Zhang Rong, Wang Hongjiang, Jia Haiying, Yang Yuan, Sun Bo, Yang Kai, Gao Junfeng, Gao Chuan, Strategic Support Force Characteristic Medical Center, Research Department, Special Service Health Center, Aerospace Clinical Medicine Department, Shenzhou Medical Team
Two-photon microscope is a high-resolution microscopy technique that uses two low-energy photons to excite a sample at the same time, resulting in three-dimensional, high-definition images. Compared with traditional microscopy techniques, two-photon microscopy is widely used in biomedical research due to its advantages such as larger penetration depth, lower background fluorescence, and less cell damage. The Shenzhou-15 astronaut crew used the space station two-photon microscope to carry out an on-orbit verification experiment and achieved a number of firsts:
- For the first time in the world: Two-photon microscope is operating normally in orbit.
- For the first time in China: femtosecond lasers are operating normally in orbit.
- International first: observation of astronaut cell structure and metabolic composition information in orbit.
The space station two-photon microscope project of the in-orbit verification experiment, led by Cheng Heping, academician of the Chinese Academy of Sciences, provides a new tool for the future research of astronauts' on-orbit health detection, and also provides an important technical means for the future use of the Chinese space station platform to carry out brain science research.
The space station's two-photon microscope looks similar to a printer. The Shenzhou-15 astronaut crew used the space station two-photon microscope to carry out a successful in-orbit verification experiment. It is the world's first known two-photon microscope to obtain three-dimensional images of the epidermis and superficial dermis of astronauts' skin during space flight.
The Shen-15 crew uses a two-photon microscope in orbit
Two-photon microscope uses
Two-photon microscopy works by using two fairly low-frequency laser beams to pass through a lens at the same time and enter the sample. When two low-frequency laser beams converge at the focal point in the sample, only the regions that are simultaneously at the corresponding wavelengths of the two photons will be excited to emit light, resulting in the formation of a fluorescence signal that excites only the focal point of the sample. The two-photon fluorescence signal generated by this quadratic nonlinear optical process enables high-resolution imaging of deep structures within the sample. On the other hand, the two-photon excitation fluorescence signal is generated only within the focal point, reducing the damage to biological samples by phototoxic substances such as organic dyes.
Two-photon microscopy has a wide range of applications, and here are some of the main ones:
1. Cell and tissue imaging: Two-photon microscopy enables high-resolution imaging of living cells and tissues to observe molecules and structures within cells. It can provide information about the three-dimensional structure, morphology, spatial distribution and cell dynamics of cells and tissues, which is of great significance for studying the surface of cells, intranuclear structures and the function of organelles.
2. Imaging of neural activity: Two-photon microscopy can provide real-time observation of neuronal morphology, connections, and electrophysiological activity through the interaction of dyes or dyes with biomarkers. Researchers can use this technique to study the spatial distribution of neurons, synaptic transmission, synaptic structure, and neural network function.
3. Cancer research: Two-photon microscopy can observe and track the expansion and metastasis of tumor cells in vivo. Biochemical molecular expression in tumor cells or organs can be quantitatively measured by stains or fluorescent markers. At the same time, two-photon microscopy can also be used for drug discovery and evaluation, as well as for the study of tumor pathological processes.
4. Immunological research: Two-photon microscopy can observe and monitor the migration and activity of immune cells in vivo. For example, it can be used to study the interaction of immune cells with pathogens such as bacteria, viruses, parasites, etc., as well as between fixed and transitional immune cells.
5. Skin research: Two-photon microscopy can observe the physiological and pathological processes of cells and tissues, such as leprosy, urticaria, lupus erythematosus, etc., on living skin. By observing certain characteristics of cells, such as nuclear morphology, cell wall distribution, etc., it can help diagnose skin diseases.
In conclusion, two-photon microscopy combines the characteristics of high-resolution imaging and in vivo imaging to make it a powerful biomedical research tool. It provides high-resolution 3D images of cells and tissues, helping researchers gain insight into the structure and function of biological samples, thereby further advancing the development of life sciences and medicine.
Structure and physiological function of the skin
The skin is the largest organ of the human body, and it is a kind of tissue located on the surface of the human body that is in direct contact with the external environment, and plays an important protective role in the human body. It is mainly responsible for protecting the body, perspiration, feeling hot and cold, and stress. The skin covers the whole body, and it protects the various tissues and organs in the body from physical, mechanical, chemical, and pathogenic microorganisms.
The skin area of adults is 1.6-2 square meters, the total weight (including subcutaneous tissue) accounts for about 16% of the human body weight, and the skin thickness (excluding subcutaneous tissue) is about 0.5-4mm. The skin contains 18 to 20 percent of the body's water content.
The skin is made up of epidermis, dermis, and subcutaneous tissues, and contains accessory organs (sweat glands, sebaceous glands, nails, hair), blood vessels, lymphatic vessels, nerves, and muscles.
【Epidermis】
Under the microscope, the surface of the skin is uneven, deep into the surface of the skin, we see that it is composed of skin grooves, skin chus, hair and pores, etc. With the growth of age, the skin function gradually declines, the distance between the skin groove and the skin chuck will become larger, the skin texture will also become thicker, and the surface of the skin is a bit like a rolling hill, so the convex part is called the skin chu, and the concave part is called the skin groove.
The epidermis is the outermost layer of the skin, with an average thickness of 0.2 mm, according to the different stages of development and morphological characteristics of the cells, the structure of the epidermis layer can be divided into five layers from the inside to the outside: basal layer, spinous layer, granular layer, transparent layer and stratum corneum, whose characteristics and physiological functions are:
1. Basal layer
The basal lamina has a proliferative function. It is the innermost layer of the epidermal layer of the skin, the cells are single-layer cylindrical or cubic, and the basement membrane band is arranged perpendicular to the fence, it is the layer of cells with the strongest multi-growth ability, 30%-50% of the basal cells undergo nuclear division every day, the division cycle is 19 days, and the pH value of the basal cell layer is 6.8-6.9, which is weakly acidic.
Each 10 cells are a group of vertical overlapping epidermal multi-growth units, the basal center is the stem cell, located in the lower part of the epidermis, and the periphery is the transient multi-growth cell, the skin in the process of wound healing, the basal layer cells have an important regeneration and repair effect, and are also the source of life of the epidermal cells. On average, there is 1 melanocyte for every 10 basal mother cells, which determines the shade of skin tone for each person.
2. There is a spinous layer
Some cells have the function of proliferation in the spinous layer, and many receptors for antigenic substances are in this layer. Located above the basal layer, it is composed of 4-10 polygonal and large spinous cells, with lymphatic fluid flowing in the intercellular space, providing nutrients for the cells, and also having the ability to divide and multiply, but it is limited to the cells close to the basal layer in the deep layer, the cell shape is flatter and flatter the more superficial, and the pH value of the spinous cell layer is 7.3-7.5, which is weakly alkaline.
The cells contain exopolysaccharides, which are hydrophilic and adhesive. It also contains sugar conjugates, pegania receptors, glucocorticoids, epinephrine and other hormone receptors, HLA-DR antigen and epidermal growth factor receptors, etc.
3. Particle layer
The granular layer cells are tightly connected and mainly play the role of water retention. Located in the superficial part of the spinous cell layer, it is generally composed of 3-5 layers of flat cells or spindle cells, and is a cell that further differentiates into epidermal cells, and is also called a transition zone because it transitions between normal epidermal cells and dead cells.
Biochemical studies have shown that the particles are rich in histidine proteins, and the particle layer contains minerals, with calcium being the most. The "membrane" in the cells in the upper part of the granular layer is released into the intercellular space by the particles, which is not easy to separate between adjacent cells, called the waterproof barrier, so that the epidermal water is not easy to penetrate, and also prevents the extravasation of water in the body, and it is closed at room temperature to prevent water loss and bacterial infection.
When exposed to heat, it creates fissures that absorb nutrients. The skin can synthesize glycogen, the adult skin contains about 80mg of sugar, mainly distributed in the granular layer, the sugar content of the diabetic skin will increase, so it is susceptible to bacterial and fungal infections, and the human body ingests too much sugar, which will also promote the acceleration of aging.
4. Transparent layer
The transparent layer acts as a barrier. It is a precorneum stage, composed of 2-3 layers of flat cells, without nucleus, only seen in the epidermis of the palms and soles of the feet, the HE chromosome is eosinophilic, with strong refractive properties, and the cytoplasm contains microfilaments with a diameter of 7-8nm and hydrophobic protein-bound phospholipids, which have a barrier effect to prevent the passage of water, electrolytes and chemicals. In terms of electrostatics, the granulosa cell layer is a charged negative charge band, and the transparent layer is a charged yang charge band, which constitutes an important defense barrier of the epidermis.
5. Stratum corneum
The stratum corneum is the skin's protective barrier. It is the outermost layer of the epidermis, composed of multiple layers of keratinocytes and stratum corneum lipids, the cells are flat and nucleated, most of the parts are 5-15 layers, and the palms and soles can reach 40-50 layers, the cell structure is blurred, the cell membrane is thickened, the cell is full of tension silk and keratin, it has strong water absorption, makes the skin soft, prevents excessive evaporation of water in the body, can resist the invasion of external friction and defense pathogenic microorganisms, has a certain tolerance to acid, alkali and ultraviolet rays, and is the most important natural protective layer of the human body.
The desmosomes of the stratum corneum cells gradually disappear, the keratinocytes are tightly bound to laxity with each other, and continue to fall off into dandruff, while the basal layer cells continue to produce new keratinocytes to replenish one after another, this metabolism is the thickness of the epidermis to maintain a relatively stable state, there are no blood vessels in the epidermis, and the nutrition and metabolism of the cells rely on the diffusion of substances in the intercellular space to transmit.
The stratum corneum has hygroscopicity and moisturizing properties, the two are closely related, when the stratum corneum lacks moisture, even if it absorbs water, it will be lost rapidly, although the stratum corneum is very thin, generally not more than 20μm, there are two forms of water in the stratum corneum, namely bound water and free water, the bound water is in a molecular state in the stratum corneum and the combination of ions, amino acids and proteins, etc., and the softness of the stratum corneum is closely related to the bound water.
【Dermis】
The 0.5-1 micron wavy junction at the dermis and epidermis junction is called the basement membrane band. There are gaps between cells, mainly containing neutral mucopolysaccharides. The basement membrane band closely links the epidermis and is the site of material exchange between the dermis and the epidermis, which can exchange raw materials and oxygen semi-permeable membrane and barrier effect. Once damaged or structurally abnormal, epidermal detachment may occur, forming subepidermal blisters.
The dermis is located in the lower layer of the epidermis with a thickness of about 0.2 cm, and this layer contains 60% of the skin's moisture, promoting moisture and elasticity of the skin. The firmness and elasticity of the skin are mainly determined by the dermis. It stores water and blood and plays a major role in skin nutrition. The dermis is mainly divided into the papillary layer, the reticular layer, and the stroma. Its characteristics and physiological functions are:
1. Nipple layer
The thinnest layer of the dermis is composed of collagen fibers, forming many papillary bumps, giving the skin more elastic space, containing abundant nerve endings, capillaries and capillary lymphatic vessels to send nutrients to the epidermis, and the lower layer of the papilla contains more water, where the lack of water causes the epidermis to form fine wrinkles.
2. Reticulated layer
Extending parallel to the epidermis and intertwined into a network to give the skin resistance and firmness, to protect the internal tissues and organs, mainly composed of coarse collagen fibers, more elastic fibers and reticular fibers, if the collagen fibers in this layer break or harden the skin aging and wrinkles. It is rich in blood vessels and nerves that provide nutrients to the skin, and also has the ability to kill bacteria.
Collagen fibers, the main component of the dermis, are composed of collagen eggs, and their collagen is combined into bundles, so they have a certain elasticity, making the skin flexible, resisting external pulling, and preventing skin sagging. But it lacks elasticity.
Elastic fibers - good elasticity, so that the stretched collagen fibers are restored, in the dermis reticular layer is thicker, is the scaffold that constitutes the skin and its appendages.
Reticular fibers – naïve collagen fibers found only under the epidermis and around sweat glands, sebaceous glands, hair follicles and capillaries. It is produced in large quantities during trauma and promotes wound healing. Inhibition of fibroblast thickness prevents scar hyperplasia.
3. Substrate
The matrix is an amorphous, gelatinous substance composed mainly of mucopolysaccharides, proteins, electrolytes, and water. Maintain tissue moisture, provide nutrients for cell metabolism, natural barrier, prevent a large number of pathogenic bacteria from invading, and act as a bonding and lubricating effect on fiber bundles. (Therefore, the skin of the elderly is wrinkled, dry and loose)
【Subcutaneous tissue】
It is mainly composed of lipid cells and loose connective tissue, also known as subcutaneous lipid defense layer, its thickness is about 5 times that of the dermis, which is heat preservation and cold protection, energy storage, buffer external force, and protect internal tissues: rich blood vessels, lymphatic vessels, nerves, sweat glands and deep hair follicles.
Two-photon microscopy is a nonlinear optical imaging technology based on two-photon absorption and fluorescence excitation, which has the characteristics of high resolution, strong three-dimensional tomography ability, large imaging depth, in vivo non-invasive and non-labeling, and can specifically excite the second harmonic signal of collagen fibers, which is especially suitable for in vivo non-invasive microscopic imaging of the structure and function of living biological tissues. However, due to the huge size of the traditional two-photon microscope system, which cannot meet the demanding requirements of on-orbit experimental instruments and equipment in terms of reliability, volume, weight, shock and vibration resistance, etc., the on-orbit operation and application of two-photon microscopy imaging technology on the space station have not been realized in the world.
On November 12, 2022, the space station two-photon microscope was successfully transported to the Chinese space station aboard the Tianzhou-5 cargo spacecraft, becoming the world's first two-photon microscope in space. Recently, the Shenzhou-15 astronaut crew completed the installation, commissioning and first imaging test of the two-photon microscope, and successfully obtained in vivo two-photon microscopic images of the astronauts' face and forearm skin in orbit.
The two-photon microscope can clearly display the three-dimensional distribution of astronauts' skin structure and cells with sub-micron resolution, and has the ability to perform non-invasive microscopic imaging of the structure and components of the skin surface. The imaging results showed that the three-dimensional structures of the skin, such as the stratum corneum, granular layer, spinous layer, basal cell layer, and superficial dermis, were clearly identifiable.
Two-photon microscopic imaging of the skin of the astronauts of the Shen-15 crew
Tomography of two-photon microscopy results of skin
The signals for two-photon microscopy imaging on the space station are derived from autofluorescent substances in cells and extracellular matrix, as well as second harmonic signals generated by collagen fibers. These signals help to probe the mitochondrial metabolic stress response of astronaut cells. Through the quantitative observation of cell metabolites with autofluorescence, the metabolic function of astronauts can be reflected.