Surfactants, Refraction, and Microscopy in Biophysics (PDF)

SURFACTANTS Surfactants are molecules that lower surface tension of liquids. (The word is an abbreviation of surface active agent.) The most common surfactant molecules have one end that is water-soluble (hydrophilic) and the other end water insoluble (hydrophobic) (see Figure below). A...

SURFACTANTS Surfactants are molecules that lower surface tension of liquids. (The word is an abbreviation of surface active agent.) The most common surfactant molecules have one end that is water-soluble (hydrophilic) and the other end water insoluble (hydrophobic) (see Figure below). As the word implies, the hydrophilic end is strongly attracted to water while the hydrophobic has very little attraction to water but is attracted and is readily soluble in oily liquids. Many different types of surfactant molecules are found in nature or as products of laboratory synthesis.  When surfactant molecules are placed in water, they align on the surface with the hydrophobic end pushed out of the water as shown in Fig. 7.11. Such an alignment disrupts the surface structure of water, reducing the surface tension. A small concentration of surfactant molecules can typically reduce surface tension of water from 73 dyn/cm to 30 dyn/cm. 74  In oily liquids, surfactants are aligned with the hydrophilic end squeezed out of the liquid. In this case the surface tension of the oil is reduced. The most familiar use of surfactants is as soaps and detergents to wash away oily substances. Here the hydrophobic end of the surfactants dissolves into the oil surface while the hydrophilic end remains exposed to the surrounding water as shown in Figure here. REFRACTION (O p t i c s) Refraction of Light Refraction is the bending of a wave when it enters a medium where its speed is different. The refraction of light when it passes from a fast medium to a slow medium bends the light ray toward the normal to the boundary between the two media. The amount of bending depends on the indices of refraction of the two media and is described quantitatively by Snell's Law. REFRACTION Refraction occurs when light travels from one medium to another (from air to water) and changes speed. This change in speed causes the light to change direction. The Laws of Refraction First Law: The incident ray, the refracted ray, and the normal to the surface at the point of incidence all lie in the same plane. Second Law: Snell's Law describes the relationship between the angles and the indices of refraction of the two media. It can be expressed mathematically as: where, 𝑛1 = index of refraction of the first medium, 𝑛2 = index of refraction of the second medium, 𝜃1 = angle of incidence, 𝜃2 = angle of refraction INDEX OF REFRACTION The index of refraction (n) of a medium is defined as the ratio of the speed of light in a vacuum to the speed of light in that medium. Air: n ≈ 1.0003 Water: n ≈ 1.33 Glass: n ≈ 1.5 Diamond: n ≈ 2.42 The higher the index of refraction, the more the light will bend when entering that medium. CRITICAL ANGLE AND TOTAL INTERNAL REFLECTION Critical Angle The critical angle is the minimum angle of incidence at which light, traveling from a medium with a higher refractive index to a medium with a lower refractive index, is refracted along the boundary, rather than passing into the second medium. When the angle of incidence exceeds this critical angle, all the light is reflected back into the first medium instead of being refracted. The critical angle (𝜃𝑐) can be calculated using Snell's Law: where, 𝑛1 is the refractive index of the first medium (where light is coming from), 𝑛2 is the refractive index of the second medium (where light is trying to go). Total Internal Reflection Total internal reflection occurs when the angle of incidence exceeds the critical angle. Under this condition, all the incident light is reflected back into the original medium, rather than being refracted into the second medium. This phenomenon is only possible when light is moving from a medium with a higher refractive index (like water or glass) to a medium with a lower refractive index (like air). Point of intersection of the reticle light-dark area Brix scale nD scale  Brix can be approximately converted to specific gravity (SG) by a simple equation: SG = 1 +(0.004 x Brix) Brix refractometers are meant to measure the percentage of sugar in a pure sucrose solution. Brix (degree brix) and Plato (degree plato) are both defined by 1 gram of sucrose is 100 grams of total solution. Also reported as percent mass or mass fraction in lab terms. Specific gravity, ratio of the density of a substance to that of a standard substance. Specific Gravity (SG) is the ratio of relative density of a liquid in reference to water. If you filled one Liter of water at reference temperature it should weight 1.000 kg, if you weighed 1 liter of 1.040 wort, it should weight 1.040 kg. FUNDAMENTALS OF OPTICS IN BIOPHYSICS Optics is the branch of physics that studies the behavior and properties of light. In biophysics, it helps us understand how light interacts with biological molecules and structures. Light behaves as both a wave and a particle (photon), which influences its interaction with matter. Lenses Lenses are optical devices that manipulate light to create images. They are characterized by their shape and refractive properties: Convex Lenses Concave Lenses  Convex Lenses These are thicker in the center and thinner at the edges. They converge light rays to a focal point, making them useful for magnifying small objects, such as cells or microorganisms. Concave Lenses These are thinner in the center and thicker at the edges. They diverge light rays, creating a virtual image that appears smaller than the object. Concave lenses are often used in eyeglasses for nearsightedness.  The convex lens shown has been shaped so that all light rays that enter it parallel to its axis cross one another at a single point on the opposite side of the lens. (The axis is defined to be a line normal to the lens at its center, as shown in Figure below. The point at which the rays cross is defined to be the focal point F of the lens. The distance from the center of the lens to its focal point is defined to be the focal length f of the lens. The figure above shows how a converging lens, such as that in a magnifying glass, can converge the nearly parallel light rays from the sun to a small spot. 89  The figure here shows a concave lens and the effect it has on rays of light that enter it parallel to its axis (the path taken by ray 2 in the figure is the axis of the lens). The concave lens is a diverging lens, because it causes the light rays to bend away (diverge) from its axis. In this case, the lens has been shaped so that all light rays entering it parallel to its axis appear to originate from the same point, F, defined to be the focal point of a diverging lens. The distance from the center of the lens to the focal point is again called the focal length f of the lens. Note that the focal length and power of a diverging lens are defined to be negative. Image Formation by Thin Lenses Consider an object some distance away from a converging lens. To find the location and size of the Image formed, we trace the paths of selected light rays originating from one point on the object, in this case the top of the person’s head. The figure shows three rays from the top of the object that can be traced using the ray tracing rules given above. We define do to be the object distance, the distance of an object from the center of a lens. Image distance di is defined to be the distance of the image from the center of a lens. The height of the object and height of the image are given the symbols ho and hi , respectively. Image Formation by Thin Lenses To obtain numerical information, we use a pair of equations that can be derived from a geometric analysis of ray tracing for thin lenses. The thin lens equations are: Note that the minus sign causes the magnification to be negative when the image is inverted. Magnification: The Science Behind It Magnification refers to the process of enlarging the appearance of an object. It can be quantified as the ratio of the size of the image to the size of the object. Magnification is particularly important in microscopy and imaging techniques used in biophysics. Linear Magnification: Given by the formula: where: ℎ𝑖 = height of the image, ℎ𝑜 = height of the object, 𝑑𝑖 = distance from the lens to the image, 𝑑𝑜​ = distance from the lens to the object.  𝑑𝑖 21 𝑐𝑚 𝑚= = 𝑑𝑜 −3 𝑐𝑚 𝑚 = -7  How an image is formed when an object is held closer to a converging lens than its focal length. The image is on the same side of the lens as the object and is farther away from the lens than the object. This image, like all case 2 images, cannot be projected and, hence, is called a virtual image. We can see the magnified image with our eyes, because the lens of the eye converges the rays into a real image projected on our retina. Finally, we note that a virtual image is upright and larger than the object, meaning that the magnification is positive and greater than 1.  The ray diagram in Figure on right shows that the image is on the same side of the lens as the object and, hence, cannot be projected - it is a virtual image. Note that the image is closer to the lens than the object. This is a case 3 image, formed for any object by a negative focal length or diverging lens. Magnification: The Science Behind It Types of Magnification Angular Magnification Used in optical instruments like telescopes and microscopes, calculated by comparing the angle subtended by the image and the object. Total Magnification In microscopes, total magnification is the product of the magnifications of the objective and eyepiece lenses. MICROSCOPY TECHNIQUES IN BIOPHYSICS Microscopy is an essential tool in biophysics, allowing us to visualize biological samples at the cellular and molecular levels. Light Microscopy Uses visible light to magnify specimens. Brightfield Microscopy The simplest form, where light passes through the sample. Useful for observing stained or naturally pigmented samples. Microscopy Techniques in Biophysics Microscopy is an essential tool in biophysics, allowing us to visualize biological samples at the cellular and molecular levels. Phase Contrast Microscopy Enhances contrast in transparent specimens, allowing for the visualization of living cells without staining. Comparison of bright field and phase contrast microscopic appearances of stained blood smears. A: Bright field microscopy 100 x objective B: Phase contrast microscopy 100 x objective Microscopy Techniques in Biophysics Microscopy is an essential tool in biophysics, allowing us to visualize biological samples at the cellular and molecular levels. Fluorescence Microscopy Utilizes specific wavelengths of light to excite fluorescent molecules in a sample, enabling visualization of specific structures or proteins within cells. Light microscope and fluorescence microscope observation of DBM scaffolds under dynamic perfusion culture (A, B) and static culture (C, D) (scale bar=100 μm). Microscopy Techniques in Biophysics Microscopy is an essential tool in biophysics, allowing us to visualize biological samples at the cellular and molecular levels. Confocal Microscopy Uses lasers to illuminate samples at specific depths, providing high-resolution images and allowing for 3D reconstruction of samples. APPLICATIONS OF OPTICS IN BIOPHYSICS The principles of optics and lenses have several important applications in biophysics: Cell Biology Understanding cell structure and function through microscopy allows researchers to investigate cellular processes, such as division and communication. Medical Diagnostics Optical techniques are used in various imaging modalities, including endoscopy and optical coherence tomography, providing non-invasive ways to diagnose conditions. Biophysics Research Optical traps and laser tweezers utilize focused light to manipulate small biological particles, allowing for studies of molecular interactions and dynamics. Thank you for your attention QUESTIONS? 110