The current revolution in fluorescent protein technology is driving a wide spectrum of associated methodologies, including various imaging modes in fluorescence microscopy using living cells. Over the past few years, live-cell imaging has become an essential tool in many cell biology laboratories due to the wealth of dynamic information it can provide concerning the fundamental nature of cellular function. Perhaps the most interesting questions in biology, including those pertaining to growth, differentiation, division, and apoptosis as visualized in living cells, will ultimately be answered by long-term microscopy investigations using time-lapse imaging techniques. Live-cell imaging, in which the cells must be maintained in a healthy environment on the microscope stage, presents a host of challenges to ensure that the cells are kept alive and in sharp focus throughout the course of the experiment. Provided that the environmental conditions are ideal, the most significant pitfall to imaging living cells is the inherent focus drift that often occurs due to thermal gradients, vibration, mechanical instability, and a number of other factors.

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 used with the TiE-PFS inverted microscopes for focus compensation. The near-infrared 870-nanometer LED and CCD line sensor utilized by the PFS are housed in a specialized nosepiece unit (Figure 1) that does not require additional infinity space and enables the primary microscope optical train to remain dedicated for imaging. Among the most advanced features of the Nikon PFS is the 5 millisecond (200 Hz) sampling rate, which is independent of microscope and camera control software, and considerably faster than other systems that must repeatedly probe both the coverslip interface and the focal plane of interest. Utilization of the long-wavelength LED enables the TiE-PFS to be used with a large variety of fluorophores emitting in the wavelength range between 340 and 750 nanometers. Additionally, the PFS can be used with virtually all contrast-enhancing imaging modes, including brightfield, darkfield, phase contrast, Hoffman modulation contrast, DIC, widefield fluorescence, confocal, TIRFM, spinning disk, and line-scanning swept-field.

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. As discussed above, the bandpass interference filter in the PFS unit spans a broad transmission range of 340 to 750 nanometers (see Figure 2; left red curve), enabling the application of all known fluorescent proteins (including near-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 internal infrared blocking filter from the PFS unit, it can be adapted for use with laser tweezers and laser trapping applications, as well as a portion of the multiphoton excitation spectral region. Filter passbands for the visible and near-infrared region for the PFS system (red curves; 870-nanometer LED) and other focus compensating systems (blue curves; approximately 780-nanometer LED) are illustrated in Figure 2. Note that the longer wavelength LED in the PFS enables the use of fluorophores emitting at wavelengths that would conflict with focus detection in the other systems. 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.

Anatomy of the Perfect Focus System

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 onto the 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.

In operation, the investigator focuses on the plane of interest within the specimen (usually a culture of living, adherent cells in a specially designed imaging chamber) and activates the PFS unit, which then projects the near-infrared line pattern onto the reference plane and defines an offset or distance between the axial reference plane and the specimen focal plane. The PFS continuously feeds positional data back to the circuitry controlling the microscope focusing mechanism to maintain a precise relationship between the reference plane and the imaging focal point. The degree of offset is controlled by the operator, and is kept constant by a stepper motor (0.025 micrometers in the axial resolution) in the PFS unit, regardless of independent vertical fluctuations that occur to the specimen position or microscope optical train. The linear encoder registers the positional coordinates of the nosepiece as a function of the objective type and is used as a metering device to determine the position of the objective in relation to the reference plane. Thus, the axial position of the nosepiece and the focus offset can be registered and stored with the linear encoder control electronics for each objective.

The Nikon PFS unit is housed in a six-objective motorized nosepiece that is positioned between the fluorescence filter set turret and the stage on the inverted model Ti-E microscope. Electronic controllers for the system are integrated within the frame so the unit operates in stand-alone mode without requiring a software interface to the host computer. Perfect Focus can be engaged or disengaged using a lever on the housing that swings a dichromatic mirror into or out of the primary optical train in a region of parallel light waves beneath the objective. Incorporated within the PFS unit is a separate optical system containing offset lenses, beam-shaping optics, a near-infrared 870-nanometer light emitting diode, and a line-CCD detector. Individual optical trains for the LED source and detector intersect at a half-mirror in the central portion of the unit, and the near-infrared light is directed and retrieved from the main optical system using the switchable main dichromatic mirror. A unique feature of the PFS is that the image-forming and focus-detecting systems share a portion of the same optical train, but can operate independently of each other without interference due to the differences in their spectral composition.

Illustrated in Figure 3 is a schematic diagram illustrating the primary (imaging forming; the yellow ray traces) and focus detection (red ray traces) optical systems used to monitor and maintain focus. Near-infrared light emitted by the LED is first passed through a condensing lens and slit plate (having the long axis perpendicular to the page) that shares a conjugate plane with the liquid-glass interface at the coverslip. A collimating lens converts the slit-shaped light into a parallel bundle and passes it through a half-moon pupil-restricting mask that blocks a portion of the light along the central region of the optical axis. The beam is then transmitted through a half-mirror, which also serves as an intersection point to the detector optical train. The light transmitted through the half-mirror is first focused by the offset adjustment lens system, and then directed through a visible light cut filter before entering the primary microscope optical system via a dichromatic mirror that serves as the intersection to the PFS unit.

The intersecting dichromatic mirror (positioned beneath the objective), which reflects near-infrared light and passes visible light, is a primary component in the afocal infinity space (parallel light wavefronts) of both the image-forming and focus detecting optical systems. Near-infrared light from the PFS unit enters the primary optical system after reflection from the mirror surface and is focused by the objective onto the specimen chamber. Note that the objective also operates to simultaneously illuminate (in fluorescence mode) and receive image-forming wavefronts from the specimen. As illustrated in Figure 3, the image-forming components of the optical system include the objective, various dichromatic mirrors and filters, the tube lens, camera relay lens, and primary CCD image detection sensor (denoted in yellow ray traces). An infrared-cut filter can be placed in the primary optical train between the PFS intersection and the camera system to ensure that stray near-infrared light from the focus-detection unit does not interfere with image formation.

The visible and infrared cut filters depicted in Figure 3 serve to cleanse their respective optical systems (imaging and PFS) of contaminating light from each other. Thus, the visible cut filter (positioned at the entrance of the PFS optical train) ensures that fluorescence emission and transmitted broadband visible radiation does not enter the PFS optical system and disrupt the line sensor or LED light source. Likewise, the infrared cut filter that is situated beneath the intersecting dichromatic mirror serves to remove PFS-generated light from entering the imaging portion of the optical train and create noise that can be detected by the image sensor. The two separate optical systems therefore share only a small portion of the overall imaging optical train, including the intersecting dichromatic mirror, objective, and specimen chamber. Otherwise, the optical systems are able to coexist and operate independently.

The line-shaped PFS near-infrared light beam reflected from the glass-water interface in the specimen chamber is captured by the objective and converted into parallel wavefronts in the primary microscope optical system. This light is reflected by the intersecting dichromatic mirror back into the focus-detection optical train and passes again through the visible light cut filter and the offset adjustment lens assembly before being reflected into the image-forming PFS detection optical train by the half-mirror. After entering the PFS detection system, which contains a condensing (objective) lens, two relay lenses, a half-moon mask, a cylindrical lens, and a line-CCD detector, the light is converted into an image of the slit by the condensing lens. The slit image is then passed through the relay lenses and half-moon mask before entering the cylindrical line-shaping lens and being projected onto the surface of the line-CCD sensor. Note that the light shielded by the half-moon mask in the detection optical train corresponds to the same area that is shielded by the similar mask in the LED portion of the PFS optical system. The cylindrical lens compresses and shapes the slit image so that it forms a sharp line in the central portion of the CCD. The PFS system uses light shaped by the slit rather than a circular light beam in order to reduce scattering and other artifacts associated with detection of the glass-liquid interface.

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 4. 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 4(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 4. 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 4(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 4(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 4(c)), the objective focal point (F) is then moved into the central portion of the specimen. The distance between the focal points F and A 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 4, respectively), is determined by the projection of the slit image on the line-CCD sensor in the PFS unit, as presented in Figure 5. 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 5 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 5(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 5(b) through 5(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. It is important to ensure there is adequate aqueous solution (culture medium) bathing the specimen 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.

The PFS unit is designed primarily to image living cells housed in specialized imaging chambers equipped with a coverslip ranging in thickness from 150 to 180 micrometers (#1 or #1.5 coverslips). Other specimens may be far more difficult to observe with focus drift correction due to weak infrared reflectivity or excessive scattered light. These include fixed specimens that are mounted in a high refractive index medium (which more closely matches that of the coverslip). In this case, the amount of reflected light from the focus monitoring system may be insufficient to detect the interface surface. Likewise, thick tissue specimens, which scatter a considerable amount of light, are difficult to use with focus drift correction. Thick glass coverslips (greater than 180 micrometers) or plastic tissue culture dishes are also not recommended, as the boundary surface may not be detectable due to insufficient offset. Finally, dust and debris on the coverslip surface can degrade the precision of boundary detection, leading to excessive hunting or errors in focus correction.

Conclusions

Although there are several commercially available hardware-based focus drift correction systems on the market, all are designed to measure reflectance from the coverslip interface as the basis of their operation. Major differences occur in the manner for which each unit accomplishes this task, and these variables have significant implications for the speed with which the units can successfully detect focus and capture images. Another major difference lies in the capacity of the various systems to handle imaging in complex routines that require capturing images at different lateral and axial locations in a single experiment. In terms of the Nikon PFS unit, the near-infrared focus detection light does not interfere with most forms of transmitted and fluorescence microscopy and doesn't contribute to photobleaching or phototoxicity. The focus position in the specimen is controlled by the operator using the offset function and focus can be maintained for any location in the viewfield. Focus control with PFS can be implemented with virtually any scientific grade CCD or electron-multiplying CCD camera system and for imaging in a wide variety of contrast-enhancing techniques. In situations where PFS is not required, the intersecting dichromatic mirror can be easily removed from the imaging optical path to improve light throughput. When coupled to the proper environmental chambers and thermostatic control systems, the Nikon PFS system is capable of delivering unprecedented accuracy in maintaining suitable specimens in sharp focus throughout the imaging session.