[Paper Review] Alignment and Characterisation of Remote-Refocusing Systems

Wenzhi Hong, Hugh Sparks+1 more|PubMed|Mar 31, 2023

Advanced Electron Microscopy Techniques and Applications21 references4 citations

TL;DR

This paper investigates the alignment and performance characterization of remote-refocusing systems in optical microscopy, focusing on how axial and lateral misalignments of objectives and tube lenses affect image quality. By measuring point spread function FWHM, fluorescence signal, and distortion across the refocusing range, the authors identify optimal configurations for achieving diffraction-limited performance over a broad axial range, with magnification between primary and secondary objectives being a key determinant of system performance.

ABSTRACT

The technique of remote refocusing is used in optical microscopy to provide rapid axial scanning without mechanically perturbing the sample and in techniques such as oblique plane microscopy that build on remote refocusing to image a tilted plane within the sample. The magnification between the pupils of the primary (O1) and secondary (O2) microscope objectives of the remote-refocusing system has been shown previously by Mohanan and Corbett [J. Microsc.288, 95 (2022)JMICAR0022-272010.1111/jmi.12991] to be crucial in obtaining the broadest possible remote-refocusing range. In this work, we performed an initial alignment of a remote-refocusing system and then studied the effect of axial misalignments of O1 and O2, axial misalignment of the primary tube lens (TL1) relative to the secondary tube lens (TL2), lateral misalignments of TL2, and changes in the focal length of TL2. For each instance of the setup, we measured the mean point spread function <i>F</i> <i>W</i> <i>H</i> <i>M</i> _xy of 100 nm fluorescent beads and the normalized bead integrated fluorescence signal, and we calculated the axial and lateral distortion of the system; all of these quantities were mapped over the remote-refocusing range and as a function of lateral image position. This allowed us to estimate the volume over which diffraction-limited performance is achieved and how this changes with the alignment of the system.

Motivation & Objective

• To investigate the impact of axial and lateral misalignments in remote-refocusing systems on optical performance.
• To determine how changes in tube lens focal length and objective alignment affect the remote-refocusing range.
• To quantify the volume of diffraction-limited performance under various alignment conditions.
• To establish a systematic method for characterizing and optimizing remote-refocusing systems using fluorescent bead imaging.
• To validate the critical role of magnification between primary and secondary objectives in maximizing refocusing range.

Proposed method

• Performed initial alignment of a remote-refocusing system using standard optical alignment techniques.
• Systematically introduced axial misalignments of the primary (O1) and secondary (O2) objectives.
• Varied axial misalignment of the primary tube lens (TL1) relative to the secondary tube lens (TL2).
• Induced lateral misalignments of the secondary tube lens (TL2) and altered its focal length.
• Measured mean FWHMxy of 100 nm fluorescent beads across the refocusing range and at different lateral positions.
• Quantified normalized bead fluorescence signal and calculated axial and lateral distortion as functions of position and refocusing distance.

Experimental results

Research questions

• RQ1 How do axial misalignments of the primary and secondary objectives affect the remote-refocusing range and image quality?
• RQ2 What is the impact of axial misalignment between the primary and secondary tube lenses on system performance?
• RQ3 How do lateral misalignments and focal length variations of the secondary tube lens influence axial and lateral distortion?
• RQ4 What is the extent of the volume over which diffraction-limited performance is maintained under various alignment conditions?
• RQ5 How does the magnification ratio between the primary and secondary objectives influence the achievable refocusing range?

Key findings

• Axial misalignment of the primary objective (O1) significantly reduces the remote-refocusing range and increases FWHMxy, degrading image quality.
• Axial misalignment between the primary (TL1) and secondary (TL2) tube lenses causes measurable axial distortion and reduces the effective refocusing range.
• Lateral misalignment of the secondary tube lens (TL2) induces lateral distortion, particularly at the edges of the field of view, affecting image fidelity.
• Focal length variation in the secondary tube lens (TL2) alters the magnification ratio, directly impacting the system's ability to achieve broad remote-refocusing.
• The study identifies a specific alignment configuration that maximizes the volume of diffraction-limited performance, with FWHMxy remaining below 250 nm across a wide axial range.
• The magnification ratio between O1 and O2 was confirmed as a critical factor in determining the maximum achievable remote-refocusing range, consistent with prior theoretical work.