“Multimodal multiphoton nanoscopy using SPAD array detectors and adaptive optics”

Project description:

Modern biology and medicine increasingly rely on advanced imaging techniques. It is through microscopy that we have become able to observe cellular structures, follow processes occurring within tissues, and study disease at a level that was out of reach just a decade ago. Despite this remarkable progress, many imaging methods still face significant limitations, particularly when the goal is to image deep within tissue while maintaining high resolution and minimizing disturbance to the biological sample.

The aim of this project is to develop a new class of microscopy methods that enable precise imaging of biological structures at substantial depths (greater than 50 µm). The proposed system will be based on multiphoton microscopy, in which the signal is generated exclusively within a tightly focused region of laser light. This allows selective imaging of tissue interiors while reducing photodamage to surrounding areas. A key innovation of the project lies in the detection strategy. Instead of conventional point detectors, the system will employ image-scanning microscopy combined with modern SPAD (Single-Photon Avalanche Diode) array detectors. These semiconductor detectors, based on avalanche diodes, can register individual photons and provide spatial information. This enables more efficient use of the available signal and computationally enhances image resolution without increasing illumination power. The system will further incorporate adaptive optics, which dynamically corrects optical aberrations introduced as light propagates through optically inhomogeneous tissue. As a result, image sharpness and contrast can be restored directly at the plane of observation, even at large imaging depths. The combination of optical correction and array-based detection will enable high-quality imaging in situations where images would otherwise appear blurred or lack detail, while further reducing the sample's light exposure.

Building on this technological foundation, the project will also develop methods for single-molecule localization using structured illumination, enabled by adaptive optics and SPAD detectors. Carefully designed illumination patterns allow molecular positions to be determined with nanometre-scale precision, well beyond the classical resolution limits of optical microscopy. This will enable the observation of biological processes at the molecular level, such as protein motion and reorganization, while remaining compatible with deep-tissue imaging and studies of living samples.

The project “Multimodal Multiphoton Nanoscopy with SPAD Array Detectors and Adaptive Optics” is carried out within the First Team programme of the Foundation for Polish Science, co-financed by the European Union under the European Funds for Smart Economy 2021–2027 (FENG) programme.

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