Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy

With potential relevance for brain-mapping work, hydrogel-based structures can now be built from within biological tissue to allow subsequent removal of lipids without mechanical disassembly of the tissue. This process creates a tissue-hydrogel hybrid that is physically stable, that preserves fine structure, proteins and nucleic acids, and that is permeable to both visible-spectrum photons and exogenous macromolecules. Here we highlight relevant challenges and opportunities of this approach, especially with regard to integration with complementary methodologies for brain-mapping studies.

CLARITY for mapping the nervous system

The coupling of optical waveguides to ring resonators holds the promise of a new generation of switches (modulators) which employ orders of magnitude smaller switching (modulation) voltages (or control intensities). This requires a means for voltage (or intensity) control of the coupling between the waveguide and the resonator. Schemes for achieving such control are discussed.

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Critical coupling and its control in optical waveguide-ring resonator systems

Simultaneous multiview light-sheet microscopy using two illumination and two detection arms with one- or two-photon illumination is coupled to a fast data acquisition framework and analysis pipeline for quantitative imaging and tracking of individual cells and the developing nervous system throughout a living fly embryo. A related paper by Krzic et al. is also in this issue.

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy.

We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

https://www2.le.ac.uk/departments/npb/news-1/journal/nphoton

Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology.

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

By controlling the internal loss of a ring resonator near critical coupling, we demonstrate control of the transmitted power in a fiber that is coupled to the resonator. We also demonstrate wavelength-selective optical amplification and oscillation.

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Control of critical coupling in a ring resonator-fiber configuration: application to wavelength-selective switching, modulation, amplification, and oscillation.

Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos.

Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging

An apparatus for and method of performing multi-photon light sheet microscopy (MP-LISH), combining multi-photon excited fluorescence with the orthogonal illumination of light sheet microscopy are provided. With live imaging of whole Drosophila and zebrafish embryos, the high performance of MP-LISH compared to current state-of-the-art imaging techniques in maintaining good signal and high spatial resolution deep inside biological tissues (two times deeper than one-photon light sheet microscopy), in acquisition speed (more than one order of magnitude faster than conventional two-photon laser scanning microscopy), and in low phototoxicity are demonstrated. The inherent multi-modality of this new imaging technique is also demonstrated second harmonic generation light sheet microscopy to detect collagen in mouse tail tissue. Together, these properties create the potential for a wide range of applications for MP-LISH in 4D imaging of live biological systems.

Multiple-photon excitation light sheet illumination microscope

An apparatus for and method of performing orthogonal light sheet microscopy (OLM) and computer optical tomography (COT) simultaneously in a single device are provided The dual-mode imaging microscope allows for the use of both OLM and COT in a single instrument This dual-mode device will allow researchers to have access to both types of microscopy, allowing access to the widest possible selection of samples, and improved imaging results In addition, the device will reduce the high costs and space requirements associated with owning two different imagers (OLM and COT)

John M. Choi

Papers

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

Control of critical coupling in a ring resonator-fiber configuration: application to wavelength-selective switching, modulation, amplification, and oscillation.

Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging

Multiple-photon excitation light sheet illumination microscope

Simultaneous orthogonal light sheet microscopy and computed optical tomography