A practical guide to light-sheet microscopy for nanoscale imaging: Looking beyond the cell

TL;DR

This paper provides a comprehensive, practical guide to light-sheet microscopy, covering fundamental concepts, system construction, calibration, and data analysis to assist scientists in nanoscale imaging applications.

Contribution

It offers a detailed, step-by-step practical guide for implementing light-sheet microscopy, including system design, alignment, calibration, and data analysis tools.

Findings

Guidelines for constructing a home-built light-sheet microscope

Insights into alignment and calibration procedures

Introduction of custom data analysis code

Abstract

We present a comprehensive guide to light-sheet microscopy (LSM) to assist scientists in navigating the practical implementation of this microscopy technique. Emphasizing the applicability of LSM to image both static microscale and nanoscale features, as well as diffusion dynamics, we present the fundamental concepts of microscopy, progressing through beam profile considerations, to image reconstruction. We outline key practical decisions in constructing a home-built system and provide insight into the alignment and calibration processes. We briefly discuss the conditions necessary for constructing a continuous 3D image and introduce our home-built code for data analysis. By providing this guide, we aim to alleviate the challenges associated with designing and constructing LSM systems and offer scientists new to LSM a valuable resource in navigating this complex field.