# Protocol for measuring the Young’s modulus of organoids using atomic force microscopy

## TL;DR

This paper provides a detailed protocol for measuring the stiffness of organoids using atomic force microscopy, improving the reliability of biomechanical studies.

## Contribution

A standardized protocol for measuring organoid stiffness via AFM with optimized probe and force-curve analysis is introduced.

## Key findings

- A protocol combining OCT embedding and AFM improves organoid stiffness measurement reproducibility.
- Force-curve analysis using Hertz contact models enables accurate Young’s modulus quantification.
- The method addresses mechanical gaps in organoid studies, expanding biomechanical research capabilities.

## Abstract

Atomic force microscopy (AFM) is extensively applied to measure cell and tissue mechanics but lacks a standardized organoid stiffness assessment. Here, we present a protocol for quantifying the Young’s modulus of organoids via AFM, combining force-curve analysis with an optimized probe. We describe steps for preparing organoids, OCT embedding, slicing, AFM detection, and force-curve analysis. By mechanically addressing gaps in organoids, this protocol improves reproducibility and expands the capabilities of biomechanical research.

•Steps to perform atomic force microscopy on organoids•Instructions for embedding organoids with OCT gel•Guidance on analyzing AFM-measured organoid curves using Hertz contact models

Steps to perform atomic force microscopy on organoids

Instructions for embedding organoids with OCT gel

Guidance on analyzing AFM-measured organoid curves using Hertz contact models

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Atomic force microscopy (AFM) is extensively applied to measure cell and tissue mechanics but lacks a standardized organoid stiffness assessment. Here, we present a protocol for quantifying the Young’s modulus of organoids via AFM, combining force-curve analysis with an optimized probe. We describe steps for preparing organoids, OCT embedding, slicing, AFM detection, and force-curve analysis. By mechanically addressing gaps in organoids, this protocol improves reproducibility and expands the capabilities of biomechanical research.