Key Concepts in Light and Electron Microscopy

Photon

Electromagnetic radiation released as waves/ particles are the photons

Contrast

Must exist between the object and its surroundings luminance/ color difference that makes an object visible against a background

Wavelength

Affects resolution which is the ability to distinguish two points. Shorter wavelengths yield higher resolution

Magnification

Spreading the light rays allows us to collect more photons in our retina. Ability of a microscope to produce an image of an object at a scale larger (or even small) than its actual size

Absorption

Light is absorbed and the energy is transferred to the object. Reemit the energy with a longer wavelength. Physical changes dark or a different color

Reflection

The light is redirected off at an equal angle

Refraction

Light enters an object and will bend as it changes speed. Initial medium and new medium has the same refraction index light will not bend

Refractive index

Measure of light-bending ability of a medium; different mediums have different refractive index. Light refract after passing through a specimen. Immersion oil prevents the refraction through blocking air

Scattering

Light striking an object is sent into multiple directions. Detect but not resolve objects

Lens

There is objective and ocular lens. Bending the light. Focal point. Parallel rays bent inward will meet at a point. Ray crossed the image will appear backwards and upside down. Focal distance. Distance from the lens to the focal point. Determined by the curvature and refractive index

Immersion oil

Oil with the same refractive index of glass - preventing refraction from air

Stain/ dye

Coloring the sample with a dye to emphasize certain structures. Before staining smearing and fixing is needed. Smear is a thin film of material with the microorganisms spreading over a slide. Fixing attaches microorganisms to the slide either through heat or chemically. Kills the microorganisms and preserves them at a constant state-chemical fixations reserves parts of microbes with less distortion

Acidic Dye

Negative ion is the chromophore

Basic Dye

Positive ion is the chromophore

Positive stain

Taken up by the cells, darkening the cell. Cells are negatively charged - positive stains are attracted to cells

Negative stains

Are taken up by the background darkening the background causing cells to be illuminated/ outlined

Chromophore

Positive and negative ions. Chromophore containing conjugate double bonds or aromatic rings absorbing specific bands of visible light

Fluorophore

Fluorescent particles. Tagging molecules through chemical affinity to certain biomolecules or classes of molecules. Labeled antibodies. Certain affinity to certain molecules/ component antigens. DNA hybridization is a short DNA sequence is tagged with the fluorophore. Gene fusion reporters. Cells are genetically engineered to express their own fluorophores

Decolorizing

Using ethanol or acetone to wash out the dye out of cells within the cell wall. Used in gram staining- a differential stain; primary staining -> iodine mordant -> decolorizing -> safranin for secondary staining. Gram positive -> thick cell wall. Gram negative -> thin cell wall

Mordant

To combine with a dye or stain to fix the material in

Antibody

A Y-shaped protein produced by the immune system in response to the presence of a foreign substance (antigen)

Antigen

Substance that triggers the body's immune system to produce antibodies

Acid-Fast Stain

Binding to only bacteria that have a waxy material in the cell walls. Thick peptidoglycan cell wall. Primary stain - carbolfuschin, decolorizing agent - alcohol, counterstain - methylene blue. Color of acid fast: red — non-acid fast: blue

Capsular Staining

Negative staining to identify the capsule. Suspension of India ink or nigrosin contrasts with the background of the capsule. Cells are stained with a simple stain

Endospore Staining

Endospores are resistant or dormant internal structures produced by some cells-heat and acid. Primary stain malachite green with heat to help dye penetrate the endospore. Decolorize cells with water. Counterstain with safranin. Spores appear green within red or pink cells

Light Microscopy

Understand the concept of resolution and how the wavelength of light limits the resolution of a light microscope.

Ultraviolet (UV) Light

Smaller wavelength too much energy is UV.

Infrared Light

Longer wavelengths too weak to be seen is infrared.

Electron Microscopy

Uses electrons which have a smaller wavelength than photons, thus offering higher resolution.

Light Microscopy

Utilizes light to magnify images of small objects.

Refractive Indices

Light passing from one medium to another will bend if the media have different refractive indices.

Compound Microscope

A type of light microscope that has multiple lenses (objective and ocular).

Darkfield Microscope

Blocks direct light - light goes around the edges.

Phase Contrast Microscope

Light is separated; some passes through the sample while light that does not is separated.

Fluorescence Microscope

Specimen or fluorophore absorbs light of a defined excitation wavelength and emits photons of lower energy, resulting in a longer wavelength.

Total Magnification

Calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.

Staining

Includes methods such as simple, differential, Gram, acid fast, endospore, capsule, and various fluorescent tags.

Fluorescence

Emitted light is always of a specific wavelength based on the fluorophore, allowing for filtering to produce a sharp image.

Scanning Electron Microscopy (SEM)

Involves staining the surfaces of the sample and shooting electrons at the stain layer, imaging only the surface in high resolution.

Transmission Electron Microscopy (TEM)

Involves staining the entire sample and shooting electrons through it, providing high resolution detail of the interior.

Atomic Force Microscopy

Involves a physical metal probe being scanned across the surface of the sample to produce an image.

Scanning Tunneling Microscopy

Similar to atomic force microscopy, it uses a probe to detect interactions with the sample surface.

Brightfield Microscopy

Uses stained samples and is not suitable for very small organisms.

Phase-Contrast Microscopy

No fixation or staining is needed, revealing details of internal structures in living cells.

Differential Interface Contrast

Similar to phase-contrast, it provides more contrast and color to the specimen, resulting in a 3-D and brightly colored image.

Super-Resolution Microscopy

Uses specific antibodies to achieve higher resolution images.

Electron Transmission Microscopy

Focuses on internal structures of the sample.

Scanning Electron Microscopy

Focuses on the surface of the sample but kills the specimen.