[Paper Review] Simultaneous multiplane imaging with reverberation multiphoton microscopy

Devin R. Beaulieu, Ian G. Davison+2 more|arXiv (Cornell University)|Dec 12, 2018

Advanced Fluorescence Microscopy TechniquesBiochemistry, Genetics and Molecular Biology24 references3 citations

TL;DR

This paper introduces reverberation multiphoton microscopy (RMPM), a technique enabling simultaneous multiplane 3D volumetric imaging at video-rate speeds using conventional multiphoton microscopy hardware. By exploiting optical reverberation in a custom cavity to perform near-instantaneous axial scanning, RMPM achieves micron-scale 3D resolution without compromising imaging speed or performance, particularly effective in scattering tissues with low-repetition-rate lasers.

ABSTRACT

Multiphoton microscopy (MPM) has gained enormous popularity over the years for its capacity to provide high resolution images from deep within scattering samples1. However, MPM is generally based on single-point laser-focus scanning, which is intrinsically slow. While imaging speeds as fast as video rate have become routine for 2D planar imaging, such speeds have so far been unattainable for 3D volumetric imaging without severely compromising microscope performance. We demonstrate here 3D volumetric (multiplane) imaging at the same speed as 2D planar (single plane) imaging, with minimal compromise in performance. Specifically, multiple planes are acquired by near-instantaneous axial scanning while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, is well adapted for large-scale imaging in scattering media with low repetition-rate lasers, and can be implemented with conventional MPM as a simple add-on.

Motivation & Objective

• To overcome the fundamental speed limitation of conventional multiphoton microscopy in 3D volumetric imaging.
• To enable simultaneous acquisition of multiple optical planes without sacrificing spatial resolution or requiring high-repetition-rate lasers.
• To develop a practical, add-on solution compatible with standard multiphoton microscopy systems for large-scale, deep-tissue imaging.
• To maintain high imaging performance in scattering biological samples where traditional methods are too slow.

Proposed method

• The method employs a custom optical cavity that sustains multiple round trips of the excitation laser beam, creating a train of temporally spaced pulses.
• These pulses are spatially focused at different axial depths within the sample, enabling simultaneous excitation across multiple planes.
• The axial scanning is achieved through the round-trip time of the laser pulses in the cavity, effectively replacing mechanical axial scanning with optical delay.
• The technique uses a low-repetition-rate laser source, making it compatible with standard multiphoton microscopes without hardware upgrades.
• Image reconstruction is performed by synchronizing the detection with the temporal sequence of pulses in the cavity, allowing discrimination of signals from different axial planes.
• The system maintains high spatial resolution by preserving the tight focus of the excitation beam across multiple planes.

Experimental results

Research questions

• RQ1 Can multiplane 3D imaging be achieved at video-rate speeds without compromising spatial resolution in multiphoton microscopy?
• RQ2 How can optical reverberation be harnessed to enable rapid axial scanning in scattering samples?
• RQ3 Can this approach be implemented as a simple add-on to existing multiphoton microscopes without requiring high-repetition-rate lasers?
• RQ4 What is the achievable axial resolution and imaging speed when using reverberation-based axial scanning?
• RQ5 How does the technique perform in deep, scattering biological tissues compared to conventional scanning methods?

Key findings

• The technique achieves simultaneous multiplane imaging at video-rate speeds, matching the acquisition speed of standard 2D planar imaging.
• Micron-scale 3D spatial resolution is maintained across multiple planes, even in deep, scattering tissues.
• The method operates effectively with low-repetition-rate lasers, reducing photodamage and enabling compatibility with standard multiphoton microscopes.
• The use of optical reverberation enables near-instantaneous axial scanning, eliminating the need for mechanical scanning stages.
• The system demonstrates robust performance in large-scale volumetric imaging applications, particularly in scattering media.
• The approach is implemented as a simple add-on to conventional multiphoton microscopy systems, requiring minimal modification.