The Confocal Laser Scanning Microscope (CLSM) is a type of microscope which utilizes its electronic system for image processing whilst using a laser as the source of light. The optical image sections it obtains are of superior resolution. CLSM focuses on a single plane and removes the interference caused by the light arriving from the different optical fields across the thickness of the sample. As the images gathered using the microscope are digital images, higher magnifications for optical microscopy can be obtained using the software.
The images obtained using CLSM are of higher magnification as compared to the images obtained using the conventional optical microscopy. These images have volumetric and texture details and it is not possible to obtain such details with the conventional ones. The microscope also allows the study of the specimens using transmitted or reflected light meaning that those samples which are not transparent may also be analyzed. Since confocal microscope has the ability to create sharp optical sections, the data gathered from a series of optical sections at short and regular intervals along the optical axis are also used to create a true 3D reconstruction. This is made possible using software which combines the 2D images to create a 3D rendition 2.
Confocal microscopy has been widely used in medical and dental research and also for the clinical treatment of various diseases. Along with its research applications in cancer and Alzheimer’s disease, the technique is also commonly used in ophthalmology3,4, angiogenesis5, gynecology6, and gastro-intestinal systems7. CLSM is routinely used in dentistry to analyze surface roughness, analysis of dental erosion, measuring the actual profile, the numerical roughness parameters and in the studies of micro-tensile bond strength 8,9,10,11. It has been widely used for studying the surface topography and formation of bio film on dental implants and dental hard tissues 9,12. Once the data stack has been obtained in the CLSM a non-tactile, non-destructive measurement of the surface roughness is possible. It is an invaluable tool of measurement in dentistry studies. It is also important to mention that confocal microscope maintains the samples under humid conditions and thus avoids the artifacts that may take place with the use of SEM drying specimen preparation techniques. It offers improved relationship between the signal and the noise.
Confocal microscopy, like other devices has some limitations particularly the resolution and the pinhole size 13,14. It has inherent resolution limitations due to diffraction and the airy disk used in CLSM can limit the maximum resolution which can be attained using the
device. The pinhole size, once decreased in CLSM leads to reduced signal to noise ratio and thus more fluorescence from the specimen may be required.
CLSM is widely used in Europe and North America and is one of the most expensive types of microscope available in the market. The microscope is currently being employed for detailed microscopic examination of the cornea in some hospitals across Pakistan. National institute of biotechnology and genetic engineering uses CLSM for research purposes. It is a valuable tool in dental research particularly for accessing the surface roughness of dental biomaterials and the formation of biofilm on dental tissues. However, in Pakistan it is not yet employed in dental research partly because of its cost and partly because of lack of knowledge. Irrespective of financial constraints, it is important that this wonderful device is made available to the local researcher to claim its place in contemporary literature.
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