Jonathan Nylk

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. In contrast to the Bessel beam, which also provides an increased FOV, the Airy beams characteristic asymmetric excitation pattern results in all fluorescence contributing positively to the contrast, enabling a step change for light-sheet microscopy.

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. Here we present a straightforward and low-cost modification to the traditional light-sheet setup, based on the open-access light-sheet microscope OpenSPIM, to achieve Airy light-sheet illumination. This brings wide field single-photon light-sheet imaging to a broader range of endusers. Fluorescent microspheres embedded in agarose and a zebrafish larva were imaged to demonstrate how such a microscope can have a minimal footprint and cost without compromising on imaging quality.

Wavefront corrected light sheet microscopy in turbid media

Light sheet microscopy is a powerful method for three-dimensional imaging of large biological specimens. However, its imaging ability is greatly diminished by sample scattering and aberrations. Optical clearing, Bessel light modes, and background rejection have been employed in attempts to circumvent these deleterious effects. We present an in situ wavefront correction that offers a major advance by creating an “optimal” light sheet within a turbid sample. Crucially, we show that no tissue clearing or specialized sample preparation is required, and clear improvements in image quality and depth resolution are demonstrated both in Gaussian and Bessel beam-based light sheet modalities.

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. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

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. SIM images of normal glomeruli revealed curvilinear patterns of podocin densely covering capillary walls similar to podocyte foot processes seen by EM. Podocin staining of all nephrotic disease biopsies were significantly different than normal, corresponding to and better visualizing effaced foot processes seen by EM. The findings support the first potential use of SIM in the diagnosis of nephrotic disease.

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. We also used optical clearing to tune the scattering properties of the tissue and found that the improvement when using an Airy light-sheet is greater in the presence of stronger sample-induced aberrations. Finally, we used homogeneous resolution probes in these tissues to quantify absolute depth penetration in cleared samples with each beam type. The Airy light-sheet method extended depth penetration by 30% compared to a Gaussian light-sheet.

Development of a graded index microlens based fiber optical trap and its characterization using principal component analysis.

We demonstrate a miniaturized single beam fiber optical trapping probe based on a high numerical aperture graded index (GRIN) micro-objective lens. This enables optical trapping at a distance of 200μm from the probe tip. The fiber trapping probe is characterized experimentally using power spectral density analysis and an original approach based on principal component analysis for accurate particle tracking. Its use for biomedical microscopy is demonstrated through optically mediated immunological synapse formation.

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

Tailoring beams to overcome attenuation allows light-sheet microscopy to image at greater depth with enhanced contrast. 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. A key attribute of this method is that only minimal knowledge of the specimen transmission properties is required. We numerically quantify the imaging capabilities of attenuation-compensated Airy and Bessel light sheets, showing that increased depth penetration is gained without compromising any other beam attributes. This powerful yet straightforward concept, combined with the self-healing properties of the propagation-invariant field, improves the contrast-to-noise ratio of light-sheet microscopy up to eightfold across the entire field of view in thick biological specimens. This improvement can significantly increase the imaging capabilities of light-sheet microscopy techniques using Airy, Bessel, and other propagation-invariant beam types, paving the way for widespread uptake by the biomedical community.

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 excitation modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 μm diameter cellular spheroids. In addition, we show, through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams. OCIS codes (110.0180) Microscopy; (190.4180) Multiphoton processes; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy; (180.6900) Three-dimensional microscopy; (110.4100) Imaging through turbid media.

A compact optical fibre trap based on a GRIN microlens and a principal component analysis algorithm for trap characterization in noisy environments (Conference Presentation)

Widespread use of optical manipulation in combination with advanced imaging techniques will be accelerated by compact, optically simple approaches which are readily integrated into advanced microscopy platforms. For example, optical manipulation has been combined with confocal, multi-photon, and STED microscopes. However, these typically require addition of optical components into the existing beam paths of the microscope, increasing complexity and potentially compromising image quality. Optical fiber trapping (OFT) offers an ultra-compact and simple solution but compromises on trap quality due to the low numerical aperture (NA) and short manipulation distance of optical fibers. Tapered fibers can be fabricated but this further reduces the manipulation distance and requires access to specialist fabrication facilities. Here we present a compact, single-beam, high NA OFT probe design based on a graded-index (GRIN) micro-objective lens and single-mode fiber. The OFT probe uses only off-the-shelf components, enables optical trapping at a distance of 200μm from the probe tip, and is compatible with inverted imaging systems. A challenge with specialist imaging systems is the incompatibility between the specialist imaging modality of the platform and the imaging modality required for trap characterization, resulting in noisy and poor trap characterisation data. To overcome this challenge, we developed an adaptive image filter based on principal component analysis (PCA). The filter separates orthogonal degrees of motion in trap characterisation movies and strong stochastic noise can be removed before tracking, resulting in accurate characterisation. We demonstrate the use of this PCA image filter for in situ characterisation of the GRIN lens OFT probe.