A high-resolution two-photon oblique plane microscopy system for fast, deep, volumetric fluorescence imaging

Background

Modern biological research increasingly relies on advanced fluorescence microscopy techniques to visualize and quantify dynamic processes within living cells and tissues. High-resolution, volumetric imaging is essential for studying complex biological phenomena such as gene expression, neural activity, and cellular interactions in three dimensions over time. However, imaging deep within thick, scattering samples—such as organoids, embryos, or intact tissues—poses significant challenges due to light scattering, absorption, and photodamage. Traditional light-sheet and two-photon microscopy methods have been developed to address some of these issues, enabling optical sectioning and reduced phototoxicity. Nevertheless, the demand for faster, deeper, and more precise imaging continues to grow, particularly as researchers seek to resolve single molecules and track rapid biological events in their native environments. Current approaches to volumetric fluorescence imaging face several limitations that hinder their effectiveness in thick or scattering samples. Conventional two-photon point-scanning microscopes, while offering deep tissue penetration and reduced out-of-focus excitation, are inherently slow for volumetric imaging due to their sequential acquisition of each voxel. Light-sheet microscopy, on the other hand, provides rapid imaging by illuminating entire planes, but often requires complex sample mounting and suffers from degraded resolution and contrast at depth, especially when using single-objective configurations. Additionally, maintaining a thin, uniform light sheet over a large field of view is technically challenging, and one-photon excitation methods are prone to increased background and photobleaching. These trade-offs between speed, resolution, imaging depth, and sample compatibility limit the utility of existing techniques for high-throughput, high-content, and in vivo studies, underscoring the need for innovative solutions that overcome these barriers.

Technology

The two-photon oblique plane microscope (2P-OPM) is an advanced fluorescence imaging platform designed for high-resolution, volumetric imaging of biological samples, especially in thick and scattering tissues. This system uniquely combines two-photon excitation with oblique plane microscopy, using a single high-numerical aperture objective for both illumination and detection. The microscope generates an oblique light sheet by shaping and scanning near-infrared ultrafast pulses, either digitally or with specialized beam-shaping optics like a multi-ring annular “layer-cake” phase mask. This approach produces a thin, uniform light sheet across a large field of view while suppressing out-of-focus background and side lobes. The fluorescence signal is relayed via a remote-focus subsystem, preserving high detection efficiency and spatial resolution—achieving approximately 300 nm lateral and 650 nm axial resolution. The system supports rapid volumetric imaging, single-molecule sensitivity, and simultaneous optogenetic stimulation, all while accommodating standard sample mounting formats.

This technology is differentiated by its ability to overcome traditional trade-offs in light-sheet microscopy, such as balancing field of view, resolution, and imaging depth. The use of two-photon excitation with advanced beam shaping allows for deep tissue imaging with reduced photodamage and photobleaching, distributing the photon budget across an entire plane and lowering peak intensity by over tenfold compared to point-scanning systems. The “layer-cake” phase mask innovation ensures a thin, uniform light sheet over extended distances, maintaining high contrast and resolution even in challenging, scattering environments. Furthermore, the system’s compatibility with standard sample formats and its turnkey design make it accessible for a wide range of applications, from basic biological research and single-molecule tracking to high-throughput clinical screening and optogenetics.

Benefits

• High-resolution volumetric fluorescence imaging with ~300 nm lateral and 650 nm axial resolution
• Enhanced imaging depth and contrast in thick, scattering biological samples
• Over 5× faster volumetric imaging compared to conventional two-photon point-scanning microscopy
• Reduced photodamage and photobleaching due to distributed photon budget and lower peak intensity
• Single-objective design compatible with standard sample mounting formats
• High sensitivity, enabling detection of weak fluorescence signals in low-SNR conditions, including single-molecule applications
• Simultaneous optogenetic stimulation enabled by near-infrared excitation without spectral interference
• Versatile applications including live molecular imaging, clinical screening, and high-content drug discovery

Applications

• High-speed 3D live-cell imaging
• Single-molecule fluorescence detection
• Deep imaging of organoids
• Simultaneous optogenetics and imaging
• High-throughput drug screening

Additional info

This system enables high-resolution, depth-enhanced, volumetric fluorescence imaging of biological samples. It integrates two-photon excitation with oblique plane microscopy, utilizing a single high-NA objective for illumination and detection. Near-infrared ultrafast pulses form an oblique light sheet, with optional beam shaping to maintain a thin, uniform sheet over a larger field of view when needed. The system achieves ~300 nm lateral and ~650 nm axial resolution, offering rapid volumetric imaging with reduced photodamage in thick, scattering samples.