Chapter 2

Constructive vs Destructive Interference

*Constructive* 2 or more waves with crests aligned; leads to higher amplitudes *Destructive* crest of 1 meets trough of another; leads to lower amplitude The colors in a soap bubble are from simultaneous reflection of light from the inside and outside surfaces of the bubble.

Compare and contrast the contributions of van Leeuwenhoek, Hooke, and Galileo to early microscopy.

*Girolamo Fracastoro* First person to think of the idea of Microbes *Antonie van Leeuwenhoek* credited with the first observation of microbes, including protists and bacteria, with simple microscopes that he made. *Galileo Galilei* Used a compound microscope to examine insect parts. *Robert Hooke* Coined the term "cell" although what he saw weren't cells. *Hans and Zaccharias Janssen* Dutch spectacle makers who may have invented the telescope, the simple microscope, and the compound microscope during the late 1500s or early 1600s *Hans Lippershey* Other possible inventor of the telescope

Describe historical developments and individual contributions that led to the invention and development of the microscope

*Girolamo Fracastoro* regarded as the first person to formally postulate that disease was spread by tiny invisible seminaria, or "seeds of the contagion." In his book De Contagione *Antonie van Leeuwenhoek* sometimes hailed as "the Father of Microbiology," is typically credited as the first person to have created microscopes powerful enough to view microbes. First person to observe microbes, including bacteria, using a simple microscope. He called them "animalcules" and "wee little beasties." *Galileo Galilei* used a compound microscope to examine insect parts. Galileo's compound microscope was more sophisticated, passing light through two sets of lenses. *Robert Hooke* publishing in his book Micrographia; many observations using compound microscopes. Viewing a thin sample of cork through his microscope, he was the first to observe the structures that we now know as "cells"

Define hyperopia and myopia

*Hyperopia* = farsightedness (too little curvature) *Myopia* = nearsightedness (too much curvature) Controlling the distance between the object, lens, and retina as well as the curvature of the lens can manipulate an image.

Interactions of light

*Reflection:* waves bounce off; reflected color is the color we see *Absorbance:* light is captured *Transmittance:* light passes through *Phosphorescence:* when light is absorbed and then later re-emitted. I.e. Glow in the dark stuff *Interference:* light waves interacting with each other like two separate drops of water *Diffraction:* Scattering or bending of light, diffraction is relative to the size of the opening or obstacle. Scattering is larger when obstacle or opening is smaller than wavelength. It can result in interference *Dispersion:* separating white light into its rainbow component colors using a prism *Refraction:* Light waves changing direction in new medium

Compare and contrast acidic and basic dyes.

Basic dyes = positive charge Acidic dyes = negative charge

Simple Stains

Composed of single dye

Electron Microscopy

Electron microscopy focuses electrons on the specimen using magnets, producing much greater magnification than light microscopy. The transmission electron microscope (TEM) and scanning electron microscope (SEM) are two common forms.

Identify and define the characteristics of electromagnetic radiation (EMR) used in microscopy

Fluorescent dyes absorb UV or blue light and use the energy absorbed to emit photons of different colors. Electrons get to higher energy states and fall back to ground states, emitting energy. Phosphorescence happens when photons are emitted after a delay for absorption.

Why do fluorescent dyes emit a different color of light than they absorb?

Fluorescent dyes and phosphorescent materials can effectively transform nonvisible electromagnetic radiation into visible light.

Which has a higher frequency: red light or green light?

Green. "ROYGBIV" is lowest to highest

What types of specimens should be chemically fixed as opposed to heat-fixed?

Heat fixation is carried out for the microorganisms to stop their movement and metabolism. Whereas the chemical fixatives are preferable to heat fixing of tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can be uses which denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples.

The Refractive Index

How much a material slows transmission The extent to which a material slows transmission speed relative to empty space is called the refractive index of that material. 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. For example, light moves much more slowly through water than through air, so light entering water from air can change direction greatly. We say that the water has a higher refractive index than air (Figure).

Explain why dispersion occurs when white light passes through a prism.

This occurs because, for a given material, the refractive index is different for different frequencies of light.

Name the device that is used to create thin sections of specimens for electron microscopy.

Ultramicrotome

Differential Stains

Used to distinguish between bacteria.

Electromagnetic Radiation (EMR)

Visible light is part of the electromagnetic spectrum; light waves of different frequencies and wavelengths are distinguished as colors by the human eye. --- EMR: a kind of radiation including visible light, radio waves, gamma rays, and X-rays, in which electric and magnetic fields vary simultaneously.

Types of Microscopes

-Numerous types of microscopes use various technologies to generate micrographs. Most are useful for a particular type of specimen or application. -Light microscopy uses lenses to focus light on a specimen to produce an image. Commonly used light microscopes include brightfield, darkfield, phase-contrast, differential interference contrast, fluorescence, confocal, and two-photon microscopes.

Properties of light

-Visible light behaves as an electromagnetic wave. Different types of light have different properties: -Wavelength -Frequency -Amplitude

Discuss at least 2 factors that affect resolving power.

-Wavelength of light -numerical aperture of condenser lens -numerical aperture of objective lens

1. Explain the role of Gram's iodine in the Gram stain procedure. 2. Explain the role of alcohol in the Gram stain procedure. 3. What color are gram-positive and gram-negative cells, respectively, after the Gram stain procedure?

1. The iodine acts as a *mordant* to make the dye less soluble so it can adhere to the cell walls. 2. Alcohol washes away stain from the gram-negative cell walls. 3. Positive = Purple, Negative = Pink

Explain how a lens focuses light at the image point.

A lens is a medium with a curved surface that refracts and focuses light to produce an image. A prism refracts light. A convex lens refracts light to a focal point on the opposite side. A concave lens refracts light away from a focal point in the lens.

Explain how a prism separates white light into different colors.

A prism can separate the colors of white light (dispersion) because different frequencies of light have different refractive indices for a given material.

Fluorescence in situ hybridization (FISH)

A procedure using a fluorescence-labeled probe to detect specific nucleotide sequences (DNA) within intact cells attached to a microscopic slide

Explain why it is important to fix a specimen before viewing it under a light microscope.

It kills the microbes and allows attaches it to the slide for staining, rinsing, and viewing without losing the specimen.

Why is Antonie van Leeuwenhoek's work much better known than that of Zaccharias Janssen?

Leeuwenhoek can thank ample documentation of their work for their respective legacies. Leeuwenhoek began his work in obscurity, leaving behind few records. However, his friend, the prominent physician Reinier de Graaf, wrote a letter to the editor of the /Philosophical Transactions of the Royal Society/ of London calling attention to van Leeuwenhoek's powerful microscopes.

Explain how lenses are used in microscopy to manipulate visible and ultraviolet (UV) light

Light waves interacting with materials may be reflected, absorbed, or transmitted, depending on the properties of the material. Light waves can interact with each other (interference) or be distorted by interactions with small objects or openings (diffraction).

Explain the difference between magnification and resolution.

Magnification makes something look bigger. Resolution is the clarity and sharpness of an image.

Oil Immersion

Oil immersion lenses are used to improve resolution. Because immersion oil and glass have very similar refractive indices, there is a minimal amount of refraction before the light reaches the lens. Without immersion oil, light scatters as it passes through the air above the slide, degrading the resolution of the image.

What is the main difference between preparing a sample for fluorescence microscopy versus light microscopy?

Preparation for fluorescence microscopy is similar to that for light microscopy, except that fluorochromes are used.

Prisms, Concave, and Convex

Prism: disperses Convex: focuses Concave: scatters

Name two factors that affect resolution.

Resolution can be increased by shortening wavelength, increasing the numerical aperture of the lens, or using stains that enhance contrast. Wavelength: shorter = more clear Numerical aperture, which is ability to gather light

Explain the difference between resolution and contrast.

Resolution is how clearly you can see an image Contrast is how much differences you can see between objects by affecting the amount of light saturation.

Compare and contrast transmission and scanning electron microscopy in terms of procedure.

Samples for TEM require very thin sections, whereas samples for SEM require sputter-coating.

Staining

Samples must be properly prepared for microscopy. This may involve staining, fixation, and/or cutting thin sections. A variety of staining techniques can be used with light microscopy, including Gram staining, acid-fast staining, capsule staining, endospore staining, and flagella staining. Depending on the type of dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called the counterion. If the chromophore is the positively charged ion, the stain is classified as a basic dye; if the negative ion is the chromophore, the stain is considered an acidic dye. Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin, crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making them negative stains. Commonly used acidic dyes include acid fuchsin, eosin, and rose bengal.

Scanning Probe Microscopy

Scanning probe microscopy produces images of even greater magnification by measuring feedback from sharp probes that interact with the specimen. Probe microscopes include the scanning tunneling microscope (STM) and the atomic force microscope (AFM).

Compare and contrast the features of simple and compound microscopes

Simple microscopes have a single lens, while compound microscopes have multiple lenses.

What is the function of the condenser in a brightfield microscope?

The condenser fulfills two functions in the microscope. It provides an area of evenly-illuminated light in the field of view at the specimen plane and illuminates the aperture of the objective uniformly with light of sufficient yet controllable angle.

Why did the cork cells observed by Robert Hooke appear to be empty, as opposed to being full of other structures?

They likely appeared to Hooke to be filled with air because the cork cells were dead, with only the rigid cell walls providing the structure.

Why is it important to dehydrate cells before examining them under an electron microscope?

They must be extremely thin to scan. However, cells are too soft to cut thinly, even with diamond knives. To cut cells without damage. Dehydrating them thins the cell while maintaining shape.

Magnification, Resolution, and Contrast

The power of a microscope can be described in terms of its magnification and resolution. *Magnification:* the ability of a lens to enlarge the image of an object when compared to the real object. I.e. 10⨯ = 10 times the size as viewed with the naked eye. *Resolution:* the ability to tell that two separate points or objects are separate. Affected by *wavelength*; shorter waves = better resolution. an electron microscope has a much higher resolution than a light microscope, since it uses an electron beam with a very short wavelength, as opposed to the long-wavelength visible light used by a light microscope. The second factor that affects resolution is numerical aperture, which is a measure of a lens's ability to gather light. The higher it is the better the resolution. *Contrast:* Differences light intensity

Refraction index

a measure of how light bends as it passes from one substance to another

Biofilms

a surface coating colony of prokaryotic that engage in metabolic cooperation

Define: Flourescence

having the property of emitting visible light when exposed to the ultraviolet light

Gram Staining

he Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, (Gram-postive or Gram-negative). The purple, crystal-violet stained cells are referred to as gram-positive cells, while the red, safranin-dyed cells are gram-negative

Define: Phosphorescence

light emitted by a substance without combustion or perceptible heat

Immunofluorescence

method of tagging antibodies with a luminating dye to detect antigen-antibody complexes

Calculate total magnification for a compound microscope

ocular magnification × objective magnification I.e, (40×)(10×)=400× magnification