User:Dr Luis Acevedo

Introduction to Confocal Microscopy'''

Confocal microscopy is a technique in optical imaging that uses point illumination via a spatial pinhole to eliminate outof-focus signals. The excitation light in confocal microscopy is usually provided by a laser to generate high intensities of fluorescence or reflectance from the focal spot. Fluorescence confocal microscopy is the most used in dermatology to analyze ex vivo and in vitro samples. Reflectance confocal microscopy can be used for real-time microscopy and uses melanin as a natural contrast agent. Confocal microscopy has many advantages, including increasing the optical resolution and contrast of an image of a specimen; facilitating reconstruction of 3-D images; enabling collection of serial optical sections from thick specimens; and enabling in vivo imaging without the artifact induced by tissue processing (Pawley, 2006). In addition to LSCM, 3-D images of nonliving samples can also be acquired by SCEM, where an electron beam is used for illumination, resulting in higher resolution compared with onfocal microscopy. Limitations of confocal microscopy include the depth of imaging within thick samples and cost compared with conventional microscopes. The problems of fluorescent probe photobleaching and phototoxicity inherent in conventional fluorescence microscopy are also present with confocal microscopy. Multiphoton microscopy is an alternative strategy for fluorescence microscopy, which offers higher resolution, somewhat greater depth of imaging, and minimal photobleaching. Technologies for microscopy are promising and are still being improved.

ADVANTAGES OF CONFOCAL MICROSCOPY

• High-resolution, high-contrast images. • Reconstruction of 3-D images. • Absence of artifacts induced by conventional microscopy (e.g., shrinkage, loss of fat, no blood flow). • In vivo microscopy to a skin depth of about 200 μm.

LIMITATIONS

• Depth of imaging is limited by optical penetration and signal-to-noise ratio. In vivo confocal microscopes can generate high-resolution images of the entire epidermis and a superficial layer of dermis. • Photobleaching of fluorescent probes and phototoxicity of live samples are no worse with confocal microscopes than with conventional fluorescence microscopes. However, multiphoton fluorescence microscopes can nearly eliminate photobleaching compared with either confocal or conventional imaging. • High cost relative to conventional microscopy or dermoscopy.

FLUORESCENCE CONFOCAL MICROSCOPY

Fluorescence confocal microscopy is most commonly used for dermatologic research of in vitro or ex vivo studies. In general, fluorescence microscopy uses dyes that fluoresce when stimulated by light (“fluorophores”) and are added to the specimen depending on the purpose of the imaging. Fluorescence microscopy is generally much more sensitive than light microscopy. Fluorophores that specifically target and identify subcellular structures such as the cytoplasm, sarcoplasmic reticulum, nuclei, and mitochondria have been designed. Fluorophores improve sensitivity and specificity by increasing the signal-to-noise ratio and by allowing better and sharper detection of the target. The excitation light in fluorescence confocal microscopy is usually provided by a laser at a wavelength that will also excite a specific fluorophore. In some instances, more than one fluorophore can be used at the same time, and by switching the excitation light or by observing at different emission wavelengths, different parts of the specimen can be distinguished. When the excitation laser hits the target tissue, it generates high intensities of fluorescence at a welldefined focal point. Both the laser light (the excitation beam) and the resultant emission fluorescence pass through the same objective—a special mirror called a dichroic mirror that reflects the incoming, higher-energy (but shorter-wavelength) laser light, but allows the lower-energy (higher-wavelength) fluorescent light to pass through to the light detector (Figure 1). As described above for reflectance confocal microscopy, apinhole is also used in fluorescence confocal microscopy to eliminate scattered light. The end result is that light is collected from a highly focused point. Images of the scanned specimen can then be reconstructed point by point. One limitation of fluorescence microscopy is the phenomenon of photobleaching; fluorophores tend to irreversibly fade or react when exposed to excitation light. Various strategies are being explored to minimize this problem. Most confocal microscopes use this laser-scanning strategy and can be applied to the field of dermatology. For example, in vivo reflectance confocal microscopy (RCM) has been used to delineate pigmentary changes in skin due to aging or specific stimuli, such as ultraviolet radiation (UVR). In the study, investigators were able to detect UVR-induced pigmentary changes in the skin of pigmented guinea pigs, a common animal model for studying human pigment biology (Middelkamp-Hup et al., 2006). The changes could be detected well before a tanning response was clinically visible and included increase in melanocyte size, dendricity, and number and pigmentation of keratinocytes in the irradiated epidermis. Confocal microscopes that do not require laser scanning include the spinning-disk confocal microscope. Spinningdisk confocal microscopes use an alternative design, specifically a series of moving pinholes on a disk, called the Nipkow disk, to scan and obtain the confocal images. Another nonlaser strategy uses a modulator that creates a moving pattern of light focused in the virtual plane, which is then projected onto a CCD camera in which each little pixel on the camera chip acts somewhat like a pinhole.