][5] , water 6,7 and electrophoresis based 8 clearing protocols. Among them, 3-dimensional imaging of solvent cleared organs or 3DISCO is a readily applicable protocol on a variety of biological samples including central nervous system (CNS) organs, immune organs and solid tumors.…”

Section: Introductionmentioning

confidence: 99%

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Ertürk¹,

Lafkas²,

Chalouni³

2014

JoVE

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Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

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Chemical Clearing and Dehydration of GFP Expressing Mouse Brains

Cited by 266 publications

References 14 publications

Dual RNA-seq of pathogen and host

Dual RNA-seq of pathogen and host

Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

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