Confocal Laser Scanning Microscopy

Confocal microscopy is an optical imaging technique used to increase micrograph contrast and/or to reconstruct three-dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. Confocal microscopy was pioneered by Marvin Minsky in 1955 while he was a Junior Fellow at Harvard University¹. Minsky’s invention would perform a point-by-point image construction by focusing a point of light sequentially across a specimen and then collecting some of the returning rays. By illuminating a single point at a time Minsky avoided most of the unwanted scattered light that obscures an image when the entire specimen is illuminated at the same time. Additionally, the light returning from the specimen would pass through a second pinhole aperture that would reject rays that were not directly from the focal point. The remaining ‘‘desirable’’ light rays would then be collected by a photomultiplier and the image gradually reconstructed using a long-persistence screen.

The majority of confocal microscopes image either by reflecting light off the specimen or by stimulating fluorescence from dyes (fluorophores) applied to the specimen. Confocal microscopy possesses several advantages over conventional microscopy. First, confocal microscopy produces images of improved resolution, up to 1.4 times greater than standard microscopy, by eliminating out-of-focus light. Confocal microscopes also have a higher level of sensitivity compared to conventional microscopes, due to highly sensitive light detectors and the ability to accumulate images captured over time. Another key advantage of confocal microscopy is the ability to produce 3-dimensional reconstructions of specimens as mentioned above. Computer software is then used to digitally reconstruct 3D representations of the sample. Confocal microscopy is also a less invasive form of imaging. This is due to the use of high-power laser illumination and the reduction in light-scattering artifacts, allowing the non-invasive imaging of thick sections of semi-transparent tissues².

In a confocal laser scanning microscope (CLSM), a laser beam is used as a light source. The laser beam passes through a light source aperture and then is focused by an objective lens (see figure below) into a small focal volume within or on the surface of a specimen.

 

 

Image formation:
In a typical set-up for a laser scanning confocal microscope, a pinhole is placed in front of the light source to produce a distinct and spatially constrained illumination point. The light passing through this pinhole is focused on the sample, while a second pinhole is placed in front of the light detector. By varying the pinhole diameter, the degree of confocality can be adapted to practical requirements. With the aperture fully open, the image is nonconfocal. The pinhole also suppresses stray light, which improves image contrast. If the optical distance from the detector pinhole to the focal point (the point in which the light is focused) is exactly the same as that between the focal point and the illumination pinhole, only the light generated at the focal point will reach the detector since the pinhole will block out the out-of-focus light. The signal from the detector is then digitized and passed to a computer. The image of the sample is digitally built up by scanning the sample in the X and Y directions and then special software is used to reconstruct a digital image. The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the plane of interest a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The beam is scanned across the sample in the horizontal plane using one or more (servo-controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage or the objective lens. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes. By using a CLSM, it is therefore possible to exclusively image a thin optical slice out of a thick specimen (typically, up to 100 μm), a method known as optical sectioning.

Resolution Enhancement
In laser scanning confocal microscopy, a fluorescent specimen is illuminated by a point laser source, and each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning volume is determined by the spot size (close to diffraction limit) of the optical system. This is due to the fact that the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the numerical aperture of the system’s objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using wide-field illumination. However, with confocal microscopy it is even possible to overcome this resolution limit of wide-field illuminating techniques as only light generated in a small volume element is detected at a time. Here it is very important to note that the effective volume of light generation is usually smaller than the volume of illumination; that is, the diffraction pattern of detectable light creation is sharper and smaller than the diffraction pattern of illumination. In other words, the resolution limit in confocal microscopy depends not only on the probability of illumination but also on the probability of creating enough detectable photons, so that the actual addressable volume being associated with a generated light intensity is smaller than the illuminated volume. By using light creation processes with much lower probabilities of occurrence such as second harmonic generation, the volume of addressing is reduced to a small region of highest laser illumination intensity resulting in a significant improvement in lateral resolution.

References:
1. Minsky, M. Memoir on inventing the confocal microscope. Scanning 1988, 10, 128–138.
2. Matsumoto B. 2002 Cell Biological Applications of Confocal Microscopy. San Diego: Academic Press. 507 p.