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Extending the boundaries of confocal microscopy: ongoing advancements in medicine have propelled confocal microscopy into exciting new research areas.

The growing needs of researchers in a wide variety of fields continue to drive innovation in confocal microscopy (CM). Although several variations of this technology exist for ex vivo applications, which typically destroy the tissue being examined, only in vivo CM provides a noninvasive window into living skin for basic and clinical research, without damaging the tissue. Skin can be imaged in real-time, in its native state, either in vivo or freshly biopsied (ex vivo) without the fixing, sectioning and staining that is necessary for routine histology. The grayscale images, sometimes compared to ultrasound, use light and the naturally reflective properties of tissues instead of sound waves. CM provides very high-quality images with fine detail and excellent contrast, comparable to conventional microscopy at both the micro- and macro-level.

CM has traditionally been utilized in a wide range of applications in the life sciences, including research in neurophysiology, neuroanatomy and morphological studies of a wide spectrum of cells and tissues. These studies have historically been performed ex vivo using samples obtained from a living specimen or individual. Because single cells can be imaged at a scale of less than five microns, it is possible to make a definitive diagnosis of various conditions; however, due to lengthy processing times, high costs and typically destruction of the sample under study, traditional CM techniques are not routinely used in the diagnostic process.

Growing popularity of in vivo research

In recent years, the significance and utility of CM within the context of in vivo studies has become increasingly prominent. Given the advantage of observing the progression of a living specimen over a long period of time, in real-time, without damage to the tissue under investigation, in vivo applications are likely to continue growing in importance relative to traditional ex vivo research, extending the boundaries of the technology.

Not only are the advantages of in vivo CM significant; equally important are the technical advances that have facilitated its growth. One of the most important involves advances in image resolution. The traditional resolution standard for CM has been 512 square pixels, which provides a narrow field of view, typically allowing visualization of two cells per field. However, one advantage of new technology associated with in vivo studies is the ability to visualize larger areas of tissue--allowing researchers to monitor changes over time, such as the diffusion of cosmetics or chemicals through a patch of skin--without loss of resolution. For this reason, new CM devices provide 1000 square pixel resolution images, creating a larger field of view.

A related technical advance in CM with positive impact on in vivo and ex vivo studies involves the ability to create large mosaics from individual tiles or images. Traditional CM images measure in the range of 0.5 x 0.5 mm. More recently, devices have been engineered that can stitch these tiles together to produce mosaics as large as 8 x 8 and 20 x 20 mm. These expansive mosaics enable the examination of panoramic views of tissues of a magnitude never possible before.

A third advance in CM relevant to in vivo research involves the ability to combine fluorescence CM with reflectance CM. Reflectance CM enables in vivo imaging of human skin at a quasi-histologic resolution. A wide range of wavelengths can be utilized, including infrared, ultraviolet and blue/green; common wavelengths include 488, 532, 658, 785 and 830 nm. Spectral filtering serves as an additional tool to analyze an image.

For example, a new handheld reflectance confocal imager, part of the VivaScope series, offers a real-time, noninvasive optical biopsy in a lightweight, compact design, providing access to areas of the body that are not easily reached with other confocal devices. With this device, imaging the nose, ears and scalp--or anywhere on the body--is accomplished in a few seconds by touching the narrow imaging probe to the surface of the skin and freely moving it, by hand, to the area of interest. The device also makes real-time, intraoperative cellular imaging of living tissues inside the body a possibility for researchers. The never-before microscopic observation of dynamic processes in side living tissues during surgical procedures is now possible, as is tissue identification and the exploration of tissue margins. It is FDA-cleared for diagnostic use in humans and suitable for animal research.

An expanding range of applications

For contemporary researchers, CM not only has multiple applications in dermatology research, but there is also a growing body of research in cosmetics and basic skin, as well as a progression into various other areas of preclinical and small animal research. For example, increased interest in anti-aging methods mandates the development of reliable noninvasive techniques to assess skin aging, as well as the development of standardized terminology for the industry. Researchers have aimed to determine the effects of photo-aging at histopathologic resolution by means of reflectance CM to serve as a comparison to efficacy testing of anti-aging products in cosmetic research. Using this technique, it was found that among 120 subjects, sun-exposed areas of skin, such as the face and forearms, revealed more frequent irregular epidermal patterns, benign skin lesions (age spots), changes in collagen over time due to sun exposure, epidermal thickness changes and wrinkle depth values.

Other cosmetic applications utilizing the technology are also under investigation, including pigmentary disorders such as melasma, barrier function of the stratum corneum and skin hydration with different moisturization techniques. Researchers have also employed reflectance CM along with other imaging techniques to evaluate hair shafts and growth. After removal of the hair shaft with a shaving system, the hair follicle infundibula and the length of the growing hairs were measured with CM and other methods. It was determined that reflectance CM is ideally suited to the analysis of hair follicles.

Small animal research and pharmacological applications are also an emerging field for both real-time in vivo and ex vivo CM. The successful penetration of nanoparticles into the epidermis and superficial dermis in pig skin was demonstrated using in vivo and ex vivo reflectance and fluorescence CM. This research demonstrates the potential of CM in applications monitoring and tracking ingredients and drugs through the skin.

Intraoperative in vivo CM imaging has been performed on the kidneys of live rats, clearly identifying kidney structures such as glomeruli and vessels, and breast neoplasia has been studied in mouse models using ex vivo reflectance imaging. Real-time CM has been used to study conjunctiva in rabbit models and to quantify the progression of melanocytes in guinea pig models. Studies such as these provide the foundations for preclinical research in humans, as well as demonstrate the wide range of applications of real-time in vivo and ex vivo CM.

Ongoing advances in medicine necessitate better tools to diagnose, monitor and treat patients as efficiently and noninvasively as possible. There are more than 500 peer-reviewed articles, many of them pilot or clinical studies, with several demonstrating the high sensitivity and specificity for the in vivo diagnosis of skin diseases such as melanoma, basal cell carcinoma and many others. Real-time, ex vivo research in skin and breast surgeries indicates that the future of the technology may be in the rapid imaging of surgical specimens in the operating room, reducing procedure times and resection rates.

Many more examples of the application of CM in vivo could be added within various research areas. The technical advantages and versatility of CM clearly indicates a bright and promising future for this technology beyond its initial sphere of influence.


* Traditional CM procedures destroy the sample under study.

* Researchers are utilizing noninvasive techniques to study skin and hair.

* Microscopic observations inside living tissues are possible, both for humans and animals.

by William Fox, Vice President, Technology Operations and Chief Technology Officer, Caliber Imaging & Diagnostics, Rochester, N. Y.
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Title Annotation:MICROSCOPY
Author:Fox, William
Publication:Laboratory Equipment
Date:Jan 1, 2015
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