Nikon has addressed the problem of focus drift by creating a unique hardware solution, termed the Perfect Focus System (PFS), which is designed to combat axial focus fluctuations in real time during long-term imaging investigations. A wide range of dry, water, and oil immersion objectives, from 4x to 100x with varying numerical apertures and working distances, can be employed with inverted TiE-PFS instruments for focus compensation. The near-infrared 870-nanometer LED and CCD line sensor utilized by the PFS are housed in a specialized nosepiece unit that does not require additional infinity space and enables the primary microscope optical train to remain dedicated for imaging.
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. Illustrated in the optical system ray-trace diagram are the objective, imaging chamber, PFS offset lens, line-CCD, LED, and dichromatic mirror. 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. As this slider is translated, the focal region in the specimen is also changed. Simulated vibration and thermal drift can be introduced by clicking on the appropriate button. in addition, the objective type can be toggled between oil, water, and dry using the radio buttons. 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.
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. 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 system also incorporates an infrared-cut filter for diascopic (transmitted light) illumination of cell cultures using the conventional contrast-enhancing techniques listed above. 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 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.
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. Many of the commercially available focus compensation systems adjust focus prior to triggering the software for image capture, which severely limits the minimum time interval available between gathering successive images. For example, when imaging at 5 seconds per frame, most focus drift compensations systems will suffice. However, a system that requires 500 milliseconds to sample the state of focus will limit image acquisition speeds to almost a second (or more), depending upon the camera integration time. This creates a huge bottleneck for many applications in live-cell imaging. By uncoupling focus sampling from image capture, the PFS enables sequential image acquisition at speeds ranging from milliseconds to minutes without interruption. In fact, capture speeds are limited by the camera integration time rather than focus position sampling. The continuous sampling feature is especially useful in experiments that monitor several lateral specimen positions (requiring x-y stage translation) at high speed, and for investigations (such as monitoring calcium wave induction using fluorescent protein biosensors) that require rapid addition of reagents to the specimen chamber during the course of an experiment that lasts for only a few milliseconds.
Joel S. Silfies, Edward G. Lieser, and Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Tony B. Gines, Alex B. Coker, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.