Week 2: Methods in Cell Biology

Flashcards covering key terms and definitions from the 'Methods in Cell Biology' lecture, including types of research papers, microscopy techniques, and protein analysis methods.

Fluorescence

The emission of light by a substance that has absorbed light or other electromagnetic radiation, where the emitted light has a longer wavelength than the absorbed light.

Green Fluorescent Protein (GFP)

A protein originally isolated from the jellyfish Aequorea Victoria, widely used in cell biology as a marker to visualize proteins and cellular structures due to its ability to fluoresce green.

Chromophore

The part of a molecule responsible for its color and ability to absorb and emit light, for example, within GFP.

Fluorescence Microscopy

A technique where cells are labeled with a fluorescent molecule and visualized using a light microscope, which illuminates the sample with excitation light and visualizes the emitted light.

Excitation Light

The specific wavelength of light absorbed by a fluorescent molecule in fluorescence microscopy.

Emission Light

The specific wavelength of light emitted by a fluorescent molecule after absorbing excitation light, which is then visualized in fluorescence microscopy.

Phototoxicity

Damage to cells caused by exposure to bright light, a consideration in fluorescence microscopy.

Photobleaching

The irreversible photochemical destruction of fluorophores due to prolonged or intense exposure to excitation light, causing them to stop fluorescing.

Electron Microscopy (EM)

A microscopy technique that uses a beam of electrons to image a sample, allowing for much higher resolution than light microscopy.

Transmission EM (TEM)

An electron microscopy technique where electrons pass through a thinly cut sample, providing detailed internal structure.

Scanning EM (SEM)

An electron microscopy technique where the surface of a sample, coated with a conductive material, is scanned with electrons to create a 3D-like image of its surface.

Cryo EM

An electron microscopy technique where samples are supercooled and imaged with electrons, preserving their native structure without chemical fixation or staining.

Genome Engineering (for tagged proteins)

A method of introducing fluorescently tagged proteins by modifying the cell's chromosomal DNA, preserving the native gene environment but often being more complex and slower.

Plasmid Expression (for tagged proteins)

A method of introducing fluorescently tagged proteins using extrachromosomal DNA (plasmids), which is relatively fast but can lead to variable expression levels and difficulty matching native protein levels.

Protein Domains

Parts of a protein that are often compact, somewhat autonomous, and can have distinct functions and evolutionary histories, contributing to the overall protein structure and activity.

Immunofluorescence

A microscopy technique that uses antibodies to specifically label and visualize specific proteins within cells or tissues, often distinguishing them from other structures.

Antibody

Y-shaped proteins produced by immune cells that specifically recognize and bind to unique epitopes on antigens, used as experimental tools to detect specific molecules.

Primary Antibody

An antibody used in immunofluorescence that directly binds to the target antigen of interest.

Secondary Antibody

An antibody used in immunofluorescence that binds to the primary antibody and is usually conjugated with a fluorophore for visualization.

Cell Fixation

The process of adding a cross-linking agent (like formaldehyde) to cells to preserve their structure and stick cellular components together before immunofluorescence imaging.

Immunoprecipitation (IP)

A technique used to isolate a specific protein (antigen) and its interacting partners from a cell lysate by using an antibody that binds to the target protein, followed by precipitation.

Western Blotting

A laboratory technique used to detect specific proteins in a sample by separating them by size via gel electrophoresis, transferring them to a membrane, and then visualizing them using specific antibodies.

Mass Spectrometry (MS)

An analytical technique that measures the mass-to-charge ratio of ions, used to identify substances, determine their molecular weight, and analyze molecular structure, often applied to protein identification.

Tandem Mass Spectrometry (MS/MS)

A type of mass spectrometry that involves multiple steps of mass analysis, typically used to fragment ions and analyze their resulting product ions to gain more detailed structural information, especially for peptides and proteins.

Light Sheet Fluorescence Microscopy (LSFM)

A microscopy technique that uses a thin sheet of light to illuminate only the focal plane of the sample, rather than the entire volume.

Advantages of LSFM

Reduces photodamage and photobleaching; excels at fast, gentle 3D imaging of thick, transparent samples; preserves sample viability.

Disadvantages of LSFM

Technically demanding to implement and align; requires specialized sample preparation and large data storage; may have lower lateral resolution.

Epifluorescence Microscopy (Epi/WF)

A microscopy technique where both the excitation light and emitted fluorescence travel through the same objective lens, illuminating the entire sample at once.

Advantages of Epifluorescence Microscopy

Produces a wider spectrum of wavelengths to detect fainter fluorescence; simplistic and highly efficient; offers a wider field of view; better for high-speed live-cell imaging.

Disadvantages of Epifluorescence Microscopy

High background 'noise' in larger specimens; cannot isolate information from a single plane, leading to poor 3D optical sectioning; limited resolution ($200$ nm); high phototoxicity and photobleaching for live cells due to whole-field illumination.

Confocal Microscopy (CF)

A microscopy technique that uses a pinhole to eliminate out-of-focus light, allowing for high-resolution optical sectioning of fluorescently labeled samples.

Advantages of Confocal Microscopy

Improved resolution; 3D optical sectioning; higher image quality by filtering out out-of-focus light; control over depth of field; versatile for living and fixed cells.

Disadvantages of Confocal Microscopy

Slower imaging speeds; higher cost; potential phototoxicity from lasers; photobleaching of fluorophores; limited excitation wavelengths; requires specialized training; limited depth penetration in deep tissues.