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 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.
The use of ultrashort-pulsed lasers for molecule delivery and transfection has proved to be a non-invasive and highly efficient technique for a wide range of mammalian cells. This present study investigates the effectiveness of femtosecond photoporation in plant cells, a hard-to-manipulate yet agriculturally relevant cell type, specifically suspension tobacco BY-2 cells. Both spatial and temporal shaping of the light field is employed to optimise the delivery of membrane impermeable molecules into plant cells using a reconfigurable optical system designed to be able to switch easily between different spatial modes and pulse durations. The use of a propagation invariant Bessel beam was found to increase the number of cells that could be viably optoinjected, when compared to the use of a Gaussian beam. Photoporation with a laser producing sub-12 fs pulses, coupled with a dispersion compensation system to retain the pulse duration at focus, reduced the power required for efficient optical injection by 1.5-1.8 times when compared to a photoporation with a 140 fs laser output.