Abstract
We compare the performance of linear and nonlinear methods for aligning the excitation and detection planes throughout volumes of large specimens in digitally scanned light sheet microscopy. An effective nonlinear method involves the registration of four corner extrema of the imaging volume via a projective transform. We show that this improves the light collection efficiency of the commonly used three-point affine registration by an average of 42% over a typical specimen volume. Accurate illumination/detection registration methods are now pertinent to biological research in view of current trends towards imaging large or expanded samples, at depth, with diffraction limited resolution.
Highlights
Whilst confocal techniques reject the out-of-focus signal excited by the illumination source through use of a pinhole, light sheet technology is a much more efficient optical sectioning method, since signals are exclusively generated in the plane defined by the thin sheet of illumination light
Galvanometric mirrors can be used to produce a light sheet by rapidly dithering a laser beam; this is referred to as digitally scanned light sheet microscopy (DSLM) [3]
The use of a scan lens can lead to registration errors of the light sheet with respect to the imaging plane, reducing contrast, and increasing background fluorescence
Summary
Received 29 September 2017; revised 8 November 2017; accepted 12 November 2017; posted 21 November 2017 (Doc. ID 304060); published 6 February 2018. We compare the performance of linear and nonlinear methods for aligning the excitation and detection planes throughout volumes of large specimens in digitally scanned light sheet microscopy. An effective nonlinear method involves the registration of four corner extrema of the imaging volume via a projective transform. We show that this improves the light collection efficiency of the commonly used three-point affine registration by an average of 42% over a typical specimen volume. Accurate illumination/detection registration methods are pertinent to biological research in view of current trends towards imaging large or expanded samples, at depth, with diffraction limited resolution
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