This Perspective introduces the development and use of adaptive optics in correcting aberrations in deep optical imaging applications.
Deep imaging of live tissue
Watching biological processes in vivo has piqued interest for centuries. But the less-than-transparent nature of some of our favorite models and the need for imaging large volumes at high speed pose fundamental hurdles to in vivo optical imaging. Fortunately, advances in optical imaging techniques have helped overcome some of these challenges, making it possible to visualize processes such as early development in flies, beating of the heart in fish, and neural activity in the rodent brain with high spatial and temporal resolution.
This web collection features recent content from several Nature Research journals that has been selected by the editors at Nature Methods. The research papers and commentaries we highlight cover a representative, but certainly not comprehensive, set of methodological developments that facilitate imaging of biological processes within organisms and tissues. We hope you enjoy browsing this collection.
Image: E. Dewalt, Nature Research; T. Katsuki, D. Grover and R. Greenspan, University of California, San Diego; F. Cutrale and L. Trinh, University of Southern California, Los Angeles; R. Chhetri and P. Keller, Janelia Research Campus.
Reviews and Comment
Yang and Yuste review currently available technologies for optical imaging of neural circuits, comparing them to help researchers choose optimal ones for their applications.
This Review introduces the fundamental considerations for building a light sheet microscope, describes the pros and cons associated with available implementations, and offers practical advice for users.
A new set of imaging techniques that take advantage of scattered light may soon lead to key advances in biomedical optics, providing access to depths well beyond what is currently possible with ballistic light.
A maturing open hardware and open-source software movement seeks to expand DIY light-sheet microscopy.
Ji et al. review emerging microscopy technologies that enable large-volume imaging of neural circuits. Focusing on two-photon fluorescence microscopy, they explored critical factors that limit imaging speed and restrict image volume, and also discuss three-dimensional imaging methods and their applications in rapid volume imaging of neural activity.
Photoacoustic imaging (PAI) can bridge the gap between high resolution optical imaging and deep tissue imaging applications. This Review introduces PAI as well as various implementations for a range of biological applications.
Tiling light-sheet microscopy improves live cell imaging of multicellular organisms.
A combination of flexible imaging and computational methods opens new views into the dynamics of activity across large populations of neurons.
New instruments are needed to realize the potential of quantitative and systematic imaging of living samples. But what would such a microscope look like?
Light-sheet fluorescence microscopy techniques are enabling researchers to achieve dynamic, long-term imaging and three-dimensional reconstruction of specimens ranging from single cells to whole embryos.
Thin optical lattices can be used to generate light sheets in order to image dynamic processes at high spatial and temporal resolution.
vTwINS enables high-speed volumetric calcium imaging via a V-shaped point spread function and a dedicated data-processing algorithm. Song et al. apply this strategy to image population activity in the mouse visual cortex and hippocampus.
Ouzounov et al. report calcium imaging with three-photon microscopy in the mouse brain. The approach enabled noninvasive recording of activity with high spatial and temporal resolution from GCaMP6-labeled neurons located as deep as the hippocampus.
Hyper-Spectral Phasors allow unmixing of multiple signals even under conditions with low signal-to-noise ratios, and they enable highly multiplexed 5D imaging of live zebrafish embryos labeled with conventional fluorophores.
Two-photon scanning microscopy is inherently slow and thus limits volumetric calcium imaging. Prevedel et al. achieve increased volumetric imaging speed by tailoring the excitation volume via light sculpting.
Adaptive light-sheet microscopy improves imaging of live organisms by correcting for optical aberrations in real time.
Random-access line scanning enables neural activity to be monitored at high speed in neurons and dendrites that are sparsely distributed in three dimensions. The approach is demonstrated in behaving mice.
Simultaneous imaging of neural activity in large regions of the mouse brain at subcellular resolution is made possible with a wide field-of-view two-photon microscope.
Flyception is a tracking and imaging system that enables the monitoring of brain activity in freely walking fruit flies, making the analysis of calcium dynamics possible in studies of neural mechanisms such as those that underlie social behaviors.
IsoView microscopy achieves rapid isotropic-resolution imaging of large, nontransparent samples using simultaneous light-sheet illumination and fluorescence detection in four orthogonal directions.
This protocol enables microscopic imaging of organs in live mice by addressing tissue movement resulting from cardiac and respiratory cycles.
This protocol describes how to prepare plants for light-sheet microscopy. Seeds or seedlings are suspended in low-gelling-temperature agarose or they grow in Phytagel within glass capillaries or FEP tubes. This allows long-term growth and imaging of tissues with the Lightsheet Z.1 microscope.
An optical phase-locked ultrasound lens integrated into a two-photon microscope enables continuous volumetric imaging of biological processes in vivo.
Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms
A swept light-sheet microscopy scheme allows volumetric imaging of living samples at high speed.
Lecoq and colleagues introduce a two-photon microscope with two articulated arms that can image nearly any two brain regions, nearby or distant, simultaneously. They validate this new system by imaging calcium signals in two visual cortical areas in behaving mice, and find evidence suggesting activity fluctuations can propagate between cortical areas
An adaptive optics method using multiplexed light measurement and modulation in multiple pupil segments improves structural and functional in vivo imaging over large volumes in strongly scattering mouse brain with only a single aberration correction.
Near-infrared photoluminescence from carbon nanotubes makes it possible to optically image the vasculature in the brain directly through the skull.
A series of technical and analytical improvements to light sheet microscopy is described, permitting dynamic imaging of the beating zebrafish heart at cellular resolution.