An open-source alignment method for multichannel infinite-conjugate microscopes using a ray transfer matrix analysis model.

Multichannel, infinite-conjugate optical systems easily allow implementation of multiple image paths and imaging modes into a single microscope. Traditional optical alignment methods which rely on additional hardware are not always simple to implement, particularly in compact open-source microscope designs. We present here an alignment algorithm and process to position the lenses and cameras in a microscope using only image magnification measurements. We show that the resulting positioning accuracy is comparable to the axial resolution of the microscope. Ray transfer matrix analysis is used to model the imaging paths when the optics are both correctly and incorrectly aligned. This is used to derive the corresponding image magnifications. We can then extract information about the lens positions using simple image-based measurements to determine whether there is misalignment of the objective lens to sample distance (working distance) and with what magnitude and direction the objective lens needs to be adjusted. Using the M4All open-source 3D printable microscope system in combination with the OpenFlexure microscope, we validate the alignment method and highlight its usability. We provide the model and an example implementation of the algorithm as an open-source Jupyter Notebook. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Low-cost, open-access quantitative phase imaging of algal cells using the transport of intensity equation.

Phase microscopy allows stain-free imaging of transparent biological samples. One technique, using the transport of intensity equation (TIE), can be performed without dedicated hardware by simply processing pairs of images taken at known spacings within the sample. The resulting TIE images are quantitative phase maps of unstained biological samples. Therefore, spatially resolved optical path length (OPL) information can also be determined. Using low-cost, open-source hardware, we applied the TIE to living algal cells to measure their effect on OPL. We obtained OPL values that were repeatable within species and differed by distinct amounts depending on the species being measured. We suggest TIE imaging as a method of discrimination between different algal species and, potentially, non-biological materials, based on refractive index/OPL. Potential applications in biogeochemical modelling and climate sciences are suggested.

Nanodiamonds enable adaptive-optics enhanced, super-resolution, two-photon excitation microscopy.

Particles of diamond in the 5-100 nm size range, known as nanodiamond (ND), have shown promise as robust fluorophores for optical imaging. We demonstrate here that, due to their photostability, they are not only suitable for two-photon imaging, but also allow significant resolution enhancement when combined with computational super-resolution techniques. We observe a resolution of 42.5 nm when processing two-photon images with the Super-Resolution Radial Fluctuations algorithm. We show manipulation of the point-spread function of the microscope using adaptive optics. This demonstrates how the photostability of ND can also be of use when characterizing adaptive optics technologies or testing the resilience of super-resolution or aberration correction algorithms.

Adapting the 3D-printed Openflexure microscope enables computational super-resolution imaging.

We report on a 3D printed microscope, based on a design by the Openflexure project, that uses low cost components to perform fluorescence imaging. The system is sufficiently sensitive and mechanically stable to allow the use of the Super Resolution Radial Fluctuations algorithm to obtain images with resolution better than the diffraction limit. Due to the low-cost components, the entire system can be built for approximately $1200.

Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics.

When imaging through tissue, the optical inhomogeneities of the sample generate aberrations that can prevent effective Stimulated Emission Depletion (STED) imaging. This is particularly problematic for 3D-enhanced STED. We present here an adaptive optics implementation that incorporates two adaptive optic elements to enable correction in all beam paths, allowing performance improvement in thick tissue samples. We use this to demonstrate 3D STED imaging of complex structures in Drosophila melanogaster brains.

Is phase-mask alignment aberrating your STED microscope?

The performance of a stimulated emission depletion (STED) microscope depends critically upon the pupil plane phase mask that is used to shape the depletion focus. Misalignments of the mask create unwanted distortions of the depletion focus to the detriment of image quality. It is shown how the phase errors introduced by a misplaced mask are similar to coma aberrations. The effects are investigated analytically, through numerical modelling and experimental measurement. Strategies for the systematic alignment of the masks are discussed.

Solid immersion facilitates fluorescence microscopy with nanometer resolution and sub-ångström emitter localization.

Exploring the maximum spatial resolution achievable in far-field optical imaging, we show that applying solid immersion lenses (SIL) in stimulated emission depletion (STED) microscopy addresses single spins with a resolution down to 2.4 ± 0.3 nm and with a localization precision of 0.09 nm.

Femtoliter tunable optical cavity arrays.

Large arrays of uniform, precisely tunable, open-access optical microcavities with mode volumes as small as 2.2 µm(3) are reported. The cavities show clear Hermite-Gauss mode structure and display finesses up to 460, in addition to quality (Q) factors in excess of 10,000. The cavities are attractive for use in quantum optics applications, such as single atom detection and efficient single photon sources, and have potential to be extended for experiments in the strong coupling regime.

A high stability beam-scanning confocal optical microscope for low temperature operation.

We report on the design and performance of a high stability scanning confocal microscope for optical microscopy at low temperatures. By scanning the beam in a cold objective lens system, we achieve wide fields of view without compromising image quality. Photoluminescence from single nitrogen-vacancy centers in high purity diamond is used to illustrate the imaging and stability performance of the microscope.