Nature Methods, Published online: 19 May 2026; doi:10.1038/s41592-026-03080-3

StringTie3 shows enhanced performance in total RNA-seq assembly and quantification by modeling both nascent and mature transcripts, across short-, long- and hybrid-read sequencing data.

Nature Methods, Published online: 18 May 2026; doi:10.1038/s41592-026-03084-z

This Perspective discusses current challenges in macromolecular ensemble prediction and the infrastructure and methodological advances needed to overcome these barriers.

Nature Methods, Published online: 14 May 2026; doi:10.1038/s41592-026-03083-0

This Perspective provides minimum community standards and best-practice guidelines for hydroxyl radical protein footprinting–mass spectrometry, including experimental design, sample preparation and oxidation, processing of oxidized samples, and analysis and interpretation of the resulting data.

Nature Methods, Published online: 14 May 2026; doi:10.1038/s41592-026-03071-4

This resource leverages spatial and single-cell transcriptomics to investigate lncRNAs in cancer and reports 94,795 previously unannotated lncRNAs and new approaches to infer their functions.

Nature Methods, Published online: 11 May 2026; doi:10.1038/s41592-026-03092-z

A device for simultaneous photometry and electrophysiology allows recordings in freely moving mice without restriction or inhibition of movement or entanglement of the fiber and cables.

Nature Methods, Published online: 07 May 2026; doi:10.1038/s41592-026-03090-1

FILM is a mid-infrared photothermal microscopy variant that involves optical boxcar demodulation-based illumination, denoising and spectral deconvolution. Its relatively gentle nature allows imaging of metabolic processes in organelles in cell culture as well as in Caenorhabditis elegans.

Nature Methods, Published online: 07 May 2026; doi:10.1038/s41592-026-03091-0

Optical boxcar-enhanced fluorescence-detected mid-infrared photothermal microscopy (FILM), together with artificial intelligence-assisted data processing, maps the chemical composition of individual cell organelles in their native context. This technique uncovered metabolic heterogeneity of lysosomes and lysosomal changes during physiological aging and pathological conditions.

Nature Methods, Published online: 05 May 2026; doi:10.1038/s41592-026-03082-1

$${\rm{Micro}}{\mathbb{S}}{\rm{plit}}$$ M i c r o S p l i t is a computational unmixing method for multiplexed fluorescence imaging of up to four structures per imaging channel.

Nature Methods, Published online: 04 May 2026; doi:10.1038/s41592-026-03099-6

It is not certain that everything is uncertain. —Blaise Pascal

Nature Methods, Published online: 04 May 2026; doi:10.1038/s41592-026-03051-8

UDMT is a multi-animal tracker for behavioral research that is based on a transformer architecture, which negates the need for manually annotated training data. UDMT is showcased on datasets encompassing mice, rats, Drosophila, C. elegans and betta fish.

Nature Methods, Published online: 01 May 2026; doi:10.1038/s41592-026-03088-9

AI agent systems are now being built on top of large language models, and they can conduct autonomous analyses. Choosing among the offerings takes a balance of curiosity, excitement and skepticism.

Nature Methods, Published online: 30 April 2026; doi:10.1038/s41592-026-03089-8

This study investigates technical noise in Xenium spatial transcriptomics data, including transcript spillover, and introduces SPLIT to resolve mixed signals and enhance cell-type specificity.

Nature Methods, Published online: 29 April 2026; doi:10.1038/s41592-026-03086-x

eSIG-Net is an interaction language model that predicts the effects of mutations on protein interaction.

Nature Methods, Published online: 27 April 2026; doi:10.1038/s41592-026-03094-x

We observed that a stable self-localized ultrafast pencil beam can emerge inside a standard multimode fiber near the critical power. This beam enables fast, high-resolution, volumetric imaging of intact tissues and real-time tracking of protein uptake in a human blood–brain barrier model.

Nature Methods, Published online: 27 April 2026; doi:10.1038/s41592-026-03067-0

A self-localized, ultrafast pencil beam can be readily introduced into multiphoton microscopes and applied for near-diffraction-limited, high-throughput volumetric imaging in intact tissues and in vitro models.

Nature Methods, Published online: 27 April 2026; doi:10.1038/s41592-026-03079-w

Spatial 5mC-seq enables nonbiased, spatiotemporal analysis of the genome-wide methylome at a single-cell resolution.

Nature Methods, Published online: 21 April 2026; doi:10.1038/s41592-026-03068-z

This Perspective introduces the concept of multifactor authentication for extracellular vesicle (EV) analysis as an effective tool to resolve the heterogeneity problem that often confounds mechanistic understanding of EV biology.

Nature Methods, Published online: 21 April 2026; doi:10.1038/s41592-026-03061-6

NirFAP680 is a near-infrared fluorogen-activating protein that has an order of magnitude greater cellular brightness and superior photostability compared to currently available NIR FAPs and fluorescent proteins in both single- and two-photon excitation and allows robust imaging of proteins in live cells and in vivo.