Chapter 2- How we see the invisible world Flashcards
When discussing light as relevant to microscopy, how does light behave?
As a wave
Wavelength
The distance between one peak of a wave and the next peak
Amplitude
Height of each peak (or the depth of each trough)
Frequency
The rate of vibration of the wave, or the number of wavelengths within a specified time period
How do light waves interact with materials (3 ways)
Reflection, absorption, or transmission
Reflection
When a wave bounces off of a material. A red object is reflecting the red wavelength of light
Absorbance
When a material captures the energy of a light wave
Transmittance
When a wave travels through a material, like light through glass. When a materials allows a large amount of light to be transmitted, it could be thinner or more transparent. Greater transparency means less opacity
Interference
When light waves interact with each other and create complex patterns of motion
Diffraction
When light waves interact with small objects or openings by bending or scattering. Diffraction is larger when the object is smaller relative to the wavelength of the light. Light waves can interfere with each other when they diffract in different directions around an obstacle or opening
Refraction
When light waves change direction as they enter a new medium. Transparent material transmits light at different speeds. When light passes from one material to the other, it can change speed and it experiences a corresponding change in direction. The degree of change in direction depends on the angle of the incoming light. In brightfield microscopes, refraction can stop light from reaching the lens
Refractive index
The extent to which a material slows transmission speed relative to empty space. Large differences between the refractive indices of two materials will result in a large amount of refraction when light passes from one material to the other. Light moves more slowly through a material with a greater refractive index
How does the direction of light change when passing the boundary into a material with a higher refractive index?
The light slows down and therefore moves toward the normal line (perpendicular to the boundary)
Lenses
An object with a curved boundary that collects all of the light that strikes it and refracts it so that the light meets a single point, called the image point (focus).
Convex vs concave lenses
A convex lens can be used to magnify because it can focus at a closer range than the human eye, producing a larger image. Concave lenses can be used in microscopes to redirect the light path
Focal point
The image point when light entering the lens is parallel
Focal length
The distance to the focal point
How does the lens in the human eye work?
The lens helps us to see images. It focuses the light reflecting off of objects in front of the eye onto the surface of the retina (which is like a screen in the back of eye). Artificial lenses, like contact lenses, focus light before it’s focused on the retina, and manipulates the object that appears on the retina so that it looks larger
How can images be manipulated?
By controlling the distance between the object, the lens, and the screen, and the curvature of the lens. When an object is closer to the lens, the focal points are farther from the lens. Therefore, it’s necessary to manipulate these distances to create a focused image on a screen
Electromagnetic spectrum
Describes the different types of electromagnetic radiation that is all around us. EM radiation is defined in terms of wavelength and frequency. Includes radio waves, microwaves, infrared radiation, visible light, UV light, X-Rays, and gamma rays
Relationship between wavelength and frequency
Inverse relationship- waves with high frequencies have shorter wavelengths.
What type of waves transport more energy?
High frequency waves contain more energy. The energy is delivered using particles called photons, and high frequency waves deliver more photons
How do photons with different energies interact with the retina?
With visible light, each color corresponds to a specific frequency and wavelength (red is the lowest frequency, violet is the highest). We perceive white light if the retina receives visible light of many different frequencies
Dispersion
The separation of colors that occurs when white light is passed through a prism. When the light passes through the prism, different colors refract in different directions
How do fluorescent dyes work?
They absorb UV light or blue light and use that energy to emit the photons of a different color to give off light. This is because the energy absorption causes electrons to move to higher energy states, and they emit specific amounts of energy as photons once they fall back to the base energy state. The emitted photons will have less energy than the photons that were absorbed, because not all of the energy will be emitted. These dyes include Texas red (emits red light) or FITC (emits green)
Phosphorescence
Photons are emitted following a delay after absorption. This is how glow in the dark plastic works
Magnification
The ability of a lens to enlarge the image of an object when compared to the real object. 10 times magnification means that the image appears 10 times larger than it would be if viewed by the naked eye
Resolution
The ability to tell that 2 separate points or objects are separate. A low resolution object looks fuzzy. Resolution of the human retina is about 150 µm, or 1/7th of a millimeter.
Factors that affect resolution (2)
- Wavelength- a shorter wavelength means a higher resolution
- Numerical aperture- the higher the numerical aperture, the better the resolution
Numerical aperture
A measure of a lens’ ability to gather light
Contrast
Microscopes often require contrast even at high resolution because microorganisms are mostly transparent. This can be achieved using different features of light or electrons, or dyes
Girolamo Fracastoro
The first person to state that disease was spread by tiny “seeds of contagion”. Their existence was hypothetical for over a century until microscopes were invented
Antonie van Leeuwenhoek
Credited as the first person to have created microscopes powerful enough to view microbes. In 1674, he was able to observe single celled organisms using a simple microscope
Simple vs compound microscopes
In a simple microscope, light passes through one lens. Compound microscopes use 2 sets of lenses
Robert Hooke
The first to use a microscope to observe cells in a piece of cork
Light microscopy (6)
Microscopes that use light to visualize images- many microscopes fall into this category. Includes brightfield, phase contrast, differential interference contrast, fluorescence, confocal scanning laser, and two photon microscopes. The modern light microscope was invented in 1830 by Joseph Lister.
Binocular
Having 2 eyepieces
Brightfield microscope
A compound microscope with two or more lenses that produce a dark image on a bright background. Most newer brightfield microscopes are binocular. Each eyepiece contains an ocular lens, which magnifies images 10x. At the other end of the body, there are a set of rotating objective lenses. The magnification of these lenses ranges from 4x to 100x. The ocular and objective lenses work together to create a magnified image.
Total magnification
The product of the ocular magnification times the objective magnification in a brightfield microscope
Stage
The platform of a microscope, where the specimen is clipped into place on a glass slide.
x-y mechanical stage knobs
Knobs that move the slide on the surface of the stage. They don’t raise or lower the stage, just position the specimen over light.
Coarse and fine focusing knobs
The coarse focusing knob is used for large scale movements with the 4x and 10x objective lenses. The fine focusing knob is used for small scale movements, usually with the 40x and 100x lenses
How does light travel in a brightfield microscope?
Images become dimmer when magnified, so high magnification requires intense lighting. The light comes from an illuminator, which is a lightbulb below the stage. The light from the illuminator passes through the condenser lens below the stage, which focuses all of the light on the specimen. The condenser focus knob can be used to change the position of the condenser
Diaphragm
Adjusts the amount of light striking the specimen. The rheostat (a dimmer switch) can be used to adjust the intensity of light