Spinning disk confocal imaging of neutrophil migration in zebrafish

Abstract

Live-cell imaging techniques have been substantially improved due to advances in confocal microscopy instrumentation coupled with ultrasensitive detectors. The spinning disk confocal system is capable of generating images of fluorescent live samples with broad dynamic range and high temporal and spatial resolution. The ability to acquire fluorescent images of living cells in vivo on a millisecond timescale allows the dissection of biological processes that have not previously been visualized in a physiologically relevant context. In vivo imaging of rapidly moving cells such as neutrophils can be technically challenging. In this chapter, we describe the practical aspects of imaging neutrophils in zebrafish embryos using spinning disk confocal microscopy. Similar setups can also be applied to image other motile cell types and signaling processes in translucent animals or tissues.

Fig. 1. Comparison of laser scanning confocal (LSM) and spinning disk confocal (SDCM) on in vivo neutrophil imaging

Maximum intensity projection image of a neutrophil expressing GFP-UtrCH (green) and Lifeact-Ruby (magenta), which labels stable F-actin and all F-actin, respectively. (A–B) Image acquired using an LSM (Olympus). Olympus LUMFL 60×/ 1.1NA Water objective (A) or Olympus PlanApo N 60×/ 1.45NA Oil objective (B) was used with S.U. 200, optimal step size and 3.8× zoom in. (C–D) Image acquired using SDCM (Zeiss). LCI Plan-Neofluar 63×/ 1.3NA Water objective (C) or Plan Apo 63×/ 1.4NA Oil objective (D) was used with 300 EM gain, 55 msec exposure for both channels and 0.4 µm step size. SDCM images showed superior image quality compared to that of the LSM images. The difference between using an oil objective with a higher NA than the water objective is less significant. Scale bar: 10 µm.

Fig. 2. Spinning disk confocal imaging of F-actin cytoskeleton dynamics

Time-lapse maximum intensity projection image of a neutrophil undergoing random motility in a zebrafish embryo in vivo. The neutrophil expresses GFP-UtrCH (green) and Lifeact-Ruby (red), which labels stable F-actin and all F-actin, respectively. A SDCM equipped with an EMCCD camera was used for the acquisition. LCI Plan-Neofluar 63×/1.3 Water objective; 300 EM gain; 55 msec exposure (GFP-UtrCH), 36 msec exposure (Lifeact-Ruby); emission filter BP 525/50 and FF02-617/73-25; 0.4 µm step size. Acquisition was made as fast as possible and consecutive images are shown in the panel. Scale bar: 10 µm.

Fig. 3. Spinning disk confocal imaging of organelle subcellular localization

Time-lapse maximum intensity projection image of a neutrophil expressing γtubulin-GFP (green) and a nuclear probe, mCherry-histone H2B (red). The Microtubule Organizing Center (MTOC-arrow) is localized in front of the nucleus during neutrophil motility in vivo. Scale bar: 10 µm.