Multiphoton microscopy: application in dermatological practice

Inna Krulko , candidate of biological sciences, virologist, microbiologist (Ukraine)

Today, the main task that microscopic technology is designed to solve is deeper penetration into the sample in order to provide better opportunities for studying cells, organs and tissues. One of the most effective ways to achieve deep drug penetration is through the use of two-photon and multiphoton excitation in laser scanning microscopes, which are equipped with pulsed infrared lasers. Due to reduced absorption and scattering of excitation light, two- and multiphoton confocal microscopes can achieve penetration depths of approximately 400 µm.

Skin structure

The skin, with its surface area of 1.5–2 m2, is the largest organ of the human body.

The outer layer of the skin, the epidermis, is formed by epithelial cells lying on top of each other in several dozen layers. The upper part of the epidermis is in direct contact with the external environment and is called the stratum corneum - it consists of aged and keratinized cells that are constantly exfoliated from the surface of the skin and replaced by young ones migrating from the deep layers of the epidermis.

The stratum corneum is followed by several layers of living keratinocytes. Keratinocytes have different structures in different layers of the epidermis. In particular, basal keratinocytes are oval. They are smaller in size than spinous cells. In areas with a thickened epidermis, the cells of the basal layer are elongated in the vertical direction. Their plasmalemma (plasma membrane) is characterized by smooth contours and has a thickness of 7–8 nm.

In the deepest layer of the epidermis there are melanocytes - cells that produce the pigment melanin. Melanin is a heterogeneous chemical compound that is divided into two types: eumelanin and pheomelanin, each of which has different spectral properties.

The dermis is also heterogeneous. It contains many important functional elements, including hair follicles, sweat, sebaceous and apocrine glands, nerve fibers and their receptors, blood and lymphatic vessels. The structure of the hair follicle is especially important because recent research has shown that the hair follicle niches contain skin stem cells necessary for skin repair after injury.

Multiphoton microscopy

Multiphoton microscopy (English - multiphoton microscopy, hereinafter MPM - Ed.) - a technique derived from laser scanning confocal microscopy, in which fluorochromes are excited by laser radiation in the infrared or long-wave visible range, the density of which doubles or even triples at the point of focus on sample. The sample fluorophores are excited by two or three long-wavelength photons, which is equivalent to excitation by one short-wavelength photon. For example, excitation with two or three 900 nm photons is equivalent to excitation with one 450 or 300 nm photon. Multiphoton microscopy provides deeper penetration into tissue and does not require a confocal microdiaphragm, since its fluorescence occurs strictly in the focal plane.

The invention of multiphoton microscopy in the 1990s generated enormous interest. By now, this technique has become widespread in biology and medicine. A huge number of methods, approaches and publications have appeared that use multiphoton microscopy.

It is now the primary method for non-damaging, deep penetration fluorescence microscopy using thick, light-scattering samples. It is used for a wide variety of preparations: lymphatic organs, kidneys, heart, skin and brain (slices or intact organs). The advantages of deep penetration and multiphoton in vivo imaging within the specimen are used in various areas of biomedical research, such as immunology (lymphocyte tracking), embryology, oncology and especially neuroscience (including the study of calcium dynamics and neuronal plasticity).

Skin studies using MFM

Using MPM, the structure and biochemistry of the skin can be effectively studied using contrast agents such as organic fluorophores or synthesized fluorescent proteins. Most of the light reflected by the skin is generated by backscattering by the various layers.

The contribution of different layers of the skin to the processes of absorption and scattering of light is different. The main components that determine the absorption and dissipation properties of the skin are the pigmented epidermis (melanin), the dermis and the blood vessels of the dermis (hemoglobin).

• The stratum corneum reflects about 5–7% of incident radiation.
• The epidermal layer diffusely scatters incident radiation, contributing to the reflectance of the skin in the visible region of the spectrum.
• Radiation that passes through the epidermal layer is strongly absorbed by the melanin contained in it in almost the entire visible spectral range, and the absorption coefficient increases when shifted to the short-wave region of the spectrum.
• Light passing through the layer of dermis, penetrated by blood vessels, is partially absorbed by hemoglobin.
• Finally, the remaining unabsorbed portion of the radiation is diffusely reflected from the layer containing collagen fibers and, before being released, passes again through the layers of the dermis and epidermis containing hemoglobin and melanin.

Keratinocytes can be visualized by fluorescence of reduced pyridine bases or oxidized flavin proteins. It is important to note that cellular metabolism can be non-invasively studied using redox fluorimetry.

Dermal collagen and elastin are also studied based on their fluorescence. Several collagen isoforms, including type I, often cause higher second harmonic generation of the signal due to their asymmetric molecular structure and crystalline organization. This phenomenon may provide information about the receptivity of the extracellular matrix, as well as more detailed information about the organization of collagen fiber molecules.

MFM and melanoma

Due to the increasing number of melanomas in the world, scientists began to study in detail the structure of melanin using MFM imaging. Thus, recent studies have shown a significant difference between eumelanin and pheomelanin. In a mouse model, it was shown that melanoma more often developed from “invisible” nevi containing eumelanin rather than from dark ones with a large amount of pheomelanin. In vivo studies of biopsy material from human basal cell melanoma showed enhanced fluorescence of cancer tissue. In addition, morphological changes in melanocytes were observed: the cells were more elongated than healthy ones and migrated only in conglomerates. Moreover, the method allows one to distinguish ordered collagen, which is more typical of healthy skin, from disordered collagen in tumor tissue. Studying the skin using MFM may further play an important role in the early diagnosis of skin cancer.

MFM and chronic dermatoses

The skin of patients with chronic dermatoses, free from rashes, has structural and functional differences from healthy skin, especially in patients suffering from atopic dermatitis. Ultraviolet irradiation and PUVA therapy change the image parameters and functional parameters of lesions of psoriasis and atopic dermatitis, as well as rash-free skin, which makes it possible to recommend non-invasive methods for studying the morphofunctional state of the skin, in particular MFM.

The study of skin deformation in response to external mechanical impact is used in the assessment of skin aging, connective tissue lesions in scleroderma, Ehlers-Danlos syndrome, psoriasis, and the study of skin atrophy and swelling due to ultraviolet irradiation. Also, using MFM, the process of laser treatment of wrinkles after thermal “shock” of fibroblasts, which begin to produce more collagen, was studied.

MFM and the effectiveness of cosmetics

It is important to note the role of MPM in studying the effects of cosmetic products. Very often there is a lack of data on their effectiveness and safety. The advent of MPM, which allows in vivo studies of many of these products on animal models and human volunteers, will make it possible to study in more detail the effect of a cosmetic product on the skin and body, and to more accurately conduct toxicological studies.

Variations on a theme

There are also scanning MFM techniques that can be used to study the dynamics of processes in the skin.

• Thus, using FLIM (Fluorescence Lifetime Imaging Microscopy), it is possible to locally measure many different parameters inside cells or other structures, for example, ion concentration, molecular interactions, membrane potential. Thus, the researcher receives information about ongoing processes at the molecular level.
• FCS (Fluorescence Correlation Spectroscopy) is a method for quantitatively measuring concentration and diffusion rate at the single-molecule level. The data obtained by this method allows one to analyze intermolecular interactions and transport processes both inside living cells and in vitro. This makes it possible to study the dynamics of molecular systems and cellular structures.

Despite the obvious advantages of MFM, there are some limitations and difficulties in applying this method. The high cost of equipment, of course, imposes major limitations on the widespread use of the method by clinicians. Currently, attempts are being made to develop less expensive and more compact scanning technologies, which may make this examination method accessible. Time and significant effort are also required to create training programs to train specialists in interpreting the obtained images and interpreting them in order to obtain useful clinical and histological information.

In general, the development and implementation of non-invasive diagnostic methods into practice is currently relevant for all areas of medicine, including dermatology, which is primarily due to the priority of research safety for the patient. The development of modern diagnostic methods is included in the healthcare modernization program, which provides for the rapid implementation of scientific developments in practical healthcare. One of the most informative methods, approaching the information content of a traditional biopsy, is multiphoton skin microscopy, which allows layer-by-layer assessment of the skin structure without damaging it.

First published: Les Nouvelles Esthetiques Ukraine, No. 6 (88), 2014