Epifluorescent microscopy is capable of generating images of certain fluorescent samples; however, the sample will appear indistinct if the sample reaches a thickness greater than micrometers. This is because some parts of the sample lie outside of the focal plane. A new imaging approach was required to overcome this challenge.
The confocal approach was driven by Marvin Minsky’s desire to image neural networks in unstained preparations of living brains, and he is widely regarded as the inventor of the confocal microscope in 1955. The original principle is still used in the modern confocal microscope.
This form of imaging lends itself to many areas of the life sciences due to its ability to obtain high resolution images and the scanning nature of the imaging process.
Live cell imaging
When considering imaging of live cells, the illumination of the cells must be a consideration. When fluorescent cells are excited, it can lead to photobleaching, resulting in cell damage. The inherent nature of confocal imaging decreases this risk, as only the point being imaged is illuminated, which decreases excess exposure to background cells.
This method of exciting the sample leads to a limitation of cell damage. When partnered with a higher resolution, which is brought about through the elimination of out of focus glare, confocal imaging is exceptional at capturing details of live cells.
The technique has been found to be able to provide real time images of the eye, at 630x magnification, with a high enough resolution to determine anatomical detail at a cellular level.
Confocal imaging has also been shown to describe physiological processes in vivo in the cornea, kidney and liver. It has also been shown to be able to illustrate the healing of wounds in four dimensions (x, y, z, t) at a cellular level. This suggests that this method will be able to provide a novel paradigm for imaging in experimental biology.
Through the use of a fluorescent agent, fluorescein, it has been possible to directly identify areas of corneal injury. The use of confocal imaging has shown the ability to evaluate these injured areas in vivo.
This method has allowed studies to determine the irritancy of drugs and excipients to the eye. The results from this study ranked surfactants and was able to show that cationic surfactants were the most irritating and non-ionics the least.
Scientist who were investigating the number of nucleosomes in living cells were able to combine confocal imaging with fluorescence correlation spectroscopy. This combination of methods allowed them to map the resulting images from confocal imaging into calibrated nucleosome density maps.
This was done through the calibration of the high resolution image from confocal imaging with the fluorescence yield of the single fluorescent marker as derived from the fluorescence correlation spectroscopy.
Applications in dentistry
Resin-modified glass-ionomer cements are used in dentistry, however the tooth interface with these materials is poorly understood. The interface was investigated, with particular focus on the interface with dentin, with a series of the cements.
The samples of the tooth/material interface were examined using confocal microscopy. The samples showed that in some of the samples, there was an “absorption layer” which was determined to relate to the water flux between the maturing cement and the pulp in the tooth.
Confocal imaging was used to study the effects of antimicrobials on dental plaque. It was possible to quantify the plaque vitality, as a percentage, through the analysis of the images of the three plaque layers, the outer, middle and inner.
It was found that the treatment of chlorhexidine was significant in the six hour samples and only in the outer layer of the forty-eight hour samples. It was shown that this approach was possible to quantitate and visualize the effects of treatment on the biofilm.
Confocal imaging can be applied to many areas of dental research, including imaging crack propagation in dental biomaterials, high-speed dental cutting interactions and dental materials placement.
Characterization of pharmaceutical systems
Confocal laser scanning microscopy has been used in the characterization of many pharmaceutical systems, including tablets, film coatings and colloidal systems. It has also been used to study the interaction of biological barriers of the skin, eye and intestinal epithelia, and the effectiveness of dosage forms at delivering the drugs through these barriers.
The surface roughness of tablets has been investigated through the use of confocal imaging - this was found to be an effective tool when it came to investigating surface defects in film-coated tablets and the film-core interface. The comparison of confocal imaging to laser profilometry and optical roughness analysis was able to validate these results.
Confocal imaging has been utilized to investigate the structure and time-evolution of transient gels, which are formed in the mixtures of colloid-polymers. It has shown that there can be large differences within a local structure in a single system.
The use of confocal imaging in this context greatly improves upon any attempt using conventional light or fluorescence microscopy, which would make it extremely difficult to perform a study of this kind.
The transcellular pathways via which different nanocapsules penetrate the cornea were identified through a series of in vivo and ex vivo testing with confocal imaging. The imaging these pathways were able to show the influence of different coatings of the nanocapsules, on both the rate of penetration and the biodistribution.
The information gathered from this study allowed the determination of a potential strategy with relation to site specific therapeutic agents.
Reviewed by Afsaneh Khetrapal, BSc (Hons)