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Confocal Microscopy

Laser scanning confocal microscope can provide superior axial resolution relative to widefield microscope. Since the images obtained with a confocal microcope are devoid of any out of focus blur, the technique is very popular amongst biologist to image deep into a tissue without any physical insertion into the sample. Figure 1 shows a lens "L" focusing the light emitted by two point sources (red one and the blue one). The red source is considered to be in the object plane (or the sample plane) and the light from it are focused at a point where a pinhole "PH" is positioned. It is now seen that the pinhole allows more light from the red source to reach the photodetector than the light from the blue source which is behind the object plane.

Figure 1: The principle of obtaining superior axial resolution.

Similarly it is easily seen that the light from a source ahead of the object plane are also mostly blocked by the pinhole. The principle depicted in figure 1 is used in a confocal microscope to block the out of focus light from reaching the image plane. In order to form an image of a 2D area, a focused laser beam scans the target. For each position of the laser beam, light scattered or emitted by the target is descanned by the scanner and focused onto the pinhole. Signal from the photodetector is sent to a computer, which at the end of the scan forms an electronic image of the target area. Using an illumnation laser beam of wavelength λ the lateral and axial resolutions achievable with a confocal microscope are λ/2 and λ respectively.

The performance of a confocal microscope may be compromised due to the presence aberrations in the illumnation beam or some times due to the aberrations introduced by the target being imaged. By incorporating a programmable optical element in the laser beam path of a confocal microscope it is possible to have an illumination beam with a reconfigurable wavefront. Thus knowing the aberrations being introduced, it is possible with the programmable element to get a detection path that is free from aberrations. Figure 2 shows the focus spots in the sample plane of a confocal microscope with a helical wavefront illumination beam. Due to the abberations present in the laser beam the focus spot (left) is not a perfect doughnut, while the focus spot (right) is closer to a doughnut as the illumination beam has been partially corrected for aberrations using the programmable element.

Figure 2: The illumination beam spots with a helical wavefront in the presence of aberration (left) and after partial correction of aberrations (right).

Figure 3: The axial sections of the illumination beam spot with a normal wavefront (left) and with a designed wavefront (right).

In addition to correcting for aberrations the programmable diffractive element in the illumination beam path of the confocal microscope can be used to generate user defined three dimensional focus spots. Figure 3 shows axial cross-sections (the optic axis along the vertical direction) of a plane wavefront illumination beam (left) and a top hat type wavefront illumination beam (right). The top hat type wavefront, which is generated by the programmable element, results in a doughnut focus spot again, likewise the helical beam. However, the former also has two axial side lobes as clearly seen in the figure. More details about a confocal microscope with programmable beam forming optics are availble here.

The helical and top hat wavefront beams discussed above are useful in a number of applications such as optical trapping and stimulated emission depletion (STED) microscopy. STED microscopy is basically an advanced version of confocal microscopy where the stimulated emission phenomenon is used to enhance the resolution of a confocal microscope beyond the diffraction limit. The STED technique requires doughnut shaped focus spots to reduce the effective illumination/excitation volume in the sample plane. Figure 4 shows the images of flourescent beads using a conventional confocal microscope (left) and using a STED confocal microscope (right). As clearly seen in the figure the beads are more clearly seen in the STED confocal image relative to the conventional confocal image. So far the best lateral resolution acheived in practice with a STED microscope is ≈20 nm. More details about the above STED results are availble here.

Figure 4: Images of 200 nm diameter floursecent beads using (left) a conventinal confocal microscope and (right) a STED confocal microscope.