Confocal Microscope Scanning Systems

Confocal imaging relies upon the sequential collection of light from spatially filtered individual specimen points, followed by electronic signal processing and ultimately, the visual display as corresponding image points. The point-by-point signal collection process requires a mechanism for scanning the focused illuminating beam through the specimen volume under observation. Three principal scanning variations are commonly employed to produce confocal microscope images. Fundamentally equivalent confocal operation can be achieved by employing a laterally translating specimen stage coupled to a stationary illuminating light beam (stage scanning), a scanned light beam with a stationary stage (beam scanning), or by maintaining both the stage and light source stationary while scanning the specimen with an array of light points transmitted through apertures in a spinning Nipkow disk also know as a scanning disk (see Figures 1 and 2). Each technique has performance features that make it advantageous for specific confocal applications, but that limit the usefulness in others.

The stage-scanning and beam-scanning configurations are single-beam methods, while the spinning disk approach is a multibeam scanning technique. Systems employing the Nipkow disk-scanning concept have typically utilized noncoherent broad-spectrum light sources (such as arc-discharge lamps) for illumination rather than lasers, and the overall lack of brightness has severely limited their use in fluorescence applications. However, modern microlens arrays and sophisticated disk design enhancements, coupled to laser illumination, have expanded the potential applications for spinning disk confocal microscopes. Nipkow disk systems may be designed in either tandem scanning or monoscanning variations. In the former, the illumination and detection beams follow tandem pathways through separate sets of identical apertures located on diametrically opposite sides of the disk. The monoscanning system performs illumination and detection simultaneously through each rotating aperture on the disk, maintaining the coincidence that the two light paths exhibit while passing through the objective.

An additional method of single-beam scanning has experienced limited application in specialized reflected light microscopes, primarily for integrated circuit inspection. The objective itself may be scanned over a stationary specimen utilizing a stationary light source in a scanned-lens system. This configuration has similar optical advantages to stage scanning, but enables the stationary specimen to be fitted with measuring probes, or otherwise manipulated. The configuration is not amenable to rapid scanning when a relatively massive conventional objective is employed, and is therefore not widely utilized.

The modern confocal microscope is an integrated electronic system, most commonly based on a widefield epifluorescence instrument, with the addition of multiple laser illumination sources, a scan head containing electronic and optical components, a computer and monitor for image display, and associated software for control of signal acquisition, processing, and image analysis. In the basic confocal optical configuration, the objective forms an image of both the source and detector pinholes on the specimen plane. By positioning the pinholes on the microscope optical axis in conjugate focal planes, their images overlap within the specimen focal plane. Although fluorophores outside the plane of focus are excited, detection is limited to emission occurring near the focal plane by the detector pinhole aperture, which rejects out-of-focus light. The single-beam laser scanning confocal microscope functions in this point-scanning mode as a sampling device and does not form an optical (real) image. To enable image formation, the sampling spot must be moved through the specimen and the resulting signal collected and stored. The scan head controls the generation of photon signal required to construct the confocal image. The components of a typical commercial scan head are illustrated in Figure 1, and generally include one or more laser inputs, fluorescence filter sets, a raster scanning mechanism, variable pinhole apertures, and detectors (usually photomultiplier tubes, or PMTs) for multiple fluorescence wavelength detection.

To extend the confocal point-sampling principle to enable the generation of an extended specimen image field, the point focus in the specimen is scanned in a raster pattern similar to that employed to create the image on a television screen (and in other video applications; see Figure 2(a)). This mechanism requires a fast horizontal scan (the line scan) in conjunction with a slower vertical scan, or frame scan, which offsets the scanning line to sequential positions from the top to the bottom of the frame. During the history of confocal microscope development, a number of different techniques have been employed to implement point scanning, and several have been refined into current commercial versions. In single-beam laser scanning instruments, a typical raster scanning mechanism utilizes two high-speed oscillating mirrors driven by galvanometer motors, which pivot on mutually perpendicular axes. Coordination of the two mirrors, one scanning along the x-axis and the other on the y-axis, produces the rectilinear raster scan. The scanning speed of the mirrors is negligible compared to the speed of light, and consequently the emitted fluorescence can be collected by the objective and returned, or descanned, along the original illumination path to its conjugate focal plane at the detector pinhole. Variation in the signal intensity illuminating the detector aperture corresponds to variations in emission at different points in the specimen as the exciting beam is scanned.

Several characteristics of an ideal confocal scanning system, required to provide optimum imaging performance, are extremely difficult to achieve in practice. Nearly all scanning system configurations present some shortcomings in operation, and various optical and electronic design modifications have been introduced in an attempt to correct the deficiencies. Several methods of implementing point scanning reduce sensitivity or involve severe compromises in flexibility or image quality, and are not currently used in commercially produced systems. One of the most important requirements of the scanning system design is that the objective pupil (rear focal aperture) is completely filled with light during the entire scanning cycle in order to prevent illumination fall-off at the scan extremes. This is best accomplished by minimizing movement of the beam at the aperture with a scan design that pivots the beam at a stationary point that is conjugate with the objective rear aperture. Maintaining a stationary pivot point when the beam is rocked during scanning is technically challenging, and some systems compensate for a small amount of beam movement by overfilling the aperture through increased beam expansion. This has the disadvantage of wasting light and reducing the photon efficiency of the system.

An additional desirable property of the confocal scanning mechanism is to scan at the highest frame rate possible in order to provide the flexibility to adjust various scanning modes to match the imaging application. This requires minimal inertia of the moving components that produce the beam scanning, as well as minimizing the system dead time, (or interval between each scanning cycle) in which the beam is not scanning the specimen. The proportion of each full-frame scanning interval that is utilized to actually scan the specimen is referred to as the duty cycle of the system. Minimizing non-productive scanning of the beam is not only essential to achieving a high frame rate, but in some instrument designs, reduces the unnecessary specimen photon damage that results from a poor duty cycle specification.

The capability to freely rotate the scan raster around the optical axis is a very important feature for confocal imaging as a means to optimize the scan direction with respect to specimen shape or other characteristics. When imaging elongated features such as fiber bundles, orientation of the fast scan direction parallel to the long axis of the feature greatly enhances the time resolution of the specimen signal. Furthermore, the ability to rotate the raster allows specimen features to be oriented in a manner that utilizes the image field most efficiently. Scanning arrangements that do not permit rotation of the raster direction can seriously limit the practicality of the system, unless the specimen itself can be easily rotated, a much more problematic operation that is not usually feasible.

The optical arrangement required to produce a linear motion of the illumination spot in the specimen is derived from consideration of the geometrical optics of the microscope, including the fact that the objective is telecentrically corrected (a telecentric lens system positions the entrance and exit pupils at infinity). To realize the full optical correction of the objective, the image and specimen planes must remain at fixed distances from the objective, and the locations of the conjugate image planes and conjugate telecentric planes are therefore known. A critical optical property is that all light beams intersect a telecentric plane at an angle that is a function of the position of the source point in the specimen plane. Since a flat mirror is capable of changing the angle of propagation of a light beam, the placement of a mirror at a conjugate telecentric plane on the optical axis provides a mechanism by which a change in beam angle will produce a linear motion of the focal point in the specimen. Therefore, in the simplest case, the positioning of a mirror with its pivot point at the center of the objective conjugate telecentric plane produces a one-dimensional beam scanner capable of changing the position of the illuminated spot in the specimen plane as a function of the mirror's pivot angle. Any conjugate telecentric plane is an image of the objective telecentric plane. When an intermediate optical system is employed, it forms an image of the mirror in the entrance aperture of the objective, maintaining the telecentric properties.

In principle, this scanning concept can be expanded into two perpendicular axes by simultaneously scanning the mirror in two directions or by adding a second mirror, although practical considerations generally determine the type of approach taken for a particular overall system design. When two mirrors are employed to scan the beam in perpendicular directions, they should be placed in conjugate telecentric planes, or alternatively, located in close proximity to one another (close-coupled). By deflecting the beam in orthogonal directions, such a scanning system can generate the fast and slow scan motions along the x and y axes necessary to form a complete two-dimensional image.

Various arrangements of scanning system components are possible provided that the primary requirements are satisfied. To ensure diffraction-limited performance of the optical system, the objective rear focal plane (entrance aperture) must constantly be uniformly filled by a planar wave during scanning. Because the physical diameter of this aperture varies with properties of the objective, all other components, including illumination pinholes, must be matched to the objective(s) in use. Conjugate telecentric planes can be produced in required locations by the addition of auxiliary optics, and if this approach is taken, the properties of these must also be carefully considered in regard to their compatibility with the objectives chosen for use with the system. The beam properties of illuminating lasers, in particular the diameter of the Gaussian beam profile, are significant factors in adjustment of pinhole diameter and other variables related to illumination of the objective entrance aperture.

In the simplest beam-scanning confocal configuration, a scan mirror is located in the rear focal plane of a scan lens, which is conjugate with the rear focal plane of the objective. Illustrated in Figure 3(a) is a single-mirror arrangement, which includes the tube lens required by an infinity-corrected objective. Scanning on one axis is easily accomplished with this configuration. The theoretically ideal means of achieving x-y scanning is to scan a single mirror on both axes simultaneously (referred to as cardanic scanning). Two scanning mirrors are more commonly employed, and two such possible configurations are illustrated in Figures 3(b) and 3(c). If the mirrors are close coupled (Figure 3(b)), the system can function satisfactorily without requiring additional intervening optics. With greater separation distance between the scanning mirrors (Figure 3(c)), a multilens telecentric relay system must be utilized to optimize the optical performance.

Although the mechanism of accomplishing the x-y raster scan is considered the most crucial aspect of the confocal scanning system, some method of z-axis scanning is necessary in order to acquire series of optical sections for three-dimensional imaging, and to collect x-z or y-z two-dimensional images, as well as to perform any form of free-line z-scanning. Typical microscope configurations change the objective-specimen distance by translating either the objective or the microscope stage. The movement can be performed precisely with a piezoelectric driver or galvanometer device, albeit over a limited distance range. More commonly, however, a microstepper motor is used to drive the fine-focus control of the microscope, and on modern instruments the steppers are capable of focus positioning at a minimum step size on the order of 10 nanometers. For biological fluorescence applications, z-positioning to this precision is more than adequate.

Technological developments in single-beam instruments have led to the development of fast-scanning instruments that can provide imaging at video rates in order to follow dynamic processes in living cells. Rotating polygon mirror scanners can achieve very high scan speeds, and are used in many optical devices, but do not provide the illumination and detection precision necessary for implementation in high-resolution microscopy. Various configurations combining scanning mirrors with acousto-optic deflectors (AODs) have also been explored. In some arrangements, an AOD provides very rapid scanning on one axis, and a mirror scanner controls the slower axis. This approach is acceptable in some applications, but is problematic in confocal fluorescence imaging because it does not allow the longer-wavelength fluorescence emission to be descanned back through the acousto-optic modulator, which is wavelength-specific. The partially descanned signal, still oscillating on one axis, can be passed to a photomultiplier through a slit aperture, or imaged onto a linear-array CCD detector. Although the resulting images are confocal on only one axis, the characteristics are acceptable for some applications. A more common approach to achieving high frame rates in a single-beam system is to utilize rapidly oscillating resonant mirror scanners. Most major manufacturers have incorporated resonant scanners as a standard scanning option to facilitate video rate images at speeds of 30 frames per second at full field of view. By clipping down in Y, resonant scanners can enable speeds of several hundred frames per second for applications that require high temporal resolution such as capillary blood flow or calcium dynamics.

Multi-beam scanning techniques offer an alternative to the single-beam scanning configurations, although low illumination efficiency has previously limited their use in high-resolution fluorescence applications. In either the tandem or monoscanning variation, rotating-disk scanners place hundreds of holes, which function as illumination and detection pinholes, in the microscope intermediate image plane. The configuration of a typical Nipkow disk scanning system is presented in Figure 2(b). The holes in the disk are arranged so that a large number of beams uniformly scan the image field as the disk rotates, completely covering the specimen at a much higher rate than that of single-beam scanners. Because disk-scanning microscopes form a real image, a CCD or CMOS camera may be located directly in the image plane, collecting emitted signal at much greater quantum efficiency than that exhibited by photomultiplier tubes. In spite of this advantage, which allows real-time focusing and imaging of dynamic processes, several shortcomings have limited the practical utility of disk-scanning confocal systems. Previously, one of the most serious shortcomings was the typical reliance on conventional broad-spectrum light sources, and the extreme light loss that occurs at the disk. However, advances in the design of disk systems and the application of laser sources have helped overcome some of the efficiency problems. Because each disk has holes of fixed size, the pinhole diameter cannot be matched to the specific objective being used, and consequently the choice of objectives that will perform optimally with a given disk is limited. Furthermore, it is not possible to optimize the diameter of source and detector pinholes independently.

One technique for improving upon the lack of brightness that characterized early Nipkow disk systems is to utilize microlenses to intensify the source light. Older systems transmitted only approximately one percent of the illumination incident on the disk and necessitated the use of either cooled CCD or CMOS cameras to compensate for the low signal level. Modern disk scanning microscope designs incorporate a second disk containing thousands of microlenses, which spins in alignment with the Nipkow disk, and amplifies the light passing in both directions through the Nipkow disk apertures. With advancements in microlens, laser, and camera technology, disk-scanning microscopes have become an indispensable tool for live cell imaging applications.

Conclusions

Various additional scanner modifications have either been proposed or implemented in the effort to improve some aspect of the practical performance of confocal instruments. Every aspect of confocal fluorescence microscopy is fundamentally related to efficiency, and the inherent limitations of serial data collection. The efficiency with which useful signal can be acquired defines the balance that must be reached between image contrast and photodamage to the specimen, and also controls the required compromise in serial data collection between spatial resolution of the sampling scan, the signal-to-noise ratio, and the rate of image acquisition.

For high-resolution fluorescence imaging, the most refined and versatile scanning technology currently available is some variation that utilizes galvanometer scanners, and most major microscope manufacturers produce at least one confocal instrument employing this methodology. Because of the importance of photon efficiency, the relative simplicity of single- beam scanning techniques gives them definite advantages over disk scanners in most fluorescence applications. They are compatible with a wide variety of conventional microscope optical systems and video equipment, they conform well to general microscopy principles, and the flexibility in pinhole adjustment allows optimization to specific optical and specimen variables.