Nonlinear optical microscopy techniques, and particularly multiphoton fluorescence microscopy, have become popular tools for visualizing the three-dimensional cellular architecture residing hundreds of microns and up to one millimeter deep in living organisms. This is particularly valuable in neurobiology, where multiphoton techniques have become widely used for mapping, monitoring, and manipulating neural networks in the mouse cortex and other model organisms. In living neural networks, and in many other applications, fast imaging speed is required to temporally resolve dynamic processes, reduce motion artifacts, and limit the stress to the organism being studied. In this Phase II proposal, we will build upon techniques developed in Phase I to produce a bolt- on microscope module that greatly increases the imaging speed while reducing disruption to the specimen and that can be easily integrated with existing multiphoton microscope systems. The module will achieve these goals through the creative use of high-performance spatial light modulators and fully integrated software, resulting in a user-friendly system capable of being set up and used by non-expert microscopists. The use of versatile spatial light modulator technology also enables further expansion of imaging capabilities and modalities through future software updates.
Multiphoton microscopy is a widely used tool for three-dimensional imaging throughout biology and has found particularly widespread use in the exploration and mapping of the brain. Current applications, particularly in neuroscience, require increased imaging speed and a means of fast volumetric imaging that does not disturb the sample. The proposed research will develop a novel turnkey 'bolt-on' optical module capable of vastly increasing imaging speed deep in scattering tissues without any mechanical movements near the specimen, thus greatly enhancing the performance of existing multiphoton microscopes and directly addressing one of the greatest needs of the microscopy community.
| Field, Jeffrey J; Wernsing, Keith A; Domingue, Scott R et al. (2016) Superresolved multiphoton microscopy with spatial frequency-modulated imaging. Proc Natl Acad Sci U S A 113:6605-10 |
