Nanostructural Diversity of Synapses in the Mammalian Spinal Cord.

Functionally distinct synapses exhibit diverse and complex organisation at molecular and nanoscale levels. Synaptic diversity may be dependent on developmental stage, anatomical locus and the neural circuit within which synapses reside. Furthermore, astrocytes, which align with pre and post-synaptic structures to form 'tripartite synapses', can modulate neural circuits and impact on synaptic organisation.

Multi-photon attenuation-compensated light-sheet fluorescence microscopy.

Attenuation of optical fields owing to scattering and absorption limits the penetration depth for imaging. Whilst aberration correction may be used, this is difficult to implement over a large field-of-view in heterogeneous tissue. Attenuation-compensation allows tailoring of the maximum lobe of a propagation-invariant light field and promises an increase in depth penetration for imaging.

Reducing data acquisition for light-sheet microscopy by extrapolation between imaged planes.

Light-sheet fluorescence microscopy (LSFM) is a powerful technique that can provide high-resolution images of biological samples. Therefore, this technique offers significant improvement for three-dimensional (3D) imaging of living cells. However, producing high-resolution 3D images of a single cell or biological tissues, normally requires high acquisition rate of focal planes, which means a large amount of sample sections.

Light sheet microscopy with acoustic sample confinement.

Contactless sample confinement would enable a whole host of new studies in developmental biology and neuroscience, in particular, when combined with long-term, wide-field optical imaging. To achieve this goal, we demonstrate a contactless acoustic gradient force trap for sample confinement in light sheet microscopy. Our approach allows the integration of real-time environmentally controlled experiments with wide-field low photo-toxic imaging, which we demonstrate on a variety of marine animal embryos and larvae.

Three-photon light-sheet fluorescence microscopy.

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low-photodamage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime, enhancing its penetration depth.

Light-sheet microscopy with attenuation-compensated propagation-invariant beams.

Scattering and absorption limit the penetration of optical fields into tissue. We demonstrate a new approach for increased depth penetration in light-sheet microscopy: attenuation-compensation of the light field. This tailors an exponential intensity increase along the illuminating propagation-invariant field, enabling the redistribution of intensity strategically within a sample to maximize signal and minimize irradiation.

Multimode fibre based imaging for optically cleared samples.

Optical clearing is emerging as a popular approach particularly for studies in neuroscience. However the use of corrosive clearing solutions typically requires sophisticated objectives or extreme care with optical components chosen for single- or multi-photon imaging. In contrast to the use of complex, custom-made microscope objectives, we show that the use of a corrected multimode fibre (MMF) offers a route that is resistant to corrosion, can be used in clearing media, is not constrained by the refractive index of the immersion medium and offers flexible working distances.

Enhancement of image quality and imaging depth with Airy light-sheet microscopy in cleared and non-cleared neural tissue.

We have investigated the effect of Airy illumination on the image quality and depth penetration of digitally scanned light-sheet microscopy in turbid neural tissue. We used Fourier analysis of images acquired using Gaussian and Airy light-sheets to assess their respective image quality versus penetration into the tissue. We observed a three-fold average improvement in image quality at 50 μm depth with the Airy light-sheet.

Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.

A detailed microscopic analysis of renal podocyte substructure is essential to understand and diagnose nephrotic kidney disease. Currently only time consuming electron microscopy (EM) can resolve this substructure. We used structured illumination microscopy (SIM) to examine frozen sections of renal biopsies stained with an immunofluorescence marker for podocin, a protein localized to the perimeter of the podocyte foot processes and compared them with EM in both normal and nephrotic disease biopsies.

Multimode fibre: Light-sheet microscopy at the tip of a needle.

Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within.

A compact Airy beam light sheet microscope with a tilted cylindrical lens.

Light-sheet imaging is rapidly gaining importance for imaging intact biological specimens. Many of the latest innovations rely on the propagation-invariant Bessel or Airy beams to form an extended light sheet to provide high resolution across a large field of view. Shaping light to realize propagation-invariant beams often relies on complex programming of spatial light modulators or specialized, custom made, optical elements.

Light-sheet microscopy using an Airy beam.

Light-sheet microscopy facilitates rapid, high-contrast, volumetric imaging with minimal sample exposure. However, the rapid divergence of a traditional Gaussian light sheet restricts the field of view (FOV) that provides innate subcellular resolution. We show that the Airy beam innately yields high contrast and resolution up to a tenfold larger FOV.