1
Introduction to Bioimaging

Bioimaging can be defined as visualization of a biological object. The most basic bioimaging may be just "seeing" the living object using our own eyes. This function is called "vision" and the procedure is mediated by visible light. The visible light is a part of electromagnetic wave in the wavelength range of 400-700?nm, and the image information is generated by the interaction between light and object, such as reflection, scattering, and diffraction. The generated information-rich light package travels and reaches our eyes. The focused light through lens would be projected to the screen as in a camera. The retina in our eye is the screen of the image, which is composed of the two-dimensional array of optic nerves. The photon in light signal (containing the information of the object) reaches the retina and activates optical neurons, and the signal is transferred to the brain and is reconstructed into the image of the object by neuronal processing. Even though the screen is two-dimensional, the processed images via two retinas provide three-dimensional information about the shape and distance of the object. Visible light travels at a so-called speed of light (3 × 108?m/s), so the information transfer in the vision could be almost instantaneous. If there is a possible delay, it may be from the signal transition step from the optical nerve to the brain and the information processing time in the brain.

Bats live in the dark environment without enough environmental light for vision. Instead, they use ultrasound for bioimaging platform. If other conditions are same, the light vision could be million times faster than ultrasonic sensing (340?m/s) (Figure 1.1). Among all the sensors, light vision is the fastest and most information-rich system. Therefore, the invention of eye (in more general term, photoreceptor) is one of the most dramatic events in the evolution of life. Due to the high quality and also huge quantity of information, vision is the most important sense, easily accounting for more than 90% of information we receive through all other senses, including hearing, taste, smell, and touch.

1.1 Color

Visible light sensing not only generates black-and-white images, but also can provide color information. The visible light is composed of a spectrum of electromagnetic wave in the range of 400-700?nm. Human eye has three color photoreceptors, of which the maximum sensitivity is for blue (445?nm), green (535?nm), and red (575?nm) (Figure 1.2). For example, when we receive 445?nm light, we sense it as a blue color, and 575?nm light as a red color. Therefore, color recognition is the ability of sensing different wavelengths of light. And, the term spectroscopy is derived from spectrum, i.e. spectroscopy is the study of the wavelength-dependent interaction between the light and the object.

Figure 1.1 Vision through light and sound in different speed.

Figure 1.2 Three color receptors and their sensitivity to different wavelengths.

If we have three color receptors, then do we recognize only three colors? No, it is not. At least, we give seven names of color to the rainbow! Our color sensors have the maximum sensitivity to a specific wavelength, but the sensing wavelengths are broad and overlap with each other. If the eye receives 560?nm light, both green and red receptors are activated, and we sense it as a yellow color. The light with 590?nm will more strongly activate red receptors and less strongly green receptors, making the color as orange. That means our color sense is determined by the ratio of the three receptors' activation degree. Using the three-color receptors, the distinguishable colors by human eyes are more than 10,000! With this ability, we can find our food (e.g. red apple) better, also our enemy (e.g. red ant) faster (Figure 1.3). We can imagine how useful this ability is to help us survive better during the evolution process.

Figure 1.3 Comparison between black-and-white picture and colored picture.

Figure 1.4 The color spectrum of dogs and humans.

Interestingly, this three-color recognition is not common to all animals, even to mammals. Only our very close cousins such as chimpanzees and gorillas have three color receptors, but even then not all the monkeys do. Including our remote cousins, dogs and cows have only two-color receptors. It sounds trivial whether it is two or three. But, with two color receptors, the distinguishable colors are narrowed down only to the level of 100! It is the difference between two- and three-dimensional combination power (Figure 1.4). Therefore, the visions of cows and dogs would be much more boring than the colorful flowers and spectrum of rainbow we see. This is why many sensors are designed for color change to achieve maximum effect to the naked eyes. Our eyes are a wonderful color sensory system!

There is a funny story in the bull fight. The fighters use red cloth to stimulate bulls, as red color may be related to the image of blood. Funny thing here is the bull may see red color more like dark gray rather than bloody red. The red cloth is to stimulate the audience, not the bulls at all!

Color blindness arises when part of the color receptor is defunctionalized. In humans, most common type is green-red blindness, which occurs when either green or red receptor has problem. If you look at the receptor property carefully, you may realize that the maximum wavelengths of green (535?nm) and red (575?nm) receptors are rather closer, compared to that of blue (445?nm). We call the receptors green and red, but they are more like yellow and orange. To maximize the combination power in color contrast, this design may not be the optimum choice. If we design the color pixel of a computer screen, we may choose more even distribution of the colors, such as 465, 525, and 630?nm [1]. Not surprisingly, the green and red receptors are structurally closer to each other, implying that they evolved from the common ancestor. So, we can imagine, a long time ago, we also had two color receptors similar to dogs or bulls (blue and yellow), and the yellow receptor diverged to two receptors, green and red. Without this evolution of color receptors, we might not be able to enjoy the beautiful sunset!

1.2 Colorful Material

The synthetic colorful materials are mainly organic dyes and inorganic pigments. Conventionally, dye is defined as the material that imparts its color to other substances, such as fabric or tissue. Usually, dyes are soluble in solvents, but pigments are insoluble solids. For printing purposes, pigment powder needs to be dispersed into a liquid binder before use.

On the earth, the strongest light source is the sun. To minimize the background of light sensing, our visionary system adjusts our sensors to recognize the sunlight as a background, called "pure white." White light is not the status of no color, but it is the collection of all the colors included in the sunlight. The colors of the white light can be manually separated into a spectrum by a prism through a process of dispersion, which is the same mechanism of rainbow formation. Therefore, white is the combined color of all the visible light in the rainbow.

The color of the colorful materials is determined by the wavelength of the absorbed light, i.e. leftover reflected color after absorption of white light. Therefore, the appeared color is complementary to the absorbed color. The concept of complementary color has been known for a long time and is widely used in painting art for vivid color contrast. Even though the wavelength of visible light is in linear scale (400-700?nm, violet to red), our color receptors deceive our color recognition due to the tiring of receptors. The relationship of the complementary color in our color sensing system is described in a color wheel (Figure 1.5).

A chromophore is the part of the molecule which is responsible for the color. The chromophore of inorganic pigments is usually is transition metal, which has a visible light range of electron excitation energy. The chromophore of organic dyes is a long-conjugated double bond system. The light absorption had been modeled in early quantum mechanics era through a particle-in-a-box model, which later led to Schrödinger equation for atomic structure of electrons. Interestingly, the organic conjugation system could be described as a particle-in-a-box model, where the s bond electrons define the size of the box and p electrons are the particles in the box. As the box size becomes bigger, the wavelength of absorption light gets longer, through the narrowing electron transition gap. When the absorption maximum reaches the boundary of visible light (violet color), the appeared color of the material would be the complementary color of violet, yellow (colors in opposite direction in the color wheel). If the conjugation gets longer, the absorption maximum moves from violet to blue and then green. Accordingly, the appeared color changes from yellow to orange and then brown. You may recall the old books turn into the yellow color first, and then change into reddish tone. This is the result of the extension of conjugation...