Any immersion remote refocus (AIRR) microscopy

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Abstract

3D imaging is essential for studying many biological processes. In microscopy it is traditional to take a series of 2D images at different depths by moving the objective lens or the sample. To avoid aberrations we match the refractive index (RI) of the sample to the immersion medium of the objective, an awkward constraint for microscope users and designers. Here, we present a versatile approach to aberration-free 3D imaging, where an objective of any immersion medium can be combined with a sample of any RI. For example, we can use a high numerical aperture (NA) air objective (Nikon 40x0.95) to look into a watery sample (RI ~ 1.33), and use remote refocus optics to extend the accessible depth from ~6μm to ~151μm. Avoiding liquid immersion is convenient for users and great for automated microscopy. Similarly, we can extend the depth of a high NA oil objective (Nikon 40x1.30) from ~8μm to ~65μm in water, and enjoy a larger field of view and NA compared to the equivalent water immersion objective. We can apply this method to use different objectives on the same system, with maximum depth, immersion-free and maximum NA modalities. In addition, we present a dynamic zoom lens to enable fast RI tuning in the range 1.33-1.51, which maximizes performance for a diverse set of biological samples. By combining different objectives with dynamic remote refocus optics we can make an 'any immersion' microscope with a boosted focal range.