Opterra II Swept Field vs. Spinning Disk Comparison for Deep Imaging

Z-stack comparison of signal-to-noise ratio through a 200 um gel embedded with collagen fibers.  Left: spinning disk system. Right: Opterra II.  Sample courtesy of Brian Burkel, University of Wisconsin.

The Opterra pinhole architecture was designed to minimize the amount of crosstalk compared to two-dimensional pinhole arrays as found in spinning disk and other multipoint confocal systems. As excitation light is focused deeper within a sample, there is an increased chance that the fluorescence signal will be scattered. The scattered fluorescence can be collected by the objective lens at off-angles and pass to the detector through the wrong pinhole. The one-dimensional array of pinholes in the Opterra reduces the noise introduced by scattering to enable deeper imaging with greater signal to noise ratio.

The video demonstrates this concept by showing a Z-series collected with the same sample, same objective, and same laser power at the objective with a spinning disk system and Opterra II. As the images are taken deeper into the sample, the crosstalk increases significantly in the spinning disk images, causing a high amount of background noise. The Opterra is able to maintain a good signal to noise ratio throughout the deep Z-series.

In biological imaging, speed, signal intensity and spatial resolution are interweaved elements that the experimenter has to control to meet the end goals for the data. For most confocal live-cell microscopes, such as with spinning disk confocal systems, many of imaging parameters are fixed. The Opterra II system uniquely allows for the ability to balance speed, intensity and resolution as needed at a click of a mouse for any given experiment without hardware changes.


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