Fluorescence microscopy allows researchers to explore the living world at the cellular and subcellular scales with remarkable precision. However, as time passes, microscopes inevitably degrade: detectors become noisy, optical systems lose alignment, and image quality declines. This aging process can hinder long-term biological studies and quantitative analysis.

To address this challenge, a team of Engineers from IBDM and LIS (France-BioImaging Marseille node) developed μPIX, a new deep-learning algorithm based on generative artificial intelligence.

A smarter way to restore microscopy images

μPIX uses a specific type of AI called a Pix2Pix conditional Generative Adversarial Network (cGAN): this algorithm learns how to transform low-quality or noisy images into clean and high-quality ones, based on examples.

Figure: µPIX architecture is based on a Pix2Pix generative network. µPIX consists of two subnetworks: a generator, based on a UNet architecture with an EfficientNet-b0 backbone, and a discriminator (PatchGAN). During supervised training, a noisy image is input to the generator, which generate an image. This output is compared to the real clean image using a pixel-wise loss function (MSE). Pairs of real and generated images are then passed to the discriminator, which classifies them as real or fake using a binary cross-entropy loss (BCE). Both subnetworks are progressively refined through adversarial loss during training. In the inference phase, only the trained generator is used to generate clean images. (Bon, Gabriel, Sapède, Daniel, Matthews, Cédric and Daian, Fabrice. “μPIX: leveraging generative AI for enhanced, personalized and sustainable microscopy” Methods in Microscopy, 2025. https://doi.org/10.1515/mim-2024-0024)

Unlike conventional image processing algorithms, μPIX adapts its training to the characteristics of the microscope, making it personalized and highly precise.

It improves image quality while preserving fine structures and intensity relationships, which is essential for quantitative imaging.

Thanks to its capacities, it extends the usefulness of old equipment, offering a cost-effective and sustainable alternative to replacement.

Better results than existing tools

In their publication, the authors show that μPIX outperforms both traditional denoising methods and popular deep learning tools such as CARE or Cellpose3. 

It also improves downstream applications: using μPIX as a pre-processing step enhances segmentation accuracy by up to 3% compared to existing pipelines.

Reviving aging detectors

The team went one step further and applied μPIX to an ambitious task: restoring images from an outdated Multi-Alkali photodetector so they resemble those acquired with a high-performance GaAsP detector.

The results are impressive: μPIX manages to compensate for signal loss along the z-axis (represents the depth), recover structural information, and maintain a near-linear relationship between the predicted and original intensities, enabling quantitative analysis on images that would otherwise be considered obsolete.

From user-centered to hardware-centered AI

Unlike most AI tools that require users to train their own models, μPIX proposes a platform-centered paradigm: platforms train one model, tailored to their equipment, and provide it to their users. This approach reduces redundancy, improves consistency, and aligns with the principles of frugal and shared AI development.

The code and models are freely available on GitLab, and μPIX is already proving to be a useful asset for microscopy platforms seeking long-term performance with limited hardware budgets.

Read the full scientific article here: https://www.degruyterbrill.com/document/doi/10.1515/mim-2024-0024/html

Recently, Pierre Bourdoncle, head of the IMAG’IC platform at the Cochin Institute (Paris Centre Node), and his team published a new protocol for intravital imaging of calvarial bone marrow. Today, he tells us more about their research and how it can enhance the study of diseases like leukemia.

Could you tell us a little about yourself and the project?

As the head of the IMAG’IC platform at the Cochin Institute, we have consistently advanced intravital imaging through multiphoton microscopy. For the past 25 years, we have been dedicated to enhancing intravital imaging at the Cochin Institute, with a focus on improving synchronization, laser technology, and OPO (Optical Parametric Oscillator, ed.) systems.

Why is the calvarial bone marrow such an interesting model to study hematopoiesis and vascular dynamics?

The calvarial bone marrow is an interesting model for studying hematopoiesis and vascular dynamics due to its unique anatomical features. Its thin structure allows for high-resolution imaging, facilitating the observation of cellular interactions and vascular networks. Additionally, it is easily accessible, making it ideal for experimental manipulations and real-time monitoring. This model provides valuable insights into the complex processes of blood cell formation and vascular development.

Your team has developed a custom-made titanium cranial implant. What advantages does it offer compared to existing methods?

The stability of the imaging area has always been a major challenge in intravital microscopy. Indeed, the animal’s breathing and temperature variations complicate long-term acquisitions. Moreover, precise repositioning of the acquisition area over several days is essential for observing the evolution of the cellular environment. The development of titanium implants, as opposed to traditional resin 3D printing, allows for more robust fixation of the system to the microscope stage and, most importantly, limits the deformation of the implant.

What perspectives does this method open for the understanding of hematological diseases, such as leukemia?

This method opens significant perspectives for understanding hematological diseases like leukemia by enabling detailed visualization of disease progression and cellular interactions. It allows researchers to study the impact of treatments in real-time, enhancing the development of targeted therapies. Additionally, it facilitates the exploration of the bone marrow microenvironment’s role in disease pathogenesis.

What are your upcoming projects?

Following the same principle, we are collaborating with the company Ymetry to develop similar appendages adapted for soft organs. Our goal remains to maintain the acquisition area for as long as possible without any drift.

Click here to read the full scientific article!

The NeurImag cellular and molecular imaging Facility, member of the Paris Centre Node of France-BioImaging, has initiated the development of a new tool called ExoJ, in collaboration with the teams of Guillaume Van Niel (CRCI2NA, Nantes University), Frederik Verweij (Utrecht University), Thierry Galli (IPNP, Inserm, Université Paris Cité) and Junjun Liu (Shandong First Medical University).

What is ExoJ?

ExoJ is a plugin developed for the Fiji/ImageJ2 software, specifically designed to automate the reliable detection and analysis of exocytosis events from fluorescence microscopy images. Exocytosis is a cellular process where molecules or substances contained within a cell are released to the extracellular environment. This process involves the fusion of a vesicle, a membrane-bound sac, with the cell membrane. Once fused, the contents of the vesicle are expelled into the extracellular space.

How does ExoJ work?

ExoJ automatically identifies user-defined exocytosis events. It extracts key quantitative information such as the intensity, apparent size and duration of each event. ExoJ is fully parameterizable and configurable, making it suitable for studying different types of exocytosis, whatever the imaging modality (TIRF [1] and/or spinning disk [2]). ExoJ is a robust and reliable tool for analyzing large datasets!

What are the benefits of ExoJ?

ExoJ automates the detection of exocytosis events, considerably reducing analysis time compared with manual annotation. Moreover, the results obtained are reproducible, facilitating comparisons between different experiments. Finally, ExoJ is based on Fiji/ImageJ2, an open-source software widely used in the scientific community.

To read the article, click here.