Microscopy and Staining Lecture 4 Notes

Flashcards covering the principles of microscopy, including resolution, contrast, light and electron microscopy types, and various staining techniques.

Wavelength Visibility Rule

In order for an object to be visible, its size needs to be approximately $\sim 1/2$ the wavelength.

Visible Wavelength Range

The range of wavelengths considered visible, extending from $200\,nm$ to the macro scale.

Resolution

The ability to distinguish between two objects; it is improved by using shorter wavelengths.

Immersion Oil

A medium used with the $100\times$ objective to prevent refraction and improve resolution.

Contrast

The ability to differentiate the specimen from the background.

Positive Stain

A type of staining technique where the specimen itself is stained.

Negative Stain

A type of staining where the background is stained instead of the cell, often used to study characteristics that are not easily stained.

Illuminator

The light source in a light microscope.

Field Iris

A component that controls the amount of light allowed to pass through the microscope.

Condenser

A component that focuses light rays into a specific point, usually on the slide.

Objective Lens

The lens responsible for the first magnification of the specimen.

Ocular Lens

The lens responsible for the second magnification, where the image from the objective lens is further magnified and viewed by the eye.

Bright Field Microscopy

A microscopy technique where even light waves travel upward through the specimen, often resulting in low contrast unless the condenser is adjusted.

Dark Field Microscopy

A technique using a special condenser so that light does not channel upward into the objective; only light that hits the cells and refracts upward is visible.

Phase-contrast Microscopy

A technique that measures phase changes using a phase plate as light passes through cell parts of varying density.

Differential Interference Contrast (DIC)/Nomarski

A technique that uses a light polarizer to turn light $90^{\circ}$ and a prism to split light into two beams to obtain $3D$ detail without stains.

Fluorescence Microscope

A microscope that uses a fluorophore to attach to specific cell parts and visualizes them through emission wavelengths; it typically only shows a $2D$ image.

Confocal Scanning Laser Microscope

A microscope that measures fluorescence at particular levels within a specimen and uses software to compile multiple planes into a $3D$ map.

Transmission Electron Microscopy (TEM)

A microscopy method using an electron beam and electron-dense dye to achieve better resolution than light microscopy.

Scanning Electron Microscopy (SEM)

A technique using a sputterer to spray dye on specimen surfaces so an electron beam can detect scattered electrons to create a high-resolution $3D$ image.

Electron Cryotomography

A method involving staining a specimen with electron-dense dye and taking images while rotating them at different angles to build a $3D$ approximation.

Atomic Force Microscopy

A technique using a probe to detect electron density in covalent bonds, used for visualizing molecules.

Simple Stain

A process where a basic (+$脚) dye attaches ionically to the negative (-$$) phospholipid head groups on a cell surface.

Differential Stains

Staining procedures that use more than one stain to differentiate cells based on specific cell properties.

Gram Stain

A differential stain that distinguishes cells based on peptidoglycan thickness in the cell wall.

Gram Negative

Cells that appear pink and have a thin peptidoglycan layer.

Gram Positive

Cells that appear purple and have a thick peptidoglycan layer.

Acid-fast Stain

A differential stain identifying a waxy coating impervious to acidified alcohol; acid-fast cells appear red/pink, while others appear blue.