Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Hdl Handle:
http://hdl.handle.net/10675.2/686
Title:
Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy
Authors:
Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen
Abstract:
Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.
Citation:
PLoS One. 2011 Nov 28; 6(11):e28237
Issue Date:
28-Nov-2011
URI:
http://hdl.handle.net/10675.2/686
DOI:
10.1371/journal.pone.0028237
PubMed ID:
22140560
PubMed Central ID:
PMC3225396
Type:
Article
ISSN:
1932-6203
Appears in Collections:
Department of Cellular Biology and Anatomy: Faculty Research and Presentations

Full metadata record

DC FieldValue Language
dc.contributor.authorRehberg, Markusen_US
dc.contributor.authorKrombach, Fritzen_US
dc.contributor.authorPohl, Ulrichen_US
dc.contributor.authorDietzel, Steffenen_US
dc.date.accessioned2012-10-26T16:29:33Z-
dc.date.available2012-10-26T16:29:33Z-
dc.date.issued2011-11-28en_US
dc.identifier.citationPLoS One. 2011 Nov 28; 6(11):e28237en_US
dc.identifier.issn1932-6203en_US
dc.identifier.pmid22140560en_US
dc.identifier.doi10.1371/journal.pone.0028237en_US
dc.identifier.urihttp://hdl.handle.net/10675.2/686-
dc.description.abstractSecond and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.en_US
dc.rightsRehberg et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_US
dc.subjectResearch Articleen_US
dc.subjectBiologyen_US
dc.subjectAnatomy and Physiologyen_US
dc.subjectMusculoskeletal Systemen_US
dc.subjectMuscleen_US
dc.subjectBiophysicsen_US
dc.subjectCell Motilityen_US
dc.subjectMolecular Cell Biologyen_US
dc.subjectCellular Structuresen_US
dc.subjectCell Nucleusen_US
dc.subjectCellular Typesen_US
dc.subjectBlood Cellsen_US
dc.subjectMuscle Fibersen_US
dc.subjectNeuronsen_US
dc.subjectChromosome Biologyen_US
dc.subjectCentromeresen_US
dc.subjectChromatinen_US
dc.subjectNeuroscienceen_US
dc.subjectNeuroimagingen_US
dc.subjectMedicineen_US
dc.subjectAnatomy and Physiologyen_US
dc.subjectMusculoskeletal Systemen_US
dc.subjectMuscleen_US
dc.subjectCardiovascularen_US
dc.subjectCardiovascular Imagingen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBlood Cellsen_US
dc.subject.meshCell Movementen_US
dc.subject.meshChromatinen_US
dc.subject.meshFibrillar Collagensen_US
dc.subject.meshImaging, Three-Dimensionalen_US
dc.subject.meshLeukocytesen_US
dc.subject.meshMiceen_US
dc.subject.meshMice, Inbred C57BLen_US
dc.subject.meshMicroscopyen_US
dc.subject.meshMusclesen_US
dc.subject.meshNerve Fibersen_US
dc.subject.meshPeripheral Nervesen_US
dc.subject.meshSarcomeresen_US
dc.subject.meshStaining and Labelingen_US
dc.titleLabel-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopyen_US
dc.typeArticleen_US
dc.identifier.pmcidPMC3225396en_US
dc.contributor.corporatenameDepartment of Cellular Biology and Anatomy-
dc.contributor.corporatenameCollege of Graduate Studies-
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