3D Nanoscope: a highly accessible, high-performance device for live cell nanoscopy

Project Information

3DNanoScope4All

Grant agreement ID: 101180631

DOI

10.3030/101180631

Project closed

EC signature date 24 April 2024

Start date 15 May 2024

End date 14 May 2026

Funded under

• Widening participation and spreading excellence

Total cost

No data

EU contribution

€ 156 778,56

Coordinated by

FUNDACAO GIMM - GULBENKIAN INSTITUTE FOR MOLECULAR MEDICINE
Portugal

Periodic Reporting for period 1 - 3DNanoScope4All (3D Nanoscope: a highly accessible, high-performance device for live cell nanoscopy)

Reporting period: 2024-05-15 to 2026-05-14

Summary of the context and overall objectives of the project

Understanding how cells function requires imaging molecular processes that are fast, three-dimensional and extremely small. Yet current microscopy methods struggle to capture such events in living cells without damaging them, and most cannot measure not only where molecules are, but also how they are oriented or organised within the membrane or cellular environment. This limits our ability to study key biological mechanisms, from how cells communicate to how pathogens interact with their hosts.

3DNanoScope4All aims to overcome these barriers by creating a new, accessible type of nanoscope that combines fast 3D imaging with single-molecule orientation sensitivity. The project integrates three emerging technologies:

1. Light Field Microscopy (LFM), which records 3D information in a single snapshot with minimal phototoxicity,

2. Super-Resolution Radial Fluctuations (SRRF), a computational method that extracts nanoscale detail from low-illumination image sequences, and

3. Single Molecule Orientation and Localization Microscopy (SMOLM), a family of methodologies that are capable of measuring the absolute 3D position of the emitting dipoles.

By merging these approaches, the project will deliver a versatile microscope capable of visualising dynamic cellular processes in three dimensions, while simultaneously measuring molecular order and orientation—a capability rarely achievable in live-cell imaging. This opens the door to studying how membranes reorganise, how proteins align or cluster, and how nanoscale structures evolve over time within their natural, living context.

The resulting technology will be broadly applicable across the life sciences. It will enable new insights into host–pathogen interactions, including how diverse viruses or bacteria engage with cell membranes, as well as into the behaviour of cellular organelles, membrane domains, and other nanoscale structures whose function depends on both spatial organisation and molecular orientation.

Designed around affordable components and open-source software, 3DNanoScope4All will make advanced 3D nanoscopy accessible to a wide community of researchers. The project supports European priorities in open science, innovation, and democratization of scientific technologies, and is expected to create lasting impact by empowering laboratories to explore dynamic nanoscale biology in ways that have previously been out of reach.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the action, all core scientific and technical objectives were completed, leading to the development of a 3D light-field nanoscope capable of fast volumetric imaging with single-molecule orientation sensitivity.

1. Light Field Microscope (LFM) construction and calibration

A custom LFM module was fully designed, assembled, and integrated into an inverted microscope. Several micro-lens arrays (MLAs) were produced using 3D-printed refractive elements, and their optical quality was validated. The system was calibrated using axial bead scans, generating the full 3D point spread function and transformation matrices required for precise volumetric reconstructions.

2. Development of SRRF-LFM for 3D super-resolution imaging

The Super-Resolution Radial Fluctuations (SRRF) algorithm was adapted to process light-field datasets, enabling 3D reconstructions from low-illumination bursts. The resulting SRRF-LFM pipeline produced isotropic nanoscale resolution in living samples with minimal phototoxicity. The method was benchmarked on simulated and experimental LFM datasets.

3. Implementation of SMOLM-LFM for molecular orientation sensing

A physical model of dipole emission through an MLA was developed, allowing extraction of single-molecule orientation from multi-perspective light-field images. Orientation-sensitive MLAs were designed and fabricated. Calibration was performed using supported lipid bilayers on stepped glass substrates, enabling quantitative validation of orientation accuracy across the axial range.

4. Biological application of the 3D nanoscope

The system was applied to biological model systems to quantify nanoscale membrane organisation and molecular order in 3D.
Main outcomes

A functional 3D light-field nanoscope combining dynamic super-resolution with single-molecule orientation sensitivity.

Validated SRRF-LFM and SMOLM-LFM algorithms, calibration datasets, and optical designs.

Demonstrated biological measurements showing 3D nanoscale organisation and dynamic molecular orientation in living cells.

A robust technological platform enabling widespread access to advanced 3D nanoscopy.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project pushed microscopy beyond the state of the art by enabling three-dimensional single-molecule orientation measurements within a light-field microscopy framework. Existing orientation-sensitive approaches rely on engineered PSFs, high light doses, and limited axial range, making them difficult to apply in living systems. In contrast, the method developed here—SMOLM-LFM—provides robust orientation readout throughout a full 3D volume while maintaining low phototoxicity and fast acquisition. This was achieved through a new physical model describing dipole emission through a multi-lens array, the design and fabrication of orientation-sensitive MLAs using 3D-printed refractive optics, and a complete calibration workflow based on stepped lipid bilayers to validate angular accuracy across depth.

The resulting system allows simultaneous retrieval of molecular position, orientation, and dynamic behaviour in living cells. This capability opens new possibilities for studying nanoscale membrane organisation, protein alignment, supramolecular assembly, and fast structural changes in processes such as host–pathogen interactions or organelle remodelling. By making 3D orientation sensitivity compatible with accessible hardware and open analysis tools, the project establishes a practical path toward widespread adoption of advanced molecular-order imaging.

To ensure future uptake, the next steps include improving orientation accuracy in complex cellular environments, validating the method across diverse biological systems, and exploring commercial pathways for MLA production and instrument modularisation. Standardised calibration protocols and community access to the software will further support reproducibility and integration into existing microscopy platforms.