Extended depth of focus multiphoton microscopy via incoherent pulse splitting
Posted on: 16 April 2020
Preprint posted on 29 March 2020
Article now published in Biomedical Optics Express at http://dx.doi.org/10.1364/BOE.393931
Categories: biophysics, cell biology
Background
Imaging large volumes is necessary in order to understand biological phenomena that manifest themselves at the tissue- or organ-scale. However, imaging large volumes with raster scanning microscopes is very time consuming, particularly if using high NA objectives that provide a high lateral resolution and a small depth of focus. To capture volumetric imaging data, many planes have to be imaged by z-stepping each focal plane, which limits the achievable imaging speed considerably. Instead, if axial image formation can be sacrificed, extending the depth of focus (EDF) would allow the volumetric information to be projected into a single 2D image. This is especially attractive for rapid imaging of sparsely populated structures.
Key findings and developments
In their work, the authors present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold with a small penalty in lateral resolution. Previous work designed for fluorescence widefield microscopy used an incoherent superposition as the means of creating an EDF beam: namely, the pupil of an objective was segmented into multiple annuli, which became incoherent to each other after passing through the mask. In their work, the authors applied this concept to beam shaping of ultrafast laser sources, achieving two-photon EDF microscopy by using a stepped annular phase mask. The phase mask, which is acting in a Fourier plane of the imaging system, consists of multiple concentric glass disks. An ultrafast laser pulse is split by the phase mask into different annular beamlets, each of which is time-delayed, forming a focus in the front focal plane of the objective at slightly different arrival times. The resulting EDF focus is the incoherent superposition of all individual foci. Numerical simulations to compute the electromagnetic field in the front plane of an objective were performed and predicted an extension of the depth of focus that scales with the number of annular zones. Finally, the authors demonstrated the potential of the EDF mask by imaging GFP-labelled neurons in fixed mouse brains. Altogether, the numerical and experimental results demonstrated that a five-fold extension in focal depth was feasible, with only a moderate lateral resolution loss.
What I like about this preprint
This exciting development could enable simultaneous high-speed imaging of biological phenomena at multiple depths. Examples include intravital microscopy or in situ imaging in tissues or whole cell contexts. Importantly, this advance can be incorporated into existing equipment with minimal modifications.
Reference
Acknowledgement
Many thanks to all authors for their engagement in discussions and questions.
doi: https://doi.org/10.1242/prelights.18768
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