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Fluorescence-based techniques are prevalent in studies of nanomedicine-targeting to cells and tissues. However, fluorescence-based studies are rarely quantitative, thus prohibiting direct comparisons of nanomedicine-performance across studies. With this Commentary , we aim to provoke critical thinking about experimental design by treating some often-overlooked pitfalls in 'quantitative' fluorescence-based experimentation. Focusing on fluorescence-labeled nanoparticles, we cover mechanisms like solvent-interactions and fluorophore-dissociation, which disqualify the assumption that ' a higher fluorescence readout ' translates directly to ' a better targeting efficacy '. With departure in recent literature, we propose guidelines for circumventing these pitfalls in studies of tissue-accumulation and cell-uptake, thus covering fluorescence-based techniques like bulk solution fluorescence measurements, fluorescence microscopy, flow cytometry, and infrared fluorescence imaging. With this, we hope to lay a foundation for more 'quantitative thinking' during experimental design, enabling (for example) the estimation and reporting of actual numbers of fluorescent nanoparticles accumulated in cells and organs. [Display omitted] • Stresses the importance of more quantitative approaches in fluorescence-based nanomedicine studies • Provides a brief introduction to some fundamental principles of fluorescence in simple language • Treats fluorescence-based measurements in bulk solution, microscopy, flow cytometry and tissue/body imaging • Highlights often-overlooked challenges related to interactions between fluorophores and the biological environment • Presents strategies toward counting actual numbers of nanoparticles in cells and organs [ABSTRACT FROM AUTHOR]

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information. [Display omitted] • Developed a novel assay to study the modes of action of membrane-active peptides. • Our flow cytometric high-throughput assay relies on label-free peptides. • Fluorescence microscopy, DLS and calcein release studies confirmed the validity of our assay. • Six very different membrane-active peptides were evaluated in our assay. [ABSTRACT FROM AUTHOR]