In addition to the wide fluorophore emission wavelength range afforded by the PFS, the high focus-correction sampling rate is critical for experiments that require imaging at real-time speeds or faster. The wide range of objectives (over 50 models) compatible with the PFS unit includes oil and water immersion, many dry objectives of varying design and correction, phase contrast objectives, and several optional objectives having a long working distance. By removing the visible light filter, the PFS can be adapted for use with laser tweezers and laser trapping applications, as well as a portion of the multiphoton excitation spectral region. The system also incorporates an infrared-cut filter for diascopic (transmitted light) illumination of cell cultures using the conventional contrast-enhancing techniques listed above. The bandpass interference filter in the PFS unit spans a broad transmission range of 340 to 750 nanometers, enabling the application of all known fluorescent proteins (including infrared-emitting plant phototropins), quantum dots, and most of the popular synthetic fluorophores (ranging from Fura-2 to Cy5, Alexa Fluor 700, and most of the ATTO dyes). The versatility of the PFS is evident from the wide range of fluorescent probes that can be utilized for live-cell imaging with focus drift compensation. Note that the real PSF unit responds to focus drift in milliseconds and therefore the actions in this tutorial are slowed for the purposes of instructional examination. in addition, the objective type can be toggled between oil, water, and dry using the radio buttons. Simulated vibration and thermal drift can be introduced by clicking on the appropriate button. As this slider is translated, the focal region in the specimen is also changed. In order to operate the tutorial, use the Offset Lens X-axis slider to alter the PFS offset quantity, thus changing the distance between the reference plane and the focal plane. Illustrated in the optical system ray-trace diagram are the objective, imaging chamber, PFS offset lens, line-CCD, LED, and dichromatic mirror. The tutorial initializes with a schematic diagram of the Nikon PFS appearing in the main window and a view of the microscope focal plane in the Specimen Window.
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