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The technique of remote refocusing is used in optical microscopy to providerapid axial scanning without mechanically perturbing the sample and intechniques such as oblique plane microscopy that build on remote refocusing toimage a tilted plane within the sample. The magnification between the pupils ofthe primary (O1) and secondary (O2) microscope objectives of theremote-refocusing system has been shown previously by Mohanan and Corbett [JMicrosc, 288(2):95-105 (2022)] to be crucial in obtaining the broadest possibleremote-refocusing range. In this work, we performed an initial alignment of aremote-refocusing system and then studied the effect of axial misalignments ofO1 and O2, axial misalignment of the primary tube lens (TL1) relative to thesecondary tube lens (TL2), lateral misalignments of TL2 and changes in thefocal length of TL2. For each instance of the setup, we measured the mean pointspread function FWHMxy of 100 nm fluorescent beads, the normalised beadintegrated fluorescence signal, and calculated the axial and lateral distortionof the system: all of these quantities were mapped over the remote-refocusingrange and as a function of lateral image position. This allowed us to estimatethe volume over which diffraction-limited performance is achieved and how thischanges with the alignment of the system.

Alignment and Characterisation of Remote-Refocusing Systems