Imaging of Caenorhabditis elegans neurons by second-harmonic generation and two-photon excitation fluorescence

Filippidis, George; Kouloumentas, Christos; Voglis, Giannis; Zacharopoulou, Fotini; Papazoglou, Theodore G.; Tavernarakis, Nektarios

doi:10.1117/1.1886729

Cited by 22 publications

(22 citation statements)

References 46 publications

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“…In the past, multiphoton microscopy has been successfully applied in the imaging of Caernorhabditis elegans and other important biological systems. [5][6][7][8] Specifically, the nonlinear imaging modalities of fluorescence, second harmonic generation ͑SHG͒, and third harmonic generation ͑THG͒ imaging have been used to study physiological processes in Drosophila. [9][10][11][12] However, to the best of our knowledge, the combination of multiphoton auto fluorescence ͑MAF͒ and SHG imaging for the structural exploration of Drosophila larval organelles on the wholebody scale has not been demonstrated.…”

Section: 3mentioning

confidence: 99%

Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy

Lin

Hovhannisyan

et al. 2008

J. Biomed. Opt.

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Abstract. The fruit fly Drosophila melanogaster is one of the most valuable organisms in studying genetics and developmental biology. To gain insight into Drosophila development, we successfully acquired label-free, in vivo images of both developing muscles and internal organs in a stage 2 larva using the minimally invasive imaging modality of multiphoton autofuorescence ͑MAF͒ and second harmonic generation ͑SHG͒ microscopy. We found that although MAF is useful in identifying structures such as the digestive system, trachea, and intestinal track, it is the SHG signal that allowed the investigation of the muscular architecture within the developing larva. Our results suggest that multiphoton microscopy is a powerful in vivo, label-free imaging technique to examine Drosophila physiology and may be used for developmental studies.

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Section: 3mentioning

confidence: 99%

Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy

Lin

Hovhannisyan

et al. 2008

J. Biomed. Opt.

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“…Multi-photon microscopy is inherently non-linear, and thus has the ability to perform optical sectioning with negligible out-of-plane absorption and emission. This dramatically reduces photobleaching and phototoxicity [8]. This is especially significant in assays that require animals to be imaged at multiple time points.…”

Section: Applicationsmentioning

confidence: 99%

High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

et al. 2009

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In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in three Nobel Prizes in medicine or chemistry during the last six years for studies conducted on the nematode Caenorhabditis elegans (C. elegans). The availability of a wide array of speciesspecific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. We present a suite of technologies for complex high-throughput wholeanimal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize C. elegans in a well-defined geometry, an integrated chip containing individually addressable screening chambers for incubation and exposure of individual animals to biochemical compounds, and a device for delivery of compound libraries in standard multiwell plates to microfluidic devices. The immobilization stability obtained by these devices is comparable to that of chemical anesthesia and the immobilization process does not affect lifespan, progeny production, or other aspects of animal health. The high-stability enables the use of a variety of key optical techniques. We use this to demonstrate femtosecond-laser nanosurgery and three-dimensional multiphoton microscopy. Used alone or in various combinations these devices facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond-laser nanosurgery for large-scale in vivo neural degeneration and regeneration studies.

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“…The same configuration has been previously used for the nonlinear imaging of Caenorhabditis elegans neurons 16,17 . The beam from a femtosecond t-pulse laser (Amplitude Systemes product -high power femtosecond oscillator) is directed to a modified optical microscope (Nicon Eclipse ME600D) using appropriate dichroic mirrors (the last one with 99% reflectivity at 1000 nm) and tight focused into the samples by means of an objective (Nikon, 50X, NA 0.8).…”

Section: Nonlinear Microscopementioning

confidence: 99%

Combined two-photon excited fluorescence and second harmonic generation imaging microscopy of collagen structures

et al. 2006

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Collagen is known to be a very efficient producer of both second harmonic generation (SHG) and two-photon excited fluorescence and the combined use of those nonlinear signals is emerging as a new imaging probe to be used as a diagnostic tool. By recording structural information of collagen between different samples, the technique shows promising for the study of the distribution of collagen in tissue and for identifying pathologic conditions. Unique information about the molecular organization of collagen can be extracted from SHG and TPEF imaging data in several ways and we have initiated a systematic study of these issues. The main objectives of this work are to combine TPEF and SHG methodologies, in order to elucidate and quantify cross-linking and to describe a model of fibrils orientation within different samples. In this early approach we discuss fundamental principles governing SHG and TPEF and present the first results of applying these rules to collagen type I images analysis. By comparing signals between lyophilized and soluble collagen we validate that the SHG signal arises from dipolar interactions that are enhanced by the quaternary structure of collagen fibrils, while TPEF arises from fluorophores which are suggested to be products of cross-linking. Using a homogenization protocol of acid treated collagen gels we manage to produce SHG and TPEF active thin films, which characterized by means of their contrast capability. A home-built scanning microscope employing SHG and TPEF was used for the high-resolution imaging of endogenous SHG and TPEF signals, without exogenous dyes.

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Imaging of Caenorhabditis elegans neurons by second-harmonic generation and two-photon excitation fluorescence

Cited by 22 publications

References 46 publications

Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy

Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy

High-throughput on-chip in vivo neural regeneration studies using femtosecond laser nano-surgery and microfluidics

Combined two-photon excited fluorescence and second harmonic generation imaging microscopy of collagen structures

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