Due to the fact that camera exposur

Due to the fact that camera exposure times are dependent upon the intensity of fluorescence emission gathered by the specimen, and vary widely from one sample to another, the disk rotation speed must be carefully adjusted to match the camera exposure time. Thus, uniform image capture can only be performed if the exposure time is an integral multiple of the time necessary to sweep one scan frame (30 degrees of disk rotation). In cases where the exposure time is not matched (in effect, is not an exact multiple of the rotation speed), scanning will continue into the next frame and can produce a striped pattern superimposed on the image. This artifact is generally not a problem for long exposure times (greater than 100 milliseconds) where averaging occurs between frames, but can seriously hamper imaging success when exposure times are reduced to a few milliseconds. Fortunately, even though the Yokogawa disk is capable of producing 12 images from each 360-degree rotation (see Figure 2(a)), these individual segments do not have defined start and stop points so that any exposure corresponding to one twelfth of a disk rotation will capture a complete image irrespective of the point at which the exposure was started. The Yokogawa CSU-X1 features an advanced disk drive motor that allows for fine-tuning the adjustment of rotation speed to match the camera exposure time. Furthermore, the unit incorporates a dynamic balancing mechanism for the rotating parts to prevent vibrations (as well as accompanying imaging artifacts) that might occur at the highest speeds.A number of advanced accessories designed to enhance performance are available for the Yokogawa CSU-X1. The high-end scanning unit versions are equipped with a computer-controlled dichromatic mirror changer that enables the user to select between three mirrors, and the 6-position auxiliary filter wheels feature stepping motors, lightweight pulleys, and a timing belt to maintain synchronization. The brightfield light path housing can be used to sequentially capture images in a variety of contrast-enhancing modes, including widefield fluorescence generated via an arc-discharge lamp attached to the microscope. The optical train of the brightfield unit bypasses the spinning disk system and sends images generated by the microscope directly to the camera via a series of relay lenses and mirrors. However, the switching speed of this option is approximately 2 seconds, which renders it the slowest system component for many applications. Additionally, a second camera port can be installed on the scanner so that simultaneous two-color imaging can be performed without the time delay incurred by using the filter wheel. A second dichromatic mirror is installed in the rear of the unit to divide the fluorescence emission received from the primary dichromatic into two discrete wavelength bands that are transferred to the individual cameras. The two-camera configuration provides the fastest action for capturing dual-color images of specimens labeled with, for example, enhanced green fluorescent protein (EGFP) and mCherry, an orange-red fluorescent protein.The strategy of using massively parallel spinning disk illumination to visualize dynamic events on a variety of timescales in live-cell imaging has proven to be an indispensible tool in cell biology. Although the number of commercially available scanning unit designs is currently limited, this technology should continue to progress with incremental improvements in performance. The Yokogawa scanning unit concept is currently the most advanced Nipkow disk-based design yet implemented, and is capable of generating images of faintly fluorescent specimens with high temporal and spatial resolution. As the microscope companies address new designs targeted at fast live-cell imaging, there will no doubt continue to be advancements in spinning disk and related technologies. An alternative to the Nipkow disk concept is based on sweeping the specimen along both lateral axes with line-shaped excitation illumination. Termed line scanners, these instruments are also capable of high temporal resolution (but at the cost of somewhat reduced spatial resolution). Several commercial line-scanning instruments are being field tested as this promising concept also warrants further development.