Confocal Microscopy is rapidly gaining acceptance as an important technology owing to its capability to produce images free of out-of-focus information. In a conventional epi-fluorescence microscope, the entire object is exposed to excitation light and the emission collected by high NA objectives comes from throughout the specimen, whether above or below the focal plane. This seriously degrades the image by reducing the contrast and sharpness. In confocal microscopy out-of-focus information (blur) is removed. In addition, confocal microscopy provides a significant improvement in lateral resolution and the capacity for direct, non-invasive serial optical sectioning of intact, thick living specimens.
Confocal Microscopy was introduced in 1957. Most confocal microscopes are of two types: (1) stage-scanning (SSCM), and (2) laser scanning (LSCM). The SSCM is assembled on an epi-illuminated microscope employing a stationary laser as an excitation source, a photomultiplier as the detector and a specimen holder (stage) which moves and thus allows the specimen to be "rapidly" scanned in the X-Y plane. A pin-hole in the emission path coupled with a high NA (1.4) objective lens removes out-of-focus information and sharply improves the contrast. However, SSCM requires a relatively long period of time (~10 sec) to acquire a single image. Thus the SSCM can be used satisfactorily for fixed specimens or microelectronic circuits, but not for the live specimens where dynamic events are occuring.
Laser Scanning Confocal Microscopy
Many investigators designed confocal microscopes for use with live specimens to image dynamic events in which a fixed microscope stage is scanned by a laser beam using a rotating disk or mirror galvanometers. LSCM generates a clear, thin image (512 X 512) free from out-of-focus information within 2 or 3 seconds. A single diffraction-limited spot of light is projected on the specimen using a high numerical aperture objective lens and the light reflected or fluoresced by the specimen is collected by the objective and focused upon a pinhole aperture and the signal detected by a photomultiplier. Light originating from above or below the image plane strikes the walls of the pinhole and is not transmitted to the detector. To generate a two-dimensional image, the laser beam is scanned across the specimen pixel-by-pixel. To produce an image using LSCM, the laser beam must be moved in a regular two-dimensional raster scan across the specimen and the instantaneous response of the photomultiplier must be displayed with equivalent spatial resolution and relative brightness at all points on the synchronously scanned phosphor screen of a CRT monitor.
For a three-dimensional projection of a specimen one needs to collect a series of images at different Z-axis planes. The vertical spatial resolution is approximately 0.5um for a 40X 1.3 NA objective. Three-dimensional image reconstruction can be accomplished with many commercially available software systems.
The photomultiplier tube (PMT) used in LSCM has highly desirable characteristics compared to video cameras: (1) stability; (2) low noise; (3) very large dynamic range (> 1 million fold); (4) sensitivity; (5) wide range of spectral resonse; (6) rapid response; and (7) small physical size. The PMT has a low quantum efficiency (QE) of about 30% and in the red wavelengths about 3% and produces very low background noise signal. The alternative, a cooled-PIN photodiode, has a QE of 60-80% but an equivalent noise level of about 100 photons/pixel so that it is not useful for weak signals. The optimum selection of pinhole size is important in the compromise between intensity (brightness) and thickness of the slice observed. For instruments with variable pinholes, an optimum pinhole diameter should be determined empirically to provide the best combination of brightness and slice thickness.
Laser scanning confocal FRET (C-FRET) microscopy overcomes the limitation of out-of focus information owing to its capability of rejecting signals from outside the focal plane and acquire the signal in real-time (Kenworthy et al, 2000; Pozo et al, 2002; Wallrabe et al, 2003). This capability provides a significant improvement in lateral resolution and allows the use of serial optical sectioning of the living specimen (Pawley, 1995; Lemasters et al, 2001). By selecting appropriate filter combinations one can configure any commercially available confocal microscopy system for FRET imaging.
Disadvantage of LSCM
A disadvantage of this technique is that the wavelengths available for excitation of different fluorophore pairs is limited to standard lasers lines. Standard laser lines do allow C-FRET to be used for a number of fluorophore combinations including CFP-YFP or ds-RED, GFP-Rhodamine or Cy3, FITC or Alexa488-Cy3, Alexa488-Alexa555 and Cy3-Cy5 (Day et al., 2003; Elangovan et al., 2003; Kenworthy et al, 2000; Mills et al, 2003; Periasamy, 2001; Wallrabe et al, 2003).
Also, in one-photon wide-field or confocal microscopy, illumination occurs throughout the excitation beam path, in an hourglass-shaped pattern. This results in absorption along the excitation beam path, giving rise to substantial fluorescence emission both below and above the focal plane. Excitation of other focal planes contributes to photobleaching and photodamage in the specimen planes that are not being involved in imaging. This can be ameliorated by Multi-photon/2-photon microscopy.