Fully automated point spread function analysis using PyCalibrate.

Reproducibility is severely limited if instrument performance is assumed rather than measured. Within optical microscopy, instrument performance is typically measured using sub-resolution fluorescent beads. However, the process is performed infrequently as it is requires time and suitably trained staff to acquire and then process the bead images.

Understanding the limits of remote focusing.

It has previously been demonstrated in both simulation and experiment that well aligned remote focusing microscopes exhibit residual spherical aberration outside the focal plane. In this work, compensation of the residual spherical aberration is provided by the correction collar on the primary objective, controlled by a high precision stepper motor. A Shack-Hartmann wave front sensor is used to demonstrate the magnitude of the spherical aberration generated by the correction collar matches that predicted by an optical model of the objective lens.

Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model.

Rigorous record-keeping and quality control are required to ensure the quality, reproducibility and value of imaging data. The 4DN Initiative and BINA here propose light Microscopy Metadata specifications that extend the OME data model, scale with experimental intent and complexity, and make it possible for scientists to create comprehensive records of imaging experiments.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted.

Random access parallel microscopy.

We introduce a random-access parallel (RAP) imaging modality that uses a novel design inspired by a Newtonian telescope to image multiple spatially separated samples without moving parts or robotics. This scheme enables near-simultaneous image capture of multiple petri dishes and random-access imaging with sub-millisecond switching times at the full resolution of the camera. This enables the RAP system to capture long-duration records from different samples in parallel, which is not possible using conventional automated microscopes.

Sensitivity of remote focusing microscopes to magnification mismatch.

Remote focusing (RF) is a technique that greatly extends the aberration-free axial scan range of an optical microscope. To maximise the diffraction limited depth range in an RF system, the magnification of the relay lenses should be such that the pupil planes of the objectives are accurately mapped on to each other. In this paper we study the tolerance of the RF system to magnification mismatch and quantify the amount of residual spherical aberration present at different focusing depths.

Spinning disk-remote focusing microscopy.

Fast confocal imaging was achieved by combining remote focusing with differential spinning disk optical sectioning to rapidly acquire images of live samples at cellular resolution. Axial and lateral full width half maxima less than 5 µm and 490 nm respectively are demonstrated over 130 µm axial range with a 256 × 128 µm field of view. A water-index calibration slide was used to achieve an alignment that minimises image volume distortion.

Microscope calibration using laser written fluorescence.

There is currently no widely adopted standard for the optical characterization of fluorescence microscopes. We used laser written fluorescence to generate two- and three-dimensional patterns to deliver a quick and quantitative measure of imaging performance. We report on the use of two laser written patterns to measure the lateral resolution, illumination uniformity, lens distortion and color plane alignment using confocal and structured illumination fluorescence microscopes.

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation.

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation.

Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen.

Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement.

Designing a holographic modal wavefront sensor for the detection of static ocular aberrations.

The extent to which holographic modal wavefront sensing can be applied to the detection of ocular aberrations was investigated. First, the idea of extending the dynamic range of the sensor by increasing the mask bias and the collection area of the pinhole detectors used in the sensor is reviewed. Errors in the detection of single-mode aberrations owing to reduced coherence from retinal scattering, photon, readout, and quantization noise are evaluated.