Transmit 5 disk series#

The detector for LSM is typically a photomultiplier tube (PMT). However, because the galvanometer speed is fixed, the limitation on image acquisition speed in practice is the dwell time for each XY position (pixel) to produce sufficient signal over background. The speed of the LSM thus depends on the line frequency of the scanner, which is limited by the galvanometer mirrors that move the laser beams, along with the number of lines needed for the field of view imaged. One of the major differences between the acquisition speed of the LSM and SDCM is that the former collects signals from the specimen one pixel at a time through a single pinhole.

Transmit 5 disk series#

In our experience, acquiring a Z-stack time series using an SDCM system with optimal camera setup offers improved temporal and spatial resolution as compared to laser scanning confocal microscopy ( Fig. One common concern regarding high-speed imaging is the trade-off between speed and resolution. The benefits of imaging using SDCM over conventional single-point laser scanning confocal microscopy (LSM) including acquisition speed, detection efficiency, resolution, reduced photobleaching, and improved signal-to-noise ratio, both in vitro and in vivo, has been discussed elsewhere. This protocol describes the use of spinning disk confocal microscopy (SDCM) for live imaging of genetically encoded fluorescent reporters expressed in zebrafish embryos.

One thing to note is that neutrophils at this developmental stage are not terminally differentiated, since they retain the ability to undergo cell division under some conditions and possess an elongated nucleus instead of the multi-lobulated nucleus observed in human neutrophils. The ability to image both neutrophil chemotaxis and random motility in the same organism allows for the dissection of the molecular regulators required for basic motility and directional sensing. In addition, neutrophils migrate spontaneously (random migration) in the interstitial tissue within the head region (reviewed in ). Neutrophils at this stage are considered to be fully functional, as they can directionally migrate (chemotaxis) to wounds or infections and perform immune functions to fight off pathogens. At this stage of development, adaptive immunity has not sufficiently matured, allowing the direct study of innate immunity in isolation. Neutrophil imaging is usually performed at 2 to 3 days post fertilization (dpf). Here, we describe methods for in vivo live imaging of neutrophils in zebrafish embryos. Zebrafish embryos are largely translucent for the first few days of development, allowing for noninvasive in vivo imaging and studies of cell behavior in a physiologically relevant context. The zebrafish ( Danio rerio) immune system is remarkably similar to its mammalian counterpart and it has therefore gained tremendous popularity as a vertebrate disease model of both innate and adaptive immunity (reviewed in ).