Glyn Nelson’s research while affiliated with Newcastle University and other places

The protocols in this collection describe how to measure and analyze the photon conversation factor (PCF photo-electrons/count), readnoise, and dynamic range of a light microscopy detection system; which can either be a point detector or an area detector, using an in-homogeneous detector illumination scheme (as opposed to uniform illumination). The collection includes protocols on how to prepare a suitable sample for the acquisition, how to acquire data, as well as a respective analysis protocol. There are three aims a detection system can be characterized for, briefly: Aim 1 - experiment QC: Characterize the microscope performance using detection settings that match the experiment. Aim 2 - instrument QC.: Monitoring of microscope performance over time for service purposes, to maintain image quality constant and at high level. Aim 3 - system characterization: Full characterization of detection path performance under the range of settings applied by users. For a more detailed description refer to protocol 1. Introduction - Background and Aims. This protocol collection represents the collective experience of over 100 imaging scientists and industry experts. Measurements made by our working group with these protocols will be available in a public database. Please note, that this is an evolving document, to be versioned and updated, based on community feedback and new data.

This protocol is the introduction to the protocols collection "Characterization of the Photon Conversion Factor, Dark Noise, and Dynamic Range of Light Microscope Detection Systems". Here we describe in more detail the mission statement of QUAREP-LiMi Working Group 2, different aims of detection system characterization, a short guide to the protocols in the collection, and detection system basic theory underlying the protocols in this collection.

This protocol describes how to create a high dynamic range fluorescent sample using basic office and microscopy lab tools. The protocol is based on the usage of the ink of a fluorescent text marker to create a thin fluorescent layer on a microscope cover glass (Olevsko et al., 2021). The result is a carrier with a thin, homogeneous fluorescent film with abrupt edges and a thickness in the order of a few micrometers. This type of sample can be used to make images with a smooth gradient going from maximum intensity to background, as described in the protocols for determination of a detector’s photon conversion factor.

This protocol describes the measurement procedure to produce dark and inhomogeneously illuminated images with a light microscope system equipped with area detectors. The protocol can be followed according to the three different aims of microscope characterization described in the introduction of the protocol collection "Characterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems". The protocol uses the microscope slide for inhomogeneous illumination described in protocol 2 and generates data which can be analyzed with protocol 5, to obtain the photon conversion factor, readnoise, and dynamic range of the detection system.

The aim of this analysis is to obtain various calibration metrics from inhomogenous calibration images using the photon transfer method (McFadden et al., 2022 and Heintzmann et al., 2016). The analysis is not completely automated, and relies on users to interpret the data, identify problems and scrutinize the results. It is generally unaware of the underlying detector technology, which can affect the results. This guide therefore intends to provide an overview of the method, the choices that a user must make, how to interpret the data, and common pitfalls that should be avoided. Note that the method significantly differs from other calibration methods that use varying light levels over time (Mullikin et al., 1994, van Vliet et al., 1998, and Murray et al., 2013). It necessitates different definitions and the results will also differ. The algorithm is based on that which is described in the supplementary material of McFadden et al., 2022. A more detailed overview of the photon transfer method can be found in Photon transfer (Janesick, 2007).

This protocol describes the measurement procedure to produce dark and inhomogeneous illuminated images with a scanning light microscope system equipped with point detectors. The protocol can be followed according to the three different aims of microscope characterization described in the introduction of the protocol collection "Characterization of the Photon Conversion Factor, Noise, and Dynamic Range of Light Microscope Detection Systems". The protocol uses the microscope slide for inhomogeneous illumination described in protocol 2 and generates data which can be analyzed with protocol 5, to obtain the photon conversion factor, readnoise, and dynamic range of the detection system.

Together with the molecular knowledge of genes and proteins, biological images promise to significantly enhance the scientific understanding of complex cellular systems and to advance predictive and personalized therapeutic products for human health. For this potential to be realized, quality-assured image data must be shared among labs at a global scale to be compared, pooled, and reanalyzed, thus unleashing untold potential beyond the original purpose for which the data was generated. There are two broad sets of requirements to enable image data sharing in the life sciences. One set of requirements is articulated in the companion White Paper entitled Enabling Global Image Data Sharing in the Life Sciences, which is published in parallel and addresses the need to build the cyberinfrastructure for sharing the digital array data. In this White Paper, we detail a broad set of requirements, which involves collecting, managing, presenting, and propagating contextual information essential to assess the quality, understand the content, interpret the scientific implications, and reuse image data in the context of the experimental details. We start by providing an overview of the main lessons learned to date through international community activities, which have recently made considerable progress toward generating community standard practices for imaging Quality Control (QC) and metadata. We then provide a clear set of recommendations for amplifying this work. The driving goal is to address remaining challenges and democratize access to everyday practices and tools for a spectrum of biomedical researchers, regardless of their expertise, access to resources, and geographical location.

This protocol focuses on measuring the microscope's lateral and axial resolution, essential for reporting size measurements of near-resolution limit objects or distances between them. Here resolution is expressed as the Full Width at Half Maximum (FWHM) of a measured Point Spread Function (PSF) of sub-resolution size beads. PSF is highly related to objective quality and condition, but also depends strongly upon other parameters ranging from sample preparation to signal detection. Monitoring PSF over time will identify possible aberrations in the system (e.g., damaged, unclean objective, defective or not adapted oil, etc.). We define test sample preparation, image acquisition, and data analysis protocols for point scanning and spinning disk confocal microscopes.