Deep and fast live imaging with two-photon scanned light-sheet microscopy

Truong, Thai V.; Supatto, Willy; Koos, David S.; Choi, Joonhee; Fraser, Scott E.

doi:10.1038/nmeth.1652

Cited by 428 publications

(382 citation statements)

References 29 publications

(3 reference statements)

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“…In contrast, as with conventional TP imaging, the IR excitation light that is typically used can penetrate further into multicellular organisms and tissues than visible excitation. As a result, the TP mode is a good option when imaging deeply into scattering and/or aberrating specimens, and it can produce substantially thinner virtual light sheets than Gaussian TP beams 12 over a comparable field of view. Bessel beam structured plane illumination.…”

Section: Tp Bessel Beam Plane Illuminationmentioning

confidence: 99%

3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

et al. 2014

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3D live imaging is important for a better understanding of biological processes, but it is challenging with current techniques such as spinning-disk confocal microscopy. Bessel beam plane illumination microscopy allows high-speed 3D live fluorescence imaging of living cellular and multicellular specimens with nearly isotropic spatial resolution, low photobleaching and low photodamage. unlike conventional fluorescence imaging techniques that usually have a unique operation mode, Bessel plane illumination has several modes that offer different performance with different imaging metrics. to achieve optimal results from this technique, the appropriate operation mode needs to be selected and the experimental setting must be optimized for the specific application and associated sample properties. Here we explain the fundamental working principles of this technique, discuss the pros and cons of each operational mode and show through examples how to optimize experimental parameters. We also describe the procedures needed to construct, align and operate a Bessel beam plane illumination microscope by using our previously reported system as an example, and we list the necessary equipment to build such a microscope. assuming all components are readily available, it would take a person skilled in optical instrumentation ~1 month to assemble and operate a microscope according to this protocol. accompanied by slower imaging speed, faster photobleaching and higher photodamage, thereby limiting the number of time points for live imaging. TP fluorescence microscopy achieves optical sectioning and simultaneously avoids an out-of-focus background through nonlinear absorption of the excitation, but the imaging speed is still limited by point-scanned detection using the photomultiplier tube (PMT), and nonlinear mechanisms introduce additional photodamage. It also has a limited multicolor imaging capability. In practice, confocal and, more recently, spinning-disk confocal microscopy have been the most common methods for live 3D imaging of cellular-scale specimens. Selective plane illumination microscopy (SPIM)Although the technique is a century old 1 , the rapid development of SPIM over the last decade 2-9 was driven by the need for highspeed, low-photobleaching and noninvasive 3D live imaging. SPIM minimizes the out-of-focus excitation by using two objectives with optical axes orthogonal to each other separately for excitation and detection. The sample is illuminated from the side by sending a sheet of excitation light into the sample, so that the illumination is confined near the focal plane of the orthogonal detection objective (Fig. 1a). In comparison with epi-illumination methods, SPIM has the following advantages. First, the confined excitation provides optical sectioning automatically by producing minimal out-of-focus fluorescence background. Second, the method is as fast as conventional wide-field detection, as fluorescence is imaged across the entire excitation plane simultaneously. Third, if the sheet thickness is comparable...

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Section: Tp Bessel Beam Plane Illuminationmentioning

confidence: 99%

3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

et al. 2014

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“…Comparisons MC/DE for 〈z max |t〉, z t , 〈z max |ρ〉, z ρ , 〈z max 〉 and z . Figure 5a,b shows 〈 z max |t〉 and z t according to Eqs (17) and (30) Figure 5a shows that 〈 z max |t〉 grows rapidly for early times and more slowly for late times. Five ns are needed to penetrate at 30 mm depth in the medium.…”

Section: Comparisons Mc/de For F(z|t) and F(z|ρ)mentioning

confidence: 99%

Structural white-matter connections mediating distinct behavioral components of spatial neglect in right brain-damaged patients

Vaessen

Saj̈

Lövblad

et al. 2016

Cortex

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We propose a comprehensive statistical approach describing the penetration depth of light in random media. The presented theory exploits the concept of probability density function f(z|ρ, t) for the maximum depth reached by the photons that are eventually re-emitted from the surface of the medium at distance ρ and time t. Analytical formulas for f, for the mean maximum depth 〈z max 〉 and for the mean average depth z reached by the detected photons at the surface of a diffusive slab are derived within the framework of the diffusion approximation to the radiative transfer equation, both in the time domain and the continuous wave domain. Validation of the theory by means of comparisons with Monte Carlo simulations is also presented. The results are of interest for many research fields such as biomedical optics, advanced microscopy and disordered photonics.

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“…Therefore, in addition to the Bessel illumination, it is currently possible to use diffractive optics that generate a structured illumination convoluted to the Bessel beam (Gustafsson et al, 2008); this allows image acquisition below the diffraction limit (Gao et al, 2012). Moreover, this setup strongly benefits from a two-photon illumination with associated increased penetration and decreased photobleaching and/or phototoxicity (Truong et al, 2011); this drastically reduces the risk of adversely affecting the physiology of the event studied (Ahrens et al, 2013;Wolf et al, 2015). A recent, but main update in this field was provided by the group of Eric Betzig with the demonstration of the lattice light-sheet microscopy as an improvement of Bessel beam LSFM.…”

Section: Single Plane Illumination Microscopy (Spim)mentioning

confidence: 99%

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Follain

Mercier

Osmani

et al. 2016

Journal of Cell Science

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Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, superresolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

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Deep and fast live imaging with two-photon scanned light-sheet microscopy

Cited by 428 publications

References 29 publications

3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

Structural white-matter connections mediating distinct behavioral components of spatial neglect in right brain-damaged patients

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

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