The critical concept underlying the Nikon PFS is the accurate detection of a suitable axial (z) reference plane that can be utilized to establish an exact proximal relationship between the objective front lens element position and the focal plane of interest within the specimen. This task is accomplished using near-infrared light (870 nanometers) that is generated by an auxiliary optical system and introduced into the primary microscope optical train via a dichromatic mirror. The 870-nanometer light, which does not interfere with normal transmitted light or fluorescence observation, is focused by the objective to project a linear pattern at a refractive index boundary that resides between the glass coverslip (refractive index of 1.5) and the medium surrounding the specimen (refractive index of 1.33-1.38) when using oil immersion objectives. The aqueous refractive index boundary serves as the reference plane when water or oil immersion objectives are employed, but dry objectives use the air-glass boundary on the opposite side of the coverslip facing the objective front lens element. This tutorial explores the offset lens system that enables the operation of the Nikon PFS.
The tutorial initializes with a schematic diagram of the Nikon PFS offset lens system and line-CCD appearing in the main window along with a view of the microscope focal plane in the Specimen Window. In order to operate the tutorial, use the Offset Control 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, and this action is illustrated by changes to the line-shaped pattern broadcast to the CCD in the upper portion of the tutorial window. Although the optical system is illustrated along a horizontal axis in the tutorial, it is actually vertical in the actual microscope. 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.
A critical component of the PFS unit is the offset adjustment lens system, which is located between the half-mirror and the primary dichromatic mirror, and outlined with a schematic diagram in Figure 1. Due to its positioning, the offset lens system is shared by both the LED slit illumination and line-CCD image-forming optical trains of the PFS. The offset system operates to shift the focal position of the PFS slit image in harmony with an electronic feedback loop that controls microscope axial (z) position, thus enabling the system to create completely separate focal planes for the specimen and the glass-water interface. The specimen image plane is directed to the detector or eyepieces and the slit image is directed to the PFS line-CCD detector via the two independent optical systems.
In Figure 1(a), an abbreviated version of the optical train containing only the offset lenses and the primary microscope objective, as well as the specimen and coverslip, is presented for brevity of explanation. Additionally, the illustration presents the situation for a dry objective where the reference plane resides at the air-glass interface on the lower surface of the coverslip. Yellow ray traces refer to the image forming light waves of the primary microscope optical system. The yellow rays do not pass through the PFS optical train due to the restricting dichromatic mirror described above, which directs these image-forming wavefronts to the eyepieces or other detectors. The red ray traces outline focus-detection light waves generated and collected by the PFS unit. The offset lens components are referred to as the turret lens and the offset lens in Figure 1. These lenses shift the PFS-generated slit image, which is projected onto the coverslip interface, back and forth along the optical axis and they also simultaneously shift the reflected image up or down across the pixels of the line-CCD detector. The offset system is designed to enable the operator to select a region of the specimen that coincides with the focal plane of the objective while maintaining a fixed distance between the objective front lens element and specimen coverslip using the PFS drift-correction hardware.
The offset system turret lens is fixed on the optical axis (incorporated into a multi-lens turret), whereas the offset lens itself is allowed to translate back and forth along the optical axis to shift the position of the slit image. Upon initialization of the PFS unit, the focal point of the objective (F) and that of the PFS slit image (A) coincide at the interface region between the coverslip and the medium bathing the specimen (Figure 1(a)). This condition is referred to as zero offset. In order to introduce an offset distance between the objective focal plane and the slit reference plane, the operator can turn the PFS offset controller dial to relocate the offset lens, and thus the focal point of the slit image. In Figure 1(b), the lens is moved by a distance x, which results in a shift of the slit image focal point A to a new location closer to the objective. Because the offset lens has no effect on the focal point of the objective image-forming light waves (focal point F), the two focal points are now offset in relation to one another. Once the PFS feedback loop adjusts the objective position to relocate the slit image focal point (A) at the coverslip interface (Figure 1(c)), the objective focal point (F) is then moved into the central portion of the specimen. The distance between the focal points F andA is known as the offset quantity, and can be altered at the convenience of the operator to probe various depths in the specimen.
The offset ranges available with the PFS unit are determined by the type of objective being used, but generally range from the coverslip interface (zero millimeters) to a maximum of around 10 micrometers for oil immersion objectives, 20 micrometers for water immersion objectives, and up to 100 micrometers or more for dry objectives. Note that the offset range generally decreases with objective working distance, and therefore exhibits the most restricted values for the high numerical aperture oil immersion objectives, which feature very shallow focal depths. One of the most important aspects of successfully being able to monitor the axial position of the z reference plane is the signal strength of the near-infrared light beam reflected at the interface. When using oil immersion objectives, reflectance of the slit image at the interface between the oil and coverslip glass is effectively zero and therefore does not interfere with focus control. With water immersion objectives, the value for reflectance at this interface equals that of the upper coverslip interface (on the specimen side), but the shallow focal depth of these high magnification and numerical aperture objectives ensures that the lower reflection does not interfere with PFS control functions. In contrast, for dry objectives, the reflectance at the coverslip-air interface is 10-fold greater than that at the specimen-coverslip interface, so the former interface is used as a reference boundary for focus control. Differences in the available offset quantity necessary for using a wide range of objectives can be obtained by introducing the appropriate turret lens into the optical path.
During operation, control over the objective position, and in turn, the focal planes imaging the specimen and the PFS slit (A and F in Figure 1, respectively), is determined by the projection of the slit image on the line-CCD sensor in the PFS unit, as presented in Figure 2. In cases where the specimen imaging chamber (reference interface) shifts in the negative axial (-z) direction, the slit image is shifted along one end of the CCD pixel rows and broadened. The reverse occurs when the chamber shifts in a positive direction (away from the objective; +z) on the microscope optical axis. Once a shift occurs, the PFS controller moves the objective in a compensatory direction to restore the slit image to the center of the line-CCD sensor. Figure 2 illustrates the signal strength of the slit image on the line-CCD sensor as a function of focal position (z). When the PFS slit light is focused directly on the reference plane, the slit image overlaps the central portion of the line-CCD (Figure 2(a)). As the reference plane is displaced by increasingly greater distances in the positive axial direction, the slit image broadens and is shifted toward the edge of the line-CCD (Figures 2(b) through 2(d)). The reverse occurs when the reference plane is shifted in the negative axial direction.
The line-CCD detector is a key element in the PFS system because the signal derived from projection of the slit image onto the CCD surface is fed directly to the control electronics CPU housed in the unit, which interprets focal point information to determine the focus state of the objective with respect to the reference plane. The PFS electronics then outputs the necessary information to the linear encoder control unit (housed in the microscope frame) to drive the nosepiece into the correct position. Each different objective used with the PFS is registered with the control electronics to provide the unit with data that can be used to establish the correct nosepiece position in relation to the objective front lens. When the PFS unit is activated, the nosepiece is driven to the registered vertical position for the objective, and then closer to the specimen chamber by half the working distance (a value of approximately 65 micrometers for a typical 100x apochromatic objective). At this point, the PFS unit searches for the reference plane (termed hunting) until it is detected by the line-CCD. In cases where the reference plane cannot be detected, the search operation ceases after a pre-defined period. Excessive hunting can be a problem when the incorrect immersion oil is used or if the specimen parameters do not conform to the requirements of the PFS unit, both conditions that lead to poor signal detection by the line-CCD. In cases where the aqueous solution bathing the specimen is less than 3 millimeters thick, hunting or complete failure to detect the interface can also occur. Under ideal circumstances, the focusing precision of the PFS is usually less than one third of the objective focal depth.
Joel S. Silfies, Edward G. Lieser, and Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.