Abstract
We present a beam splitter mask that can be easily added to a multiphoton raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.
Highlights
Imaging of large volumes with raster scanning microscopes can be very time consuming as they acquire each voxel serially
The axial Full-Width Half-Maximum (FWHM) is 16.83 ± 0.29 μm for the extended depth of focus (EDF) point spread functions (PSF) and 2.90 ± 0.08 μm for the PSF without the phase mask, which represents an axial extension by roughly a factor 5.8
In the lateral beam profile, one can see that the EDF PSF gets about 32% wider, increasing the width of the diffraction limited PSF of 0.53 ± 0.03 μm to 0.71 ± 0.02 μm
Summary
Imaging of large volumes with raster scanning microscopes can be very time consuming as they acquire each voxel serially. This is true if high NA objectives are employed that provide a high lateral resolution and a small depth of focus. To capture volumetric imaging data exceeding such a small depth, many planes have to be imaged by z-stepping each focal plane, which limits the achievable imaging speed considerably. If axial image information can be sacrificed, it can be beneficial to extend the depth of focus such that the volume to be imaged can be acquired in one lateral scan. As such, extended depth of focus (EDF) imaging can be attractive to image sparsely populated structures rapidly and has found promising applications in functional imaging of neuronal activity [1,2]
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