Laser scanning confocal microscopy offers several advantages over conventional widefield fluorescence microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus by illuminating the objective through a pinhole (see Figure 6 and Figure 8(b)). An image of the pinhole in the form of a small spot is formed on the specimen by a focused laser driven by a galvanometer-based scanning system. This spot, in turn, forms a reflected epi-fluorescence image on the original pinhole. If the specimen is in focus, the light passes through the pinhole to a detector (usually a photomultiplier). When the specimen is not in focus, the light reflected from it is defocused at the pinhole and very little passes through. Thus, fluorescence emission returning from the specimen to the detector is spatially filtered. As the pinhole aperture is reduced in size, it blocks more of the stray light from being detected but also lowers the total signal level. Although the absolute signal value is far less than observed with the widefield microscope configuration, rejecting the light from other focal planes increases the specific signal-to-noise ratio for the features of interest.
Additional advantages of laser scanning confocal microscopy include the ability to adjust magnification electronically by varying the area scanned by the laser without having to change objectives. This feature is termed the zoom factor, and is usually employed to adjust the image spatial resolution by altering the scanning laser sampling period. Increasing the zoom factor reduces the specimen area scanned and simultaneously reduces the scanning rate. The result is an increased number of samples along a comparable length, which increases both the image spatial resolution and display magnification on the host computer monitor. Confocal zoom is typically employed to match digital image resolution with the optical resolution of the microscope when low numerical aperture and magnification objectives are being used to collect data. The zoom control should be used with caution due to the fact that high zoom factors lead to increased photobleaching. In general, successful confocal imaging is a tradeoff between obtaining suitable zoom factors and imaging the specimen (especially when acquiring optical sections) without incurring significant photobleaching levels.
The major disadvantage of conventional laser scanning confocal microscopy for imaging cells and tissues is that the image is gathered by raster-scanning the specimen, which is relatively slow and often requires several seconds or longer per image. There is also a high degree of risk from irradiating the specimen with intense laser light that can produce phototoxicity in living cells. In most cases, the dwell time of the laser beam at any position on the specimen is only a few microseconds. Therefore, the laser excitation energy must be high enough to generate useable signal during that dwell time, often leading to complete saturation of all the fluorophores residing in the spot. Under these conditions, rapid photobleaching occurs, and gathering images with a high intrascene dynamic range (a significant number of gray levels) is challenging. For live-cell imaging over prolonged periods, the laser intensity should be reduced as much as possible. The major challenge for live-cell imaging using laser scanning confocal microscopes is to generate sufficient contrast while reducing phototoxicity, especially in cases where the cells are imaged for prolonged periods.
Spinning Disk Confocal Microscopy
A variation of the galvanometer-based confocal microscope is the spinning disk confocal microscope, which can operate in real time (30 frames per second) or even faster to capture dynamic events in a wide spectrum of timescales. The principle is based on a Nipkow-style disk that is opaque with the exception of thousands of drilled or etched pinholes, often covered with miniature focusing lenses, arranged in interleaved Archimedean spiral patterns. Each illuminated pinhole on the disk is imaged by the objective to a diffraction-limited spot on the specimen. The fluorescence emission reflected from the specimen can be observed and recorded after it has passed back through the Nipkow disk pinholes. Several thousand points on the specimen are simultaneously illuminated by the disk achieving, in effect, several thousand confocal microscopes, all running in parallel. Spinning the disk fills in the spaces between the holes and creates a real-time confocal image that can be directly observed with the naked eye, as with the standard microscope.
An alternative to the Nipkow disk involves recent technology that offers high light throughput using a disk that is etched with a pattern of perpendicular slits, rather than pinholes, providing greater transmission while maintaining an acceptable level of confocality (Figure 7). Unlike the Nipkow-style disks, these slit-pattern disks are produced with varying slit widths that match different objective numerical apertures, magnifications, and specimen thicknesses. Thus, the slit disks enable confocal images to be acquired at high resolution using objectives ranging in magnification from 10x to 100x. Both spinning disk designs were developed primarily for live-cell imaging applications where a compromise exists between the needed increase in image acquisition speed versus a slight loss in axial resolution (Figure 8(c)). Most spinning disk microscopes are illuminated with lasers or arc lamp sources and are coupled to sensitive electron-multiplying CCD cameras in order to image faintly fluorescent specimens.
Several manufacturers have introduced confocal systems using laser light shaped into a line, rather than a point, to scan the specimen, but with a substantial decrease in image acquisition time at the expense of resolution (usually a factor of 1.2 in one dimension). Unlike the case with spinning disk microscopy, the degree of confocality can be controlled by the user by invoking changes to the line shape and size. Line scanning microscopes are used with CCD camera systems rather than photomultipliers and are generally much faster in acquisition speed than spinning disk or laser scanning confocal microscopes. In summary, there has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and its great number of applications in many areas of current research interest.
Multiphoton Microscopy
Excitation in multiphoton microscopy is a non-linear process that occurs only at the focal point of a diffraction-limited microscope, providing the ability to optically section thick biological specimens in order to obtain three-dimensional resolution. Individual optical sections are acquired by raster scanning the specimen in the x-y plane, and a full three-dimensional image is composed by serially scanning the specimen at sequential axial (z) positions, similar to the case in confocal microscopy (to produce optical sections). Because the position of the focal point can be accurately determined and controlled, multiphoton fluorescence is especially useful for probing selected regions beneath the specimen surface. The highly localized excitation energy serves to minimize photobleaching of fluorophores to those residing in the focal plane and thus reduces phototoxicity, which increases sample viability and the subsequent duration of experiments that investigate the properties of living cells and tissues. In addition, the application of near-infrared excitation wavelengths permits deeper penetration into biological materials and reduces the high degree of light scattering that is observed at shorter wavelengths. These advantages enable researchers to conduct experiments on thick living tissue samples (see Figure 8(d)), such as brain slices and developing embryos that would be difficult, if not impossible, to image with other microscopy techniques.
