Following the previous article, here we describe the first field demonstration of the ELVIS system, performed at Newport Beach, CA. We examined ocean water to detect microorganisms using the combined holographic and light-field fluorescence microscope and successfully detected both eukaryotes and prokaryotes. The shared field of view provided simultaneous bright-field (amplitude), phase, and fluorescence information from both chlorophyll autofluorescence and acridine orange staining. The entire process was performed in a nearly autonomous manner using a specifically designed sample processing unit (SPU) and custom acquisition software. We also discuss improvements to the system made after the field test that will make it more broadly useful to other types of fluorophores and samples.

There is often a need to locate the same cellular structure of interest in light and electron microscopy, which can be a difficult task. Here we present a method that uses only commercially available reagents and standard epi-fluorescence and transmission electron microscopy (TEM) technology to make correlative light and electron microscopy (CLEM) available to a large group of researchers without specialized CLEM hardware. This was achieved by seeding cells on photo-etched gridded cover slips and staining the protein to be localized with a secondary antibody coupled to both a fluorophore and 10 nm gold. The presence of the grid allowed for the alignment of light microscopy images with TEM images and the double-labeled antibody revealed co-localization of the fluorophore with gold particles.

Based on technologies capable of data collection between the millimeter and nanometer scales, correlative imaging has been transforming how researchers obtain molecular and spatial information from specimens. Attempts to combine multidimensional data are often met with the challenge of overcoming suboptimal sample conditions such as reduced fluorescence signal, poor specimen preservation, anisotropic specimen deformation, and low specimen contrast. These issues motivated the development and use of enhanced sample preparation procedures, as well as specialized imaging software to overcome such challenges. In this work we present three simple methods to correlate optical and scanning electron microscopy images.

Technological advancements in the areas of sample illumination, image acquisition, and image processing have significantly improved the speed and sensitivity of fluorescence microscopy. In particular, light emitting diodes (LEDs) coupled to transistor-transistor logic (TTL) circuits have reduced photo-bleaching and photo-toxicity by limiting sample illumination to the camera exposure time. Unfortunately, many microscopes still rely on bulb-based light sources that cannot be configured with TTL. Moreover, even when TTL is enabled in conventional microscope software, hardware and software delays can still contribute to photo-toxicity and lead to additional delays between subsequent images, introducing errors in time lapse image recordings. The goal of the present article is to highlight the significance of these issues.

In microscopy and analytical instrumentation, a researcher or clinician may need to excite several fluorophores in a sample in order to generate a useful fluorescence map of the cell or tissue of interest. This typically would include a nuclear marker such as DAPI, a green-emitting fluorophore such as FITC or GFP, and a red-emitting fluorophore such as mCherry, TRITC, or Texas Red. These red-emitting dyes can be efficiently excited using an arc lamp with a strong peak in the 550 nm region and another at 580 nm. When technology in microscope light sources originally moved to light-emitting diodes (LEDs) in the early 2000s, fluorescence work in the green gap (540 to 590 nm) excitation range was a challenge with no solution. This article explains how technology has adapted to provide LEDs that match the excitation of fluorophores typically used in multiplex fluorescence imaging.

Biological imaging tools continue to increase in speed, scale, and resolution, often resulting in the collection of gigabytes or even terabytes of data in a single experiment. In comparison, the ability of research laboratories to store and manage this data is lagging greatly. This leads to limits on the collection of valuable data and slows data analysis and research progress. Here we review common ways researchers store data and outline the drawbacks and benefits of each method. We also offer a blueprint and budget estimation for a currently deployed data server used to store large datasets from zebrafish brain activity experiments using light-sheet microscopy. Data storage strategy should be carefully considered and different options compared when designing imaging experiments.

Electron diffraction is an essential characterization tool for materials scientists. When using the transmission electron microscope (TEM) to perform diffraction experiments, setting up the microscope for both calibration standards and unknown materials in a consistent method ensures that dependable results are obtained. Care must also be exercised to protect digital cameras from intense transmitted and diffracted beams to avoid damage. In Parts 1 and 2 of this series, procedures were presented for recording high-quality, well-calibrated, digital SAED patterns. In Part 3, aspects of diffraction analysis software packages and the reliability of data are discussed.

The smartphone microscope, or mobile microscope, is an interesting tool that is attracting attention in various fields. We report a model of mobile microscope that extends from the Leeuwenhoek-type of single microscope with a simple lens. The new mobile microscope is called the “L-eye mobile microscope” and is compact and easy to handle. The L-eye mobile microscope may become a hobby tool in everyday life for people of all ages but may also be a tool suitable in the education of medical or paramedical students. To test its potential as an educational tool, its performance was examined using samples such as permanent specimens of tissue sections from several organs used in histology and a standard target for a microscopy resolution test. As a control, we used a conventional education-use microscope that is mounted with a smartphone for image capture. From the performance comparison of the two kinds of microscope systems, the L-eye mobile microscope could take images with almost the same quality as those obtained with the conventional microscope at moderate magnifications. This result suggests the usefulness of the L-eye mobile microscope for education.