In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

Chu, Shi Wei; Chen, Szu Yu; Tsai, Tsung Han; Liu, Tzu-Ming; Lin, Cheng Yung; Tsai, Huai Jen; Sun, Chia‐Liang

doi:10.1364/oe.11.003093

Cited by 141 publications

(122 citation statements)

References 25 publications

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“…Using the nonlinear optical properties of the tissue, third-and second-harmonic generation (THG, SHG) are produced at the focus of a short-pulsed laser beam, revealing cellular structure with three-dimensional resolution. Implementations of combined SHG and THG imaging have often used excitation wavelengths around 1200 nm [2][3][4]. This choice was influenced by the relatively low absorption of this wavelength by biological tissues and the convenient placement of the SHG and THG around 600 and 400 nm, respectively, permitting the use of standard optics and detectors.…”

Section: Introductionmentioning

confidence: 99%

“…One application area where HGM has shown promise is in imaging for developmental biology [3,2,4]. In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, aberrations are introduced by the specimens themselves, particularly by thick specimens to which HGM is often applied [5]. As the harmonic generation is nonlinearly dependent on the excitation intensity, these factors could have a significant effect on the imaging performance.One application area where HGM has shown promise is in imaging for developmental biology [3,2,4]. In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida.…”

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Adaptive harmonic generation microscopy of mammalian embryos

et al. 2009

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Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Both the second-and third-harmonic intensity signals are used as the optimization metric. Aberration correction is performed to compensate both system-and specimen-induced aberrations by using an efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an improved signal level and resolution.Harmonic generation microscopy (HGM) was proposed as a method for label-free imaging of biological specimens [1]. Using the nonlinear optical properties of the tissue, third-and second-harmonic generation (THG, SHG) are produced at the focus of a short-pulsed laser beam, revealing cellular structure with three-dimensional resolution. Implementations of combined SHG and THG imaging have often used excitation wavelengths around 1200 nm [2][3][4]. This choice was influenced by the relatively low absorption of this wavelength by biological tissues and the convenient placement of the SHG and THG around 600 and 400 nm, respectively, permitting the use of standard optics and detectors. A disadvantage of this excitation wavelength is that high-numerical-aperture objectives required for such imaging are not typically specified for this wavelength. One should therefore expect reduced performance both in terms of transmission and aberrations. Furthermore, aberrations are introduced by the specimens themselves, particularly by thick specimens to which HGM is often applied [5]. As the harmonic generation is nonlinearly dependent on the excitation intensity, these factors could have a significant effect on the imaging performance.One application area where HGM has shown promise is in imaging for developmental biology [3,2,4]. In early-stage mouse embryos, THG images reveal cellular structure and sub-cellular features, whereas SHG has shown mitotic spindles and the zona pellucida. It has also been shown that large aberrations are induced when focussing through embryos [5]. When combined with the system aberrations arising from the nonoptimal objective lenses, the effects can cause a significant reduction in signal level and resolution. Adaptive optics has been used to compensate for aberrations in various microscopes [6]. In this Letter we demonstrate the use of adaptive optics for the correction of system and specimen-induced aberrations in the HGM of live mouse embryos. OCIS codes: 180.0180, 180.4315, 110.1080, 170.3880, 180.6900, 190.4160. A schematic of the adaptive microscope is shown in Fig. 1. The chromium forsterite laser (Mavericks, Del Mar Photonics) emits 65 fs pulses at a repetition rate of 100 MHz, center wavelength λ=1235 nm and output power around 200 mW. The expanded beam was steered by two-axis galvanometer mirrors that are imaged onto a deformable membrane mirror (MIRAO 52-e, Imagine Eyes). The deformable mirror (DM) was imaged onto the pupil plane of the microscope objective. The illumination power in the focus was approximately 30 mW. The harmonic emiss...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Adaptive harmonic generation microscopy of mammalian embryos

et al. 2009

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“…However, THG microscopy brings forward all interfaces between cellular structures with different nonlinear properties. Plasma and nuclear membranes emerge in THG microscopy images (8), as well as membrane-based organelles such as mitochondria (9). It consequently presents an unspecific image of the cellular morphology, without any information on the molecular composition (10).…”

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confidence: 99%

Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy

Hellerer

Axäng

Brackmann

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

289

290

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Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent antiStokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-␤) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.lipid metabolism ͉ nonlinear microscopy ͉ obesity

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“…Confocal laser scanning microscopy (CLSM) [67], two-photon fluorescence microscopy (2P-FM) [68][69][70][71][72], and higher harmonic generation (HHG) microscopy [73,74] are some examples of optical methods that have been applied in different fields of biological research. CLSM have significant axial and lateral resolutions, but it is limited to ~100 µm penetration depth due to high attenuation of visible/ultraviolet excitation light.…”

Section: Introductionmentioning

confidence: 99%

Novel Method of Noninvasive Detection and Assessment of Gas Emboli and DCS

Larin¹

2010

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information SSOCT has been developed. The system has an axial resolution of 10 µm, phase sensitivity of 0.03 radians, imaging depth of up to 6 mm in air, and in-depth scanning speed of 20 kHz for a single A-line. The performance of the sensing system was carefully evaluated in optical phantoms containing gas microbubbles with different diameter and tissues in vivo. Obtained results demonstrate that bubbles with diameter greater than 10 µm could be detected by both structural imaging and phase response whereas bubbles with diameters less than 10 µm could be detected by the phase response of the SSOCT with high sensitivity.Noninvasive, optical, microbbules, decompression AbstractNoninvasive functional imaging, monitoring and quantification of microbubbles forming in blood and tissues upon rapid changes in barometric pressure are extremely important for effective therapy and diagnostics of several diseases as well as for imaging and drug delivery projects. However, current techniques are unable of imaging and efficient detection of bubbles with diameter less than 50 micrometers. The goal of this proposal was to develop novel PhaseSensitive Swept Source Optical Coherence Tomography (PhS-SSOCT) technique capable of real-time, sensitive, accurate, and noninvasive imaging, monitoring, and quantification of microbubbles in tissues. During these studies, a novel phase resolved system based on Swept Source Optical Coherence Tomography (SSOCT) has been developed. The system has an axial resolution of 10 µm, phase sensitivity of 0.03 radians, imaging depth of up to 6 mm in air, and in-depth scanning speed of 20 kHz for a single A-line. The performance of the sensing system was carefully evaluated in optical phantoms containing gas microbubbles with different diameter. Obtained results demonstrate that bubbles with diameter greater than 10 µm could be detected by both structural imaging and phase response whereas bubbles with diameters less than 10 µm could be detected by the phase response of the SSOCT with high sensitivity. The accuracy for measurement of the diameter of gas microbubbles is limited to 10 µm in structural imaging and 0.01 µm in phase-sensitive monitoring. Preliminary studies were also performed in animals in vivo for the rapid assessment of the circulating microbubbles. The in vivo results demonstrate capability of the developed instrument to image bubbles with diameter above 8 µm.The results from the study indicate that the developed SSOCT system could be used to detect fast-moving microbubbles and warr...

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In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

Cited by 141 publications

References 25 publications

Adaptive harmonic generation microscopy of mammalian embryos

Adaptive harmonic generation microscopy of mammalian embryos

Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy

Novel Method of Noninvasive Detection and Assessment of Gas Emboli and DCS

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