Unit 2: Microscopes

Flashcards reviewing key concepts about microscopes.

What are the learning objectives for the microscope lab?

To prepare wet mounts and hanging drop slides of live specimens; to identify microscope parts and their functions; to focus samples; to use, care for, clean, and store microscopes.

Who was Robert Hooke?

Observed microbes in 1665 and published illustrations in Micrographia, used a compound microscope with ocular and objective lenses.

How is the total magnification of a compound microscope calculated?

The total magnification is the product of the ocular lens magnification and the objective lens magnification.

Who was Antonie van Leeuwenhoek?

A cloth merchant from Holland, often called the 'Father of Microbiology,' who examined various samples under a simple microscope and kept detailed notes.

What is a simple microscope?

A microscope with a single lens, essentially a magnifying glass.

What concepts are important to understand the limitations of a light microscope?

Resolution, wavelength, and numerical aperture.

What is the Abbé equation?

d = 0.5λ / n sin θ, where λ is the wavelength of light, d is the minimal distance to distinguish between two close objects, and n sin θ is the numerical aperture.

What is resolution in microscopy?

The ability of a lens to distinguish two objects that are close together.

What are the six types of light microscopes?

Bright-field, dark-field, phase contrast, differential interference contrast (DIC), fluorescence, and confocal scanning laser microscope (CSLM).

What is a bright-field microscope?

A standard microscope where the specimen is illuminated by a light source and magnified by objective and ocular lenses. Staining is often required to visualize unstained bacteria.

How does a dark-field microscope work?

It makes use of a dark-field stop, an opaque disk that blocks light directly underneath the specimen so that light reaches it from the sides.

How does a phase-contrast microscope work?

It uses an opaque ring or annular stop with a transparent ring that only releases light in a hollow cone. Phase differences are translated into differences in brightness.

How does a differential interference contrast (DIC) microscope work?

It operates by taking advantage of the differences in refractive index of a specimen and its surroundings but uses polarized light split into two beams.

How does a fluorescence microscope work?

It utilizes light emitted from a specimen, generated by a mercury-arc lamp and filtered to produce a specific wavelength of light directed at the specimen.

What are fluorochromes?

Fluorescent dyes that bind to specific cell components.

How does a confocal scanning laser microscope (CSLM) work?

Uses a laser for illumination that is directed at dichromatic mirrors that move, “scanning” the specimen. Information acquired by the detector can be compiled by a computer to create a single 3D image entirely in focus.

Why is staining important in microscopy?

Providing contrast between the microorganism and its background to make it easier to see.

What are the types of staining techniques?

A simple stain uses a single dye to stain cells directly (direct stain) or stain the background (negative stain). Differential stains combine stains to differentiate organisms based on characteristics.

What are some examples of differential stains?

Gram stain (differentiates bacteria based on cell wall type), acid-fast stain (for bacteria with specialized cell walls), and stains targeting specific bacterial structures.

What are electron microscopes?

Electron microscopes replace light with electrons for visualization. Resolution increases to around 0.5 nm, with magnifications over 150,000x.

What are the two types of electron microscopes?

Transmission electron microscope (TEM) and scanning electron microscope (SEM).

How does a transmission electron microscope (TEM) work?

It utilizes an electron beam directed at the specimen. Dense areas scatter electrons, resulting in a dark area on the image.

How does a scanning electron microscope (SEM) work?

The image is formed from secondary electrons released from the specimen's surface and collected by a detector.

How do scanning probe microscopes work?

They move a probe over a specimen’s surface in the x-z planes, allowing computers to generate an extremely detailed 3D image. Resolution is high because the probe size is much smaller than the wavelength of visible light or electrons.

What are the two types of scanning probe microscopes?

Scanning tunneling microscope (STM) and atomic force microscope (AFM).

How does a scanning tunneling microscope (STM) work?

Maintains a constant voltage with the specimen surface, allowing electrons to travel between them. The tunneling current is maintained by raising and lowering the probe.

How does an atomic force microscope (AFM) work?

Utilizes a cantilever with an extremely sharp probe tip that maintains a constant height above the specimen, typically by direct contact.