研究目的

To review the history and fundamentals of CSLM briefly, to summarize some of the most important advantages and disadvantage of the confocal microscopy, as well as to highlight the applications of CLSM for studying nanoencapsulated food ingredients.

研究成果

Monitoring of coating thickness and morphology of nanocarriers is one of the most important steps to control the nanoencapsulation procedure. CLSM, as a nondestructive and ultra-sensitive method, shows a great potential for characterizing various nanocarriers, evaluating the bioactive compounds attached onto the active film surfaces, measuring cellular uptake efficiency of bioactive-loaded nanocarriers, and tracking the biological fate of nanoparticles in stimulated gastrointestinal conditions. Reduction of out-of-focus blurring of the microscopy images through scattering of light and ability of optical sectioning are two main advantages of CLSM over conventional microscopy. Furthermore, image processing can offer a great advantage to the CLSM users. Image alignment, contrast enhancement, and sharpening and smoothing of images are some of these benefits. Visualization of a distribution map of nanomaterials can give us a great deal of information about quantity and location of encapsulated compounds as well as the composition and thickness of nanocarrier materials and calculation of the distance, volume, contact angle, and surface of nanoparticles in a more rapid and accurate manner. On the other hand, this method suffers from some limitations such as selection of suitable lasers with efficient fluorophore excitations, destructive effect of high intensity laser illumination on some samples, selection of objective lenses, working distances between the top of sample and front lens, and the necessity of pretreatment(s) such as staining or quenching of autofluorescence on specimens to be visible for CLSM. Therefore, further researches are needed to improve the applicability of this technique to evaluate nanocarrier in vivo and in vitro.

研究不足

The main limitation of CLSM is the selection of suitable lasers with efficient fluorophore excitations. Moreover, the destructive effect of high intensity laser illumination for some samples, especially viable tissues and the fluorophore is another challenge of CLSM. Many fluorophores are sensitive to light intensity of laser-illumination within the time of imaging and fluorophore is revealed as a photobleaching in the x, y, and z planes. The selecting of objective lenses and their working distances are another critical issue in using CLSM. The working distance between the top of the sample and front lens is usually restricted to about 100 μm. A longer working distance decreases the image resolution. Application of illumination systems with short-pulsed double photons results in an emission with higher energy photons. This can increase the penetration power of the beam into the specimen and reduction of the photobleaching. However, such lasers are expensive and require adjusting before they can be used. The next limitation is that most specimens need some pretreatment(s), such as staining or quenching of autofluorescence, to be visible for CLSM. Such treatment are usually carried out in liquids at room temperature, which can led to artefacts like dissolving and swelling of particles. Autofluorescence of biological samples is another challenge of CLSM. Therefore, optically sectioning thick specimens can resolve this problem. Thick samples are adequately transparent to the excitation and emission light wavelengths and, consequently, they do not scatter the light strongly, as they are comparatively free of autofluorescence.