Confocal Microscopy and the use of laser

In 1957, Marvin Minsky applied for a patent for a microscope that used a stage-scanning confocal optical system. Misnky’s particular design introduced for very first time the possibility to obtain “sectioning effect” in a microscope 1)).
One of the key elements in a confocal microscope is the so called pinhole, a spatial filter that permits the acquisition
of different sections of the specimen. The consequences of using the pinholes are very unique and are reflected in the possibility to reduce the blurring of the image, increase the effective resolution and improve the signal to noise ratio, permitting also unusually clear examination of thick light-scattering objects. The technique used a laser as a light source. If the contrast mode used is fluorescence light the technique is called Scanning Confocal fluorescence microscopy.
The method is simple, quick and sturdy and therefore it has a great variety of uses.

1)) Since the integral of the point spread function for the conventional wide field microscope is constant as a function of depth,
conventional widefield microscopes do not have sectioning capability, something that can be obtained using confocal microscopy.

Laser scanning through a liposome (left) through a series of multiple planes, where all of which emits a fluorescent contour (middle) and then can be reconstructed into a three-dimensional picture of the liposome (right). This liposome is formed by a mixture of fats which is similar to that of the human skin.

Ordinary Confocal Microscopy could also be called single-photon fluorescent because fluorescent molecules are excited when they are struck by a single photon with a certain wavelength from the laser. After a finite lifetime of particular molecules (particularly conjugated system)in the excited state, the excess of energy gained by absorption of one photon is released as a form of light (fluorescence). In this linear process the wavelength of the emission light is always longer than the excitation light. It is important to mention that the excitation process can be generated also for the simultaneous absorption of two-photons instead of one. In the case of the two photon excitation process each photon delivers half of the excitation energy. In this particular case the wavelength of the fluorescence emission light will be shorter than the excitation, so the process is non-linear. This particular process requires very high photon density in a small area. In 1990 Denk and collaborators (Science, 248:73-76) combine the strong focusing capabilities of the microscope objective and the non linear quadratic dependence of the two photon excitation process. Because of the nonlinear quadratic dependence of the two photon excitation process the excitation is confined in the focal volume 2)) and inherent spatial resolution is obtained due to this particular excitation mode. Doing this means there is no absorption above and below the focal plane and a pinhole is not required to obtain a section of the specimen. In term of resolution no substantial differences are between confocal and two photon excitation microscopy and in both cases 3D images can be easily reconstructed (because the sectioning effect). The main advantages of the two photon excitation microscopy over confocal microscopy are high penetration in thick specimen (because IR light is used to excite the molecules and the scatter coefficient in tissues for IR light is low compared with visible light), bleaching and photodamage of the specimen are reduced (very important in the study of the living tissue) and different fluorophores (that will require more than one wavelength of excitation in one photon mode) can be used at the same time at a single excitation wavelength (because the one and two photon absorption process are different).

2)) Notice that in one photon excitation this dependence is linear and absorption of light will occur in the focal volume but also above and below along the z axis (that is the reason why a pinhole in the emission path is needed to obtain sectioning capabilities in one photon excitation mode).