Background

Fluorescence microscopy serves as an essential tool in a variety of research fields, offering the flexibility to analyze specimens ranging from in vitro to in vivo, with resolutions spanning from micrometers to nanometers [1,2]. An advantage of this technique is the ability to detect individual fluorescent targets and either simultaneously or sequentially visualize multiple fluorescent markers. Applications include the characterization of fluorescently labeled purified proteins, as well as the examination of proteins and genes within 2D/3D cell culture systems, tissues, and whole organisms. Fluorescence microscopy is utilized in various in vitro studies, such as investigating the liquid–liquid phase separation of proteins using fluorescently labeled purified proteins [3,4], examining spindle formation through droplet assays [5], and employing fluorescence resonance energy transfer (FRET) to analyze protein–protein interactions [6] and measure mechanical tension [7,8]. Its utility in developmental biology extends to research across a variety of models, including plants, Drosophila, Xenopus, C. elegans, zebrafish, and mice [9]. Moreover, fluorescence microscopy is a cornerstone in cell biology research, enabling the investigation of cellular processes in organisms from yeast to humans, across both 2D and 3D culture systems, as well as in living and fixed tissue samples [10,11]. This technique allows researchers to accurately determine spatial locations, relative localizations, dimensions, and shapes at the nanoscale. For instance, it has been demonstrated that standard confocal microscopy can achieve <5 nm precision in determining 2D/3D protein architecture within cells using a dual-color fluorescence approach [12,13]. Furthermore, fluorescence microscopy excels in quantifying protein copy numbers and assessing relative protein concentrations with high accuracy [14–16]. The resolution of fluorescence microscopy is constrained by the point spread function (PSF), which represents the 3D diffraction pattern of light emitted from an infinitely small point source [17]. An ideal fluorescence microscope, equipped with a high numerical aperture (NA) objective lens (NA > 1.4) and a high-resolution camera, can achieve resolutions of approximately 250 nm laterally and 600 nm axially [18,19]. However, the maximum resolution is also contingent on the signal-to-noise ratio (SNR) of the specimens. These resolution limits can be enhanced without altering the optical setup through expansion microscopy (ExM), which physically expands specimens without changing the optics setup, or through post-imaging processing techniques, such as deconvolution [18,20,21]. Additionally, ongoing developments in super-resolution microscopy significantly improve resolutions compared to conventional fluorescence microscopes, enabling the further detailed study of nano-scale cellular structures [1,22–24].

All fluorescence microscopes have the potential to achieve their theoretical resolution maximum and quantitative accuracy. However, this potential heavily relies on the components and the conditions under which microscopy is conducted. To understand the current conditions of the imaging system, it is critical to routinely perform calibrations. We have recently developed the 3D-Speckler software, which enables precise quantification of fluorescence puncta in both biological and non-biological samples [18]. Utilizing fluorescence beads of known sizes in conjunction with 3D-Speckler allows for the comprehensive evaluation of resolution limits, size quantification accuracy, chromatic aberration determination, and the flatness of illumination produced by imaging systems. The aim of this article is to present a comprehensive guide on utilizing the 3D-Speckler software for calibrating imaging systems. This guide is intended for a broad audience interested in fluorescence microscopy, rather than experts in optics and mathematics. Consequently, this protocol minimizes the explanation of the coding and mathematical principles behind 3D-Speckler. For detailed information, please refer to our original manuscript [18]. Briefly, 3D-Speckler identifies fluorescence particles primarily based on their relative signal intensities against the background and determines particle sizes through a full-width-at-half-maximum (FWHM) calculation based on a 2D or 3D Gaussian profile fitted to the intensity profile. The intensity measurements for fluorescence beads can be performed either without background correction or with a local background correction for each bead [14]. 3D-Speckler can determine the center of fluorophores with ~2 nm accuracy, which allows it to accurately identify chromatic aberrations, an unavoidable image distortion that occurs between different wavelengths, by measuring the distance between the centers of fluorophores with different wavelengths within a single bead. This process is designed to evaluate their current performance and ensure their integrity, accuracy, and reproducibility in quantification tasks.