BIO 405 Chapter 9 Visualizing Cells and Their Molecules Key Terms

MCB flashcards from Norton's Molecular Biology of the Cell, 7th Ed

Antibodies

Proteins produced by the vertebrate immune system that recognize and bind to specific target molecules (antigens). They can be coupled to fluorescent dyes or electron-dense particles to serve as highly specific probes for locating macromolecules in cells.

Bright-Field Microscopy

A standard form of light microscopy in which the image is formed directly by light transmitted straight through the specimen.

CLEM (Correlative Light and Electron Microscopy)

A technique that combines fluorescence microscopy (to locate specific tagged molecules) with electron microscopy (to reveal the surrounding cellular ultrastructure) on the same specimen, allowing for contextual localization of molecules.

Confocal Microscope

An advanced light microscope that produces sharp optical sections by using a focused laser beam to illuminate a single point in the specimen and a pinhole aperture to block out-of-focus light from reaching the detector. A complete image is built by scanning the beam across the specimen.

Contrast

The difference in brightness or color that makes an object distinguishable. In microscopy, it can be generated by staining, exploiting phase shifts (phase-contrast/DIC), or using fluorescence.

Cryo-electron Microscopy (Cryo-EM)

A form of electron microscopy where specimens are rapidly frozen in vitreous (non-crystalline) ice to preserve their native structure without fixation or staining. It is used for high-resolution 3D reconstruction of macromolecules via single-particle reconstruction.

Dark-Field Microscopy

A light microscopy technique where oblique lighting is used so that only light scattered by the specimen enters the objective lens. This makes unstained objects appear bright against a dark background.

Deconvolution

A computational image-processing technique that mathematically removes out-of-focus blur from a stack of images. It uses the microscope's point spread function to calculate and reverse the blurring effect, resulting in sharper optical sections.

Differential-Interference-Contrast (DIC) Microscopy

A light microscopy technique that, like phase-contrast, converts differences in the refractive index of a specimen into visible contrast. It highlights edges where there is a steep change in refractive index, creating a pseudo-3D relief image.

Diffraction Limit

The fundamental limit to the resolution of a conventional light microscope, set by the wavelength of light. It dictates that two objects closer than approximately 0.2 μm (200 nm) cannot be resolved as distinct entities.

Electron Microscope (EM)

A type of microscope that uses a beam of electrons instead of light for illumination. Due to the much shorter wavelength of electrons, it can achieve significantly higher resolution (down to the sub-nanometer scale) than a light microscope.

EM Tomography

A technique for generating a 3D reconstruction of a specimen in an electron microscope. This is achieved by collecting multiple 2D images of the specimen as it is tilted at various angles and then computationally combining these views.

Expansion Microscopy (ExM)

A specimen preparation technique where a fixed sample is embedded in a swellable polymer gel. After proteins are digested, the gel is expanded 4-10 times in water, physically separating fluorescent labels that were previously below the diffraction limit, thereby allowing for higher-resolution imaging with a conventional microscope.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy)

A 3D electron microscopy technique where a focused ion beam mills away sequential thin layers from the surface of a frozen or embedded specimen block. After each layer is removed, an SEM images the newly exposed block face, allowing for the reconstruction of a large volume at high resolution.

Fixation

A process that preserves cells and tissues in a life-like state by using chemicals (e.g., glutaraldehyde) to cross-link proteins and other macromolecules, stabilizing them for subsequent microscopy preparation.

Fluorescence Microscopy

A form of light microscopy that uses fluorescent molecules (fluorochromes) to label specific cellular components. The specimen is illuminated with light at an excitation wavelength, and the image is formed from the longer-wavelength light emitted by the fluorochromes, which appear bright against a dark background.

Fluorescence Recovery After Photobleaching (FRAP)

A technique to measure the mobility and dynamics of fluorescently tagged proteins in living cells. A laser is used to bleach the fluorescence in a small region, and the rate at which unbleached molecules move into that region is monitored over time.

Fluorescent Proteins (e.g., GFP)

Proteins, such as the Green Fluorescent Protein (GFP), that are inherently fluorescent. Their genes can be attached to the gene of a protein of interest, creating a fusion protein that allows its location and dynamics to be tracked in living cells.

FRET (Förster Resonance Energy Transfer)

A technique for monitoring the proximity of two molecules (e.g., proteins) in a living cell. It relies on the transfer of energy from an excited "donor" fluorochrome to a nearby "acceptor" fluorochrome, which will only occur if they are within ~5 nm of each other.

Immunocytochemistry

A method for localizing specific molecules in cells using antibodies. In indirect immunocytochemistry, a primary antibody binds to the target molecule, and its location is visualized by using multiple labeled secondary antibodies that bind to the primary antibody, amplifying the signal.

Immunogold Labeling

An electron microscopy technique analogous to immunofluorescence. Antibodies are coupled to electron-dense colloidal gold particles, which appear as black dots in the EM, allowing for the precise localization of specific proteins.

Light-Sheet Microscopy

A fluorescence microscopy technique for imaging large, thick specimens with minimal photodamage. A thin sheet of laser light illuminates only the focal plane being imaged, and the emitted fluorescence is detected by an objective lens placed perpendicular to the light sheet.

Negative Staining

An EM preparation technique where a heavy-metal salt solution is used to surround and embed isolated macromolecules. The stain creates a dark background, and the molecules, which exclude the stain, appear as bright (negative) images, revealing their shape and subunit structure.

Numerical Aperture (NA)

A measure of a microscope objective's ability to gather light and resolve fine specimen detail at a fixed object distance. Higher NA values result in higher resolution and brighter images.

Phase-Contrast Microscopy

A light microscopy technique that enhances contrast in unstained living cells by converting small phase shifts in the light passing through the specimen into changes in amplitude (brightness).

Resolution The smallest distance between two points that allows them to be distinguished as separate objects. It is the primary measure of a microscope's image clarity.

Scanning Electron Microscope (SEM)

A type of electron microscope that generates a 3D-like image of a specimen's surface. It scans a focused electron beam over a metal-coated specimen and creates an image from the electrons that are scattered or emitted from the surface.

Single-Molecule Localization Microscopy (SMLM)

A class of super-resolution techniques (including PALM and STORM) that bypass the diffraction limit by sequentially activating and imaging sparse subsets of photoswitchable fluorescent molecules. By precisely locating the center of each molecule over thousands of cycles, a high-resolution map is constructed.

STED (Stimulated Emission Depletion) Microscopy

A super-resolution fluorescence technique that achieves resolutions down to ~20 nm. It uses a second, donut-shaped laser beam to switch off fluorescence in the periphery of the excitation spot, leaving only a very small central region to emit light, thereby shrinking the effective point spread function.

Structured Illumination Microscopy (SIM)

A super-resolution fluorescence technique that uses a patterned light grid to illuminate the sample. The resulting interference patterns (moiré patterns) contain high-frequency information that can be computationally reconstructed to create an image with approximately double the resolution of a conventional microscope (~100 nm).

Super-resolution Microscopy

A collection of light microscopy techniques (e.g., SIM, STED, SMLM) that are able to achieve a resolution greater than the diffraction limit of ~200 nm, allowing for the visualization of subcellular structures in much greater detail.

TIRF (Total Internal Reflection Fluorescence) Microscopy

A fluorescence microscopy technique that provides high-contrast images of events at or near the cell surface. It uses an evanescent wave generated by total internal reflection of a laser at the cover slip to excite only the fluorophores within ~200 nm of the surface, eliminating background fluorescence from deeper in the cell.

Transmission Electron Microscope (TEM)

The standard form of electron microscope. It forms an image by passing a high-voltage electron beam through an extremely thin specimen; electron-dense areas scatter more electrons and thus appear darker in the final image, revealing fine internal ultrastructure.