Design and manufacturing of a laser light-sheet fluorescence add-on module for upright optical microscopes (Invited Paper)

To achieve high-resolution deep imaging in thick live biological specimens with minimal acquisition times, light-sheet fluorescence microscopy is often the preferred method. Unfortunately, only a small community of biologists and microscopists can benefit from this approach, as commercially available light-sheet microscopes are still very expensive, bulky stand-alone systems. We designed and manufactured a compact light sheet illumination module suitable for implementation within upright water-dipping optical microscopes. Two illumination wavelengths (488nm and 638nm) emitted by fiber coupled laser diodes are guided towards the microscope using fiber couplers and wavelength combiners. The optical design of the light-sheet module itself consisted of an extensive investigation of the most optimal combination of reflective and/or refractive off-the-shelf available optical components. The best performing design uses a reflective parabolic collimator, a cylindrical achromatic lens and a water-dipping fluorite objective. A light-sheet with 7µm height and 700µm width is obtained with excellent optical quality, achieving a Strehl ratio after tolerancing of 0.93. A compact optomechanical design was then developed to fit the module onto a standard Nikon microscope, allowing the imaging of spheroids attached to a rotating capillary. Laboratory tests confirmed the expected size of the light-sheet, while colorectal and breast cancer cells were successfully imaged using the integrated system.