ZEISS Lightfield 4D: Instant Volume Acquisition for High-Speed and Gentle Imaging

Technology Note

Authors: Julia König, Tiemo Anhut, Thomas Egloff, Miriam Gaissmaier, Stanislav Kalinin, Volodymyr Kudryavtsev, Terra Kuhn, Annette Bergter

Carl Zeiss Microscopy GmbH, Germany

Date: November 2025

*Introduction of this innovative imaging technology: Applications, technological background, and seamless integration into a user-friendly, turn-key microscope system*

Introduction

One of the fundamental tasks of fluorescence microscopy is to capture multi-dimensional information that represents biological processes as closely to reality as possible. This involves several key parameters, including accurate localization (spatial resolution), identification of structures or proteins of interest (labeling specificity, spectral separation), and tracking dynamic processes (temporal resolution). Additionally, it is crucial that the specimen under investigation is minimally affected by the entire procedure, making gentleness a priority to minimize any disruption to the biological processes being studied. Ultimately, imaging serves as the foundation for generating data for subsequent analysis (e.g. particle / cell tracking, organismal and tissue structure and development, cellular organization and activation etc.).

The experimental procedures of microscopy have become so standardized and integrated into scientific workflows that it is easy to forget that generating high-quality datasets of 3D samples through optical sectioning entails specific compromises and costs. This principle applies not only to imaging single image frames (2D imaging) but also to capturing entire sample volumes (3D imaging). When temporal resolution is crucial, particularly for physiological processes like blood flow, heartbeat, or other rapid muscular movements, volumetric information may be entirely excluded and 2D imaging is performed at high frame rates.

If 3D information is acquired, until now, all high-quality volumetric fluorescence imaging has relied on acquiring a series of 2D images along an axis as the microscope moves the sample. Typically, this axis is the focus drive (z-axis) which moves the sample perpendicular to the (x, y) frame. Consequently, the most common imaging volume is a "z-stack", literally a stack of 2D images along the z-axis (e.g. LSM, widefield, structured illumination), whereby the z-axis is defined by the spatial direction being perpendicular to the cover glass on which the sample is mounted. There are of course exceptions, e.g. Lattice Lightsheet, with an image stack acquired along the x-axis. However, the acquired images must be transferred into a z-stack for traditional display and analysis.

Regardless of the method used, image information is gathered sequentially to construct the volume information of a sample, which requires time. As a result, each acquired volume includes time shifts between consecutive 2D images, which may be insignificant for fixed samples, but can lead to missed and low-quality data when studying living processes. These temporal shifts during z-stack acquisition can become visible as smear artefacts by a moving object while a z-stack is acquired. Because information is only accessible from one image point or 2D frame at a time, we remain unaware of any activities occurring within the sample but outside this frame at that point in time. Such movements can become apparent when objects noticeably "jump" between timepoints or alter their inner state, e.g. represented by Ca2+ signaling. Moreover, we may simply miss these events, leading to incomplete data.

ZEISS Lightfield 4D, based on light-field microscopy, overcomes the need for sequential acquisition of image frames to generate volumetric z-stacks and provides simultaneous volume information [LIPPMANN 1908, LEVOY 2006, LEVOY 2008, BROXTON 2013]. This means that every volume is captured without any time delay – all processes within the volume are captured in the same instant. Time series are captured at the appropriate speed simply by regulating the exposure time (of the volume) and the interval in between timepoints. This results in the unique opportunity to capture extremely fast processes within organs or organisms in their entirety. Each volume is acquired with a single illumination event of the sample; therefore, this imaging method is extremely gentle, enabling one to take advantage of its fast acquisition speed, for capturing fast dynamic processes, or to follow processes for hours and days, without fear of damaging the sample.

This document will describe the potential applications perfectly supported by the easy-to-use software interface and workflow of Lightfield 4D. Furthermore, information on the technical background and optical setup is provided as well as the methods used for efficient volume reconstruction (Lightfield 4D processing).