Media 1: Equilibrium orientations and positions of non-spherical particles in optical traps

Originally published in Optics Express on 04 June 2012 (oe-20-12-12987

Supplement 1: Brownian fluctuations of an optically rotated nanorod

Supplement 1 Originally published in Optica on 20 July 2017 (optica-4-7-746

Media 1: Driving corrugated donut rotors with Laguerre-Gauss beams

Originally published in Optics Express on 11 August 2014 (oe-22-16-19692

Acquisition and tracking of secretory vesicles in chromaffin cells and categorization of their motion.

<p>Chromaffin cells expressing hGH-GFP were imaged by confocal microscopy. (<b>A</b>) Six optical slices were acquired to obtain a 3 µm z-stack encompassing 20% of the total height of a chromaffin cell (<b>B</b>). (<b>C</b>) Time-coded prototypical trajectories of vesicles tracked for 100 sec illustrating the three different types of vesicular movement as indicated. (<b>D</b>) Time-coded prototypical vesicle trajectory showing a switch from caged behaviour (blue) to directed motion. (<b>E</b>) Comparison of percentages of vesicles in each of the three different motion pools in control conditions and during nicotine (10 µM) stimulation (N = 7 cells, n = 1159 tracked vesicles). Note the significant increase in directed motion and the parallel decrease in the percentage of vesicles undergoing free diffusion. **p<0.01 (paired <i>t</i>-test), NS: not significant.</p

Blocking actin and microtubule polymerization interferes with the replenishment mechanism.

<p>(<b>A, B, C</b>) Chromaffin cells were incubated with cytochalasin D (10 µM) for 20 minutes before imaging and stimulation. (<b>A</b>) Comparison of percentages of vesicles lying in the three different pools in control conditions and during nicotine stimulation (10 µM) (N = 5 cells, n = 978 vesicles). (<b>B</b>) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. (<b>C</b>) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, relative to their distance from the plasma membrane. For analysis purposes, the cells have been divided in 0.5 µm zones. The central part of cells is not represented due to insufficient data and uncertainties regarding the closest membrane direction. (<b>D</b>, <b>E</b>, <b>F</b>) Chromaffin cells were incubated with nocodazole (20 µM) for 20 minutes before imaging and nicotine stimulation. (<b>D</b>) Comparison of percentage of vesicles lying in the three different pools in control conditions and during stimulation of exocytosis following nicotine (10 µM) stimulation (N = 4 cells, n = 629 vesicles). (<b>E</b>) Histogram of accumulated vesicle positions depending on their motion and distance from the membrane. (<b>F</b>) Data from stimulated cells were used to generate maps of the average percentage of vesicles in a given motion state, based on their distance from the plasma membrane.</p

Change in secretory vesicle instant speed in the subcortical conveyor and the cortical exchange zones.

<p>For all the tracked vesicles, instant speed at all tracking points of the vesicle was recorded with Imaris and sorted depending on the cellular location of the vesicle and its type of motion (subcortical transfer zone is 1.5–2.5 µm from the membrane, and cortical exchange zone is 0–1.5 µm from the membrane). The average instant speed of the vesicles in each given state was then calculated in the two areas. Note the significant change in instant speed of directed vesicles in both areas of the cell. Mean ± S.E.M.</p>*<p>p<0.05,</p>***<p>p<0.0001, ^NS: not significant.</p

Mapping of secretory vesicle behaviour and trajectories relative to the plasma membrane.

<p>(<b>A</b>) Example of a single confocal image of a chromaffin cell expressing hGH-GFP with an edge detection algorithm applied to every frame of the 3D movies to retrieve membrane position at all times. (<b>B</b>) Example of the map of the same cell generated after nicotine (10 µM) stimulation (only 3 min of acquisition is displayed) with colour-coded vesicle trajectories. The external black line represents the average detection of the plasma membrane for this cell and the internal lines denote the edges of the 0–1.5 µm and 1.5–2.5 µm zones. (<b>C</b>) Data from 7 stimulated cells (n = 724 vesicles) were used to generate maps of the average percentage of vesicles in a given motion state, based on their distance to the plasma membrane. Note that the chosen cell shape used for representation is prototypical. For analysis purposes, the cells have been divided into 0.5 µm zones. The central part of the cell is not represented due to insufficient data and uncertainties regarding the closest membrane. (<b>D</b>) Histogram of accumulated vesicle positions depending on their motion and distance to the membrane.</p