Attenuation of optical fields owing to scattering and absorption limits the penetration depth into tissue. Whilst aberration correction may be used this is difficult to implement over a large field of view in heterogeneous tissue. Recently, the novel approach of attenuation-compensation of propagation-invariant light fields has shown increase in depth penetration for light-sheet microscopy. Here we show this powerful approach may be implemented in a facile manner utilizing a graded neutral density filter circumventing the need for expensive beam shaping apparatus. A ‘gold standard’ system utilizing a spatial light modulator for beam shaping is used to benchmark our low-cost implementation.
The use of exotic optical modes is becoming increasingly widespread in microscopy. Particularly, propagation-invariant beams, such as Airy and Bessel beams and optical lattices, have been particularly useful in light-sheet fluorescence microscopy (LSFM) as they enable high-resolution imaging over a large field-of-view (FOV), possess a resistance to the deleterious effects of specimen induced light scattering, and can potentially reduce photo-toxicity.
Although these propagation-invariant beams can resist the effects of light scattering to some degree, and there has been some interest in adaptive-optical methods to correct for beam aberrations when they cannot, scattering and absorption of the illuminating light-sheet limit the penetration of LSFM into tissues and results in non-uniform intensity across the FOV.
A new degree of control over the intensity evolution of propagation-invariant beams can overcome beam losses across the FOV, restoring uniform illumination intensity and therefore image quality. This concept is compatible with all types of propagation-invariant beams and is characterised in the context of light-sheet image quality.
Another property to control is the wavelength of light used. Optical transmission through tissue is greatly improved at longer wavelengths into the near-infrared due to reduced Rayleigh scattering and two-photon excitation has proved beneficial for imaging at greater depth in LSFM. Three-photon excitation has already been demonstrated as a powerful tool to increase tissue penetration in deep brain confocal microscopy, and when combined with beam shaping can also be a powerful illumination strategy for LSFM.
Recent progress in shaping optical fields for LSFM will be presented.
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.
Light-sheet microscopy (LSM) is an emergent fluorescence microscopy technique showing great promise for biomedical research. LSM enables rapid, high-contrast imaging of large specimens with high spatiotemporal resolution and minimal photo-damage. When imaging large specimens, the intensity of the light-sheet reduces across the field-of-view (FOV) due to absorption. This results in an image with spatially-variant intensity, affecting quantitative measurements, and ultimately limits the penetration depth of the illumination. Some existing approaches to alleviate this issue involve illuminating the sample from multiple directions or rotating the sample. These approaches are not always practical and restrict specimen choice.
Separately, propagation-invariant light modes have been used to develop high-resolution LSM techniques as they can overcome the natural divergence of a Gaussian beam, producing a thin and uniform light-sheet over long distances. Most notably, Bessel and Airy beam-based LSM techniques have been implemented.
For propagation-invariant beams, there exists a mapping between the transverse coordinate in the pupil plane of a lens, and the axial propagation in the focal plane. Spatially-variant amplitude modulation therefore offers control of the intensity of the beam with propagation.
In this paper, we report that such amplitude modulation in the pupil plane of an Airy LSM can yield a system which counteracts absorption of the light-sheet and gives uniform intensity across the FOV with a single acquisition and without restricting specimen choice. This technique is an alternative to, and may be complimented by, wavefront correction. We demonstrate this technique through numerical simulations and experimental validation in absorbing tissue phantoms.
Light-sheet fluorescence microscopy is quickly becoming one of the cornerstone imaging techniques in biology as it provides rapid, three-dimensional sectioning of specimens at minimal levels of phototoxicity. It is very appealing to bring this unique combination of imaging properties into an endoscopic setting and be able to perform optical sectioning deep in tissues.
Current endoscopic approaches for delivery of light-sheet illumination are based on single-mode optical fibre terminated by cylindrical gradient index lens. Such configuration generates a light-sheet plane that is axially fixed and a mechanical movement of either the sample or the endoscope is required to acquire three-dimensional information about the sample. Furthermore, the axial resolution of this technique is limited to 5um.
The delivery of the light-sheet through the multimode fibre provides better axial resolution limited only by its numerical aperture, the light-sheet is scanned holographically without any mechanical movement, and multiple advanced light-sheet imaging modalities, such as Bessel and structured illumination Bessel beam, are intrinsically supported by the system due to the cylindrical symmetry of the fibre.
We discuss the holographic techniques for generation of multiple light-sheet types and demonstrate the imaging on a sample of fluorescent beads fixed in agarose gel, as well as on a biological sample of Spirobranchus Lamarcki.
Light-sheet microscopy (LSM) has received great interest for fluorescent imaging applications in biomedicine as it facilitates three-dimensional visualisation of large sample volumes with high spatiotemporal resolution whilst minimising irradiation of, and photo-damage to the specimen. Despite these advantages, LSM can only visualize superficial layers of turbid tissues, such as mammalian neural tissue. Propagation-invariant light modes have played a key role in the development of high-resolution LSM techniques as they overcome the natural divergence of a Gaussian beam, enabling uniform and thin light-sheets over large distances. Most notably, Bessel and Airy beam-based light-sheet imaging modalities have been demonstrated. In the single-photon excitation regime and in lightly scattering specimens, Airy-LSM has given competitive performance with advanced Bessel-LSM techniques. Airy and Bessel beams share the property of self-healing, the ability of the beam to regenerate its transverse beam profile after propagation around an obstacle. Bessel-LSM techniques have been shown to increase the penetration-depth of the illumination into turbid specimens but this effect has been understudied in biologically relevant tissues, particularly for Airy beams. It is expected that Airy-LSM will give a similar enhancement over Gaussian-LSM. In this paper, we report on the comparison of Airy-LSM and Gaussian-LSM imaging modalities within cleared and non-cleared mouse brain tissue. In particular, we examine image quality versus tissue depth by quantitative spatial Fourier analysis of neural structures in virally transduced fluorescent tissue sections, showing a three-fold enhancement at 50 μm depth into non-cleared tissue with Airy-LSM. Complimentary analysis is performed by resolution measurements in bead-injected tissue sections.
Nephrotic disease is a group of debilitating and sometimes lethal diseases affecting kidney function, specifically the loss of ability to retain vital proteins in the blood while smaller molecules are removed through filtration into the urine. Treatment routes are often dictated by microscopic analysis of kidney biopsies. Podocytes within the glomeruli of the kidney have many interdigitating projections (foot processes), which form the main filtration system. Nephrotic disease is characterised by the loss of this tightly interdigitating substructure and its observation by electron microscopy (EM) is necessitated as these structures are typically 250500nm wide, with 40nm spacing. Diagnosis by EM is both expensive and time consuming; it can take up to one week to complete the preparation, imaging, and analysis of a single sample. We propose structured illumination microscopy (SIM) as an alternative, optical diagnostic tool. Our results show that SIM can resolve the structure of fluorescent probes tagged to podocin, a protein localised to the periphery of the podocyte foot processes. Three-dimensional podocin maps were acquired in healthy tissue and tissue from patients diagnosed with two different nephrotic disease states; minimal change disease and membranous nephropathy. These structures correlated well with EM images of the same structure. Preparation, imaging, and analysis could be achieved in several hours. Additionally, the volumetric information of the SIM images revealed morphological changes in disease states not observed by EM. This evidence supports the use of SIM as a diagnostic tool for nephrotic disease and can potentially reduce the time and cost per diagnosis.
Light sheet microscopy has seen a resurgence as it facilitates rapid, high contrast, volumetric imaging with minimal sample exposure. Initially developed for imaging scattered light, this application of light sheet microscopy has largely been overlooked but provides an endogenous contrast mechanism which can complement fluorescence imaging and requires very little or no modification to an existing light sheet fluorescence microscope. Fluorescence imaging and scattered light imaging differ in terms of image formation. In the former the detected light is incoherent and weak whereas in the latter the coherence properties of the illumination source, typically a laser, dictate the coherence of detected light, but both are dependent on the quality of the illuminating light sheet. Image formation in both schemes can be understood as the convolution of the light sheet with the specimen distribution. In this paper we explore wavefront shaping for the enhancement of light sheet microscopy with scattered light. We show experimental verification of this result, demonstrating the use of the propagation invariant Bessel beam to extend the field of view of a high resolution scattered light, light sheet microscope and its application to imaging of biological super-cellular structures with sub-cellular resolution. Additionally, complementary scattering and fluorescence imaging is used to characterize the enhancement, and to develop a deeper understanding of the differences of image formation between contrast mechanisms in light sheet microscopy.
High density micron sized aerosols from liquid surfaces were generated using surface acoustic wave (SAW)
nebulisation. The SAWs are made from a set of interdigitated electrodes (IDT) deposited on a lithium niobate (LiNbO3)
substrate and are designed to operate around 10MHz. RF powers of ~235mW are used to achieve nebulisation. Power
below this results in droplet motion across the substrate surface. The nebulisation process generated aerosols of a narrow
size distribution with diameter ranging from 0.5-2 μm. We consider ways in which these aerosols can be loaded into
optical traps for further study. In particular we look at how SAW nebulisation can be used to load particles into a trap in
a far more robust manner than a conventional nebuliser device. We demonstrate trapping of a range of particle types and
sizes and analyse the size distribution of particles as a function of the applied frequency to the SAW device. We show
that it is simpler to load, in particular, solid particles into optical traps using this technique compared to conventional
nebulisation. We also consider the possibilities for loading nanoparticles into aerosol optical tweezers.
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