No abstract

Electrophysiological studies of symmetry have found a difference wave termed the Sustained Posterior Negativity (SPN) related to the presence of symmetry. Yet the extent to which the SPN is modulated by luminance-polarity and colour content is unknown. Here we examine how luminance-polarity distribution across the symmetry axis, grouping by luminance polarity, and the number of colours in the stimuli, modulate the SPN. Stimuli were dot patterns arranged either symmetrically or quasi-randomly. There were several arrangements: ’segregated’-symmetric dots were of one polarity and randomly-positioned dots were of the other; ‘unsegregated’-symmetric dots were of both polarities in equal proportions; ‘anti-symmetric’-dots were of opposite polarity across the symmetry axis; ‘polarity-grouped anti-symmetric’-this is the same as anti-symmetric but with half the pattern of one polarity and the other half of opposite polarity; multi-colour symmetric patterns made of two, three to four colours. We found that the SPN is: (i) reduced by the amount of position-symmetry, (ii) sensitive to luminance-polarity mismatch across the symmetry axis, and (iii) not modulated by the number of colours in the stimuli. Our results show that the sustained nature of the SPN coincides with the late onset of a topographic microstate sensitive to symmetry. These findings emphasise the importance of not only position symmetry, but also luminance polarity matching across the symmetry axis.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

No abstract

This paper demonstrates multiphoton excited fluorescence imaging through a polarisation maintaining multicore fiber (PM-MCF) while the fiber is dynamically deformed using all-proximal detection. Single-shot proximal measurement of the relative optical path lengths of all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.

Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of 1.41 over standard widefield imaging. By scanning point--wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non--standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co--ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and by pinholing around the detected maxima, recover the signal across the whole field of view.

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.