Instantaneous 4D micro-particle image velocimetry (µPIV) via multifocal microscopy (MUM).

Multifocal microscopy (MUM), a technique to capture multiple fields of view (FOVs) from distinct axial planes simultaneously and on one camera, was used to perform micro-particle image velocimetry (µPIV) to reconstruct velocity and shear stress fields imposed by a liquid flowing around a cell. A diffraction based multifocal relay was used to capture images from three different planes with 630 nm axial spacing from which the axial positions of the flow-tracing particles were calculated using the image sharpness metric. It was shown that MUM can achieve an accuracy on the calculated velocity of around (0.52 ± 0.19) µm/s. Using fixed cells, MUM imaged the flow perturbations at sub-cellular level, which showed characteristics similar to those observed in the literature. Using live cells as an exemplar, MUM observed the effect of changing cell morphology on the local flow during perfusion. Compared to standard confocal laser scanning microscope, MUM offers a clear advantage in acquisition speed for µPIV (over 300 times faster). This is an important characteristic for rapidly evolving biological systems where there is the necessity to monitor in real time entire volumes to correlate the sample responses to the external forces.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis.

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

A 'pocket guide' to total internal reflection fluorescence.

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single-molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a 'one stop shop' explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

Simultaneous widefield single molecule orientation and FRET microscopy in cells.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy.

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser.

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.