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
Chromophores
Pigments that absorb and reflect particular wavelengths of light. Different colors behave differently when they interact with chromophores in the specimen. Chromophores can be artificially added using stains to increase contrast
How can resolution be compromised at high magnifications?
At high magnifications, light passes through the small amount of air between the specimen and the lens. This is because there is a large difference between the refractive indices of air and glass, and air scatters the light rays before the lens can focus them. An oil immersion lens can be used to fix this. Oil has a similar refractive index to glass, so it increases the maximum angle at which light leaving the specimen can strike the lens, so more light can be collected.
Darkfield microscope
Similar to a brightfield microscope, but there is a small, opaque disk placed between the illuminator and the condenser lens called an opaque light stop. It blocks most of the light from the illuminator as it travels toward the objective lens, and therefore produces a hollow cone of light focused on the specimen. Light only reaches the lens if it has been refracted or reflected by the specimen. Therefore, the image shows bright objects on a dark background.
Advantages of darkfield microscopes
Creates a high contrast, high resolution image without the use of stains. This is useful for viewing live specimens that could be killed or harmed if stains are used. The thin spirochetes of the bacteria that cause syphilis can be viewed with a darkfield microscope
Phase-contrast microscopes
Use refraction and interference caused by structures in a specimen to create image of high contrast and resolution without stains. It alters the wavelengths of the light passing through the specimen by using an annular stop in the condenser. The annular stop produces a hollow cone of light focused on the specimen. The objective lens contains a phase plate with a phase ring. Therefore, light from the illuminator passes through the phase ring and light refracted or reflected by the specimen passes through the plate. This makes waves traveling through the ring to be a half wavelength out of the phase with light passing through the plate (specimen). The out of phase wavelengths create destructive interference. Therefore, structures that refract light appear dark against a bright background of unrefracted light. Structures with different features, like different refractive indices, will differ in levels of darkness
Advantages of phase contrast microscopes
Increases contrast without requiring stains. Organelles in eukaryotic cells and endospores in prokaryotic cells are best visualized with phase contrast microscopy
Differential interference contrast (DIC) microscopes
Similar to phase contrast microscopes, they use interference patterns to create contrast between different features of a specimen. Creates two beams of light in which the direction of wave movement (polarization) differs. When the beams pass through the specimen or through specimen-free space, they are recombined. The specimen causes differences in the interference patterns generated by recombining the beams
Fluorochromes
Fluorescent chromophores. They absorb energy from a light source and emit this energy as visible. Includes naturally fluorescent substances like chlorophylls and fluorescent stains like Texas red and FITC
Fluorescence microscopes
Transmits an excitation light toward the specimen, the chromophores absorb the excitation light (usually UV light) and emit visible light with longer wavelengths. The excitation light has to be filtered out because UV light is harmful to the eyes, so only visible light passes through the ocular lens. This produces a bright colored specimen against a dark background
Uses of DIC microscopy
Creates high contrast images of living organisms with a 3D appearance. This is useful for looking at structures in live, unstained specimens.
Uses of fluorescence microscopes
Used in clinical microbiology- can identify pathogens and find the locations of specific molecules or structures in a cell. They can distinguish between alive and dead cells based on whether they take up specific fluorochromes. Multiple fluorochromes can be used in the same specimen to show different structures or features
Immunofluorescence
Identifies disease causing microbes by observing whether antibodies bind to them. Can use either DFA or IFA
Antibodies
Protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them
Direct immunofluorescence assay (DFA)
One approach to immunofluorescence. Specific antibodies are stained with a fluorochrome. If the specimen contains the targeted pathogen, you can observe the antibodies binding to the pathogen under the fluorescent microscope. The stained antibodies attach directly to the pathogen, so it is considered a primary antibody stain
Immunofluorescence assay (IFA)
One approach to immunofluorescence. Secondary antibodies are stained with a fluorochrome. These antibodies bind to primary antibodies rather than directly attaching to the pathogen. In this technique, unstained primary antibodies bind to the pathogen and stained secondary antibodies are observed binding to the primary antibodies. IFA makes it easier to visualize a specimen because multiple secondary antibodies will attach to one primary antibody.
Confocal microscopes
Use a laser to scan multiple z-planes (depths) at once. This produces a lot of 2D high resolution images at various depth. A computer compiles the 2D images to create a 3D image. Fluorescent stains are often used to enhance contrast and resolution. An aperture eliminates light not from the z-plane and enhances image clarity.
Uses of confocal microscopes
Used to examine thick specimens like biofilms- they can be examined alive and unfixed
The 2 photon microscope was invented to resolve which problems with fluorescent and confocal microscopes?
When viewing thick specimens, the sensitivity of a fluorescent microscope was limited by out of focus flare, causing low resolution. A confocal microscope used a confocal pinhole to get rid of out of focus fluorescence. However, these microscopes still did not have good resolution for looking a thick tissue samples.
Two photon microscope
Uses a scanning technique, fluorochromes, and long wavelength light (like infrared) to visualize specimens. Long wavelength light has low energy, so two photons have to strike a location at the same time to excite a fluorochrome. Long wavelength light is less damaging to cells and more easily penetrates thick specimens.
Pros and cons of two photon microscopes
Pros- useful for examining living cells in intact tissues- brain slices, embryos, whole organs, or entire animals.
Cons- these microscopes are very expensive, and the lasers use to excite the dye are also very expensive
Electron microscope
Uses short wavelength electrons beams instead of light to increase magnification and resolution. Similar to light, electrons can behave as waves, but they have a much shorter wavelength. This is an advantage because resolution is limited by wavelength- shorter wavelengths are better. EM produces sharp images magnified up one hundred thousand times. There are 2 types of electron microscopes- transmission electron microscopes and scanning electron microscopes
Pros and cons of electron microscopes
Pros- can be used to view subcellular structures and some molecular structures, like single strands of DNA
Cons- electron microscopes can’t be used on living material due to the methods used to prepare specimens.
Transmission electron microscope (TEM)
Similar in function to a brightfield microscope. However, it uses an electron beam from above the specimen that is focused using a magnetic lens (rather than a glass lens) and projected through the specimen onto a detector. Electrons pass through the specimen and then the detector captures the image. The image is produced because of varying opacity in various parts of the specimen.
For TEM, what characteristics are required for the specimen?
The specimen has to be extremely thin (20-100 nm thick). Electron dense stains using heavy metals can be used to enhance opacity and produce an image. The beam and specimen have to be in a vacuum and the specimen also has to be dehydrates.
Scanning electron microscopes (SEM)
Forms images of surfaces of specimens, usually from electrons that are knocked off of specimens by a beam of electrons. It creates highly detailed images with a 3D appearance that are displayed on a monitor. Specimens are dried and prepared with fixatives to prevent the shriveling that can be caused by the drying process. The specimen is then coated with a thin layer of metal such as gold.
Uses of scanning electron microscopes
Used to view the surfaces of larger objects (such as pollen grain) as well as the surfaces of very small samples. It can magnify up to 2 million times.
Scanning probe microscope
Uses sharp probes that are passed over the surface of the specimen and interact with it directly. Does not involve light or electrons. The information that is produced can be assembled into images with magnifications up to one hundred million times. Can be used to observe individual atoms for research purposes. There are 2 types- scanning tunneling microscope and atomic force microscope
2 types of scanning probe microscope
- Scanning tunneling microscope
- Atomic force microscope
Scanning tunneling microscope (STM)
Uses a probe that is passed just above the specimen as a constant voltage bias creates the potential for an electric current between the probe and the specimen. The current occurs via quantum tunneling of electrons between the probe and the specimen. The intensity of the current depends on the distance between the probe and the specimen. The probe moves horizontally above the surface and the intensity of the current is measured. It is used to map the structure of surfaces at the resolution at which individual atoms can be detected
Atomic force microscope (AFM)
Has a thin probe that passes above the specimen. AFM establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen. Forces between atoms causes the tip to move up and down as it moves horizontally. Information regarding deflection of the probe tip is used to construct images of the surface of the specimen with resolution at the atomic level.
Wet mount
The specimen is placed on the slide in a drop of liquid. Liquid specimens can be deposited on the slide using a dropper without additional liquid. A coverslip is placed on top of the liquid
Fixation
The process of attaching cells to a slide. This is done by heating or chemically treating the specimen. Fixation also kills the microorganisms in the specimen while preserving the integrity of their cellular components for observation
Smear
A thin layer of a specimen spread on a slide
How is a sample fixed to a slide?
A smear of a specimen is placed on a slide and the slide is heated over a heat source. Chemical fixatives are usually preferred over heat for tissue specimens, with acetic acid, ethanol, formaldehyde, and others being used to denature proteins, stop biochemical reactions, and stabilize cell structures.
Staining
Used to color certain features of a specimen before examining it under a light microscope. Stains contain salts that have a positive and a negative ion. Either ion could be the chromophore depending on the type of dye, with the non colored ion being called the counterion.
Basic vs acidic dye
Either the positive or negative ion could be the chromophore depending on the type of dye. If the chromophore is the positively charged ion, the stain is considered basic dye. If the negative ion is the chromophore, the stain is considered acidic dye
Positive stain
A dye that will be absorbed by the cells or organisms being observed. This means color will be added to objects of interest so they will stand out. This is preferrable in most cases. Cells usually have negatively charged cell walls, so the positive chromophores in basic dyes tend to bind to the cell walls, making the stains positive.
Negative stain
Absorbed by the background but not the cells or organisms in the specimen. This produces an outline of the organism against a colorful background
Simple staining
A single dye is used to emphasize specific structures. Usually, it will make all of the organisms appear as the same color, even if there are multiple types of organisms in the sample.
Differential staining
Distinguishes organisms based on their interactions with multiple stains. Two different organisms in a sample may appear as 2 different colors in this case. In clinical settings, differential techniques include Gram staining, acid-fast staining, endospore staining, and others
What is the purpose of Gram staining?
To distinguish between bacteria with different types of cell walls
Gram stain procedure (4 steps)
- A primary stain called crystal violet to applied to a heat fixed smear, making all of the cells purple
- A mordant called Gram’s iodine is added. It acts like a trapping agent that mixes with the crystal violet, making the crystal violet-iodine complex clump together and stay inside the layers of peptidoglycan in the cell walls
- A decolorizing agent like ethanol is added. It doesn’t really affect cells with thick peptidoglycan layers in their cell walls, but cells with thin cell walls become colorless
- A secondary counterstain (usually safranin) is added. The decolorized cells stain pink, but the dye is less noticeable in the cells that still contain the crystal violet dye
Mordant
A substance used to set or stabilize stains or dyes, makes the color stick
Interpreting Gram staining results
Purple crystal violet stained cells are gram positive, red safranin stained cells are gram negative. However, using older bacterial cells or leaving on decolorizer for too long can affect the results and make them unreliable
Implications of gram staining results
Gram staining helps clinicians to classify bacterial pathogens in a sample into categories associated with specific properties. Gram negative bacteria tend to be more resistant to certain antibiotics than gram positive bacteria
Acid-fast stain
Tuberculosis and leprosy bacteria is gram positive but doesn’t stain with the Gram stain, so we use a special type of stain. Differentiates between 2 types of gram positive cells- cells that have mycolic acid in their cell walls and cells that don’t. Uses carbolfuchsin as the primary stain. Acid fast cells retain the carbolfuchsin after a decolorizing agent (ethanol) is applied. A secondary counterstain (methylene blue) is applied, and causes non-acid fast cells to turn blue. There are 2 methods- the Ziehl-Neelsen technique and the Kinyoun technique
Ziehl-Neelsen technique vs the Kinyoun technique
The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells. The Kinyoun technique does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB)
How to tell if acid fast bacteria (AFB) are present in a tissue sample
If AFB are present in a tissue sample,
their red or pink color can be seen clearly against the blue background of the surrounding tissue cells. The bacteria that cause tuberculosis are AFB.
What is a capsule?
Certain bacteria and yeasts have a protective outer structure. The presence of a capsule is directly related to a microbe’s virulence (its ability to cause disease).
Capsule staining
Determining whether a microbe has a capsule can be an important diagnostic tool. Capsules can’t absorb most dyes, so a negative staining technique (staining around the cells) is used. The dye creates a halo around the cell border. Alternatively, positive and negative staining techniques can be combined to visualize capsules: The positive stain colors the body of the cell, and the negative stain colors the background but not the capsule, leaving halo around each cell.
Endospores
Structures produced within certain bacterial cells that allow them to survive harsh conditions
Endospore staining
Uses 2 stains to differentiate endospores from the rest of the cell. The Schaeffer-Fulton method uses heat to push the primary stain (malachite green) into the endospore. Water is used to decolorize the cell, but the green stain remains in the endospore. Then, a pink counterstain (safranin) is used. The image shows the shape and location of endospores (in green) if they are present.
Which types of bacteria is endospore staining used to identify?
Important for identifying 2 genera of bacteria- bacillus and clostridium. B. anthracis (which causes anthrax) has been of particular interest because of concern that its spores could be used as a bioterrorism agent. C. difficile is a particularly important species responsible for the typically hospital acquired infection known as “C. diff.”
Flagella
Tail like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. They are viewed using flagella staining because they are so thin
Flagella staining
Thickens the flagella by applying mordant (tannic acid) which coats the flagella, then the specimen is stained (usually with pararosaniline). Helps with seeing the location and number of flagella. Used by microbiologists to classify and identify bacteria in a sample
Preparing specimens for electron microscopy (3)
- Cells are embedded in plastic resin to hard them so they can be cut into thin slices. They are dehydrated through soaking in ethanol solution, removing water and allowing resin to enter the cell
- Thin sections are cut using an ultramicrotome
- Samples are fixed to fine copper wire or carbon fiber grids and stained with electron dense heavy metal substances like uranyl acetate or osmium tetroxide
How does preparing a specimen for SEM differ?
The specimen has to be drier than it does for TEM. Critical point drying with inert liquid carbon dioxide under pressure is used. After drying, the specimen is sputter coated with metal by knocking atoms off of a palladium target with energetic particles. This prevents specimens from becoming charged by the SEM’s electron beam.
How are samples prepared for fluorescence and confocal microscopy?
Prepared similarly to light microscopy, but the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells (immunofluorescence), others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH)
FISH
Fluorescence in situ hybridization- causes cells to be stained based on whether they have a specific DNA sequence. Dyes attach to the DNA molecules.
How are samples prepared for 2 photon microscopy?
Similar to fluorescence microscopy, but infrared dyes are used. Specimens for STM need to be on a very clean and atomically smooth surface. They are often mica coated with Au (111). Toluene vapor is usually used as a fixative.
Streptococcus
Considered streptococci if there are more than 4 of the coccus shape on a chain. Diplobacillus is when there are 2 bacilli adjacent to each other
How is wavelength related to resolution?
The white light has a mixture of wavelengths and cannot resolve structures less than 0.2µm. Resolution of Leeuwenhoek’s microscope was 1µm- therefore, we need a shorter wavelength to resolve between objects
Conditions needed to resolve an object from its surroundings (3)
- Contrast between the object and its surroundings
- Wavelength smaller than the object. If larger the image will be blurred
- A detector with sufficient resolution for the given wavelength
Ways for light to interact with an object (4)
- Absorption- light goes into the object- object gains energy and may get hot
- Reflection- light bounces off the surface
- Refraction- light bends
- Scattering- light scatters in all directions when it hits the object
How does lens quality of a brightfield microscope maximize detail?
Minimizes shape defect (aberrations), constructs a series of lenses that multiply each other’s magnifications, and corrects for aberrations. Want to make sure the light is going through. Compound microscopes contain multiple lenses to correct or compensate for aberration
Gram negative cells
Single layer of peptidoglycan, outer membrane containing LPS/endotoxin. Escherichia coli Proteus species is an example
Gram positive cells
Multiple layers of peptidoglycan. Crystal violet binds to peptidoglycan. The mordant creates a complex with crystal violet inside the peptidoglycan. Ethanol shrinks the peptidoglycan to trap the crystal violet-mordant complex. No outer membrane, no LPS/endotoxin. Staphylococcus aureus Streptococcus is an example
