WG 3 – Objectives
Illumination field uniformity is critical for quantitative imaging when comparing fluorescence intensities across a field-of-view (FOV) or a large tile of images capturing an entire sample. If the illumination is not constant over a large area, the fluorescence intensities will not represent the inherent fluorescence but rather the location within the image. Thus, WG3 aims at defining a set of universal protocols to assess the uniformity of illumination (i.e., “flatness” of field) over the FOV of any photon-based imaging system and allow for correction of any non-uniformity. These protocols will identify the necessary tools, the procedures required to perform the measurements, and the analysis methods required for their interpretation. WG3 will also define criteria regarding the cut-off for acceptability and the need for correction. A database will be created with ideal images of uniform fields-of-view from different microscope modalities and settings to be used as a reference by the community and validate the protocol and criteria.
WG 3 – Co-Chairs
WG 3 - Latest News
New QUAREP-LiMi Protocol: Monitoring Field Homogeneity
Working Group 3 (Field Homogeneity) is pleased to share a new open-access protocol: "Monitoring Field Homogeneity", authored by colleagues from institutions across Europe, Canada, and beyond.
Field homogeneity — the uniformity of illumination and detection across the field of view — is a cornerstone of quantitative fluorescence imaging. Changes in field homogeneity over time can be an early warning sign of problems in the optical path, such as misaligned light sources, a dirty or damaged objective, or aging light guides.
This protocol offers a straightforward, low-cost approach to measuring field homogeneity and tracking it over time, using widely available fluorescent sample materials. It covers recommended standard samples, sample preparation, QC image acquisition, quantification using the free MetroloJ QC Fiji plugin, and recommended testing frequency — and is broadly applicable across objective types and magnifications.
Importantly, this protocol is designed to monitor consistency on a single instrument over time, rather than for direct benchmarking across different microscopes.
Read the full protocol
QUAREP-LiMi WG3 was pleased to host a webinar presented by Daniel Schröder on the topic of the "Universal Laser Engine" with an improved homogeneous illumination profile.
The webinar introduced a versatile laser excitation platform designed for advanced fluorescence microscopy and highlighted its applications across multiple imaging modalities.
Abstract
The Universal Laser Engine is a user-friendly laserbox designed to provide a versatile excitation for advanced fluorescence microscopy. In a single box, it holds up to 6 laser wavelengths operating in modes ranging from picosecond-pulsed to continuous-wave, enabling its use across a wide range of imaging modalities. The Universal Laser Engine has been successfully tested in widefield microscopy, super-resolution localization microscopy, total internal reflection fluorescence (TIRF), and fluorescence lifetime imaging microscopy (FLIM). In addition, the Universal Laser Engine incorporates a novel speckle-suppression technology that generates a highly homogeneous flat-top hat profile that is applicable for high-speed imaging at exposure times down to microseconds. The system provides various trigger inputs and two fiber outputs for seamless integration into both existing and new microscopes. By combining flexibility, compact design, and state-of-the-art illumination uniformity, the Universal Laser Engine offers a powerful and adaptable solution for simplifying and modernizing fluorescence microscopy instrumentation.
We welcome a new member to our working groups 2, 3, 10, 11 and 12. Mr. Aleš Kladnik from, the Biotechnical Faculty of University of Ljubljana in Slovenia just joined QUAREP-LiMi.
Last meeting: 12th March 2026, 5:00 pm
Memberlist WG 3