Chapter 8-9 | Analyzing Cells, Molecules, and Systems; Visualizing Cells and Their Molecules

Cell culture

Taking cells from a living organism and growing them in a lab

Solid medium

e.g. pGLO plates

Liquid culture

Cell suspension growing in a liquid medium

Primary cell culture vs secondary cell culture

Made directly from the living tissue of an organism; made from an already existing culture

Established cel lines

Mostly derived from multicellular vertebrates and used for medicinal purposes. They raise in vivo vs in vitro ethical considerations because they are considered living tissue

Ethical/ownership question of human cell lines

e.g. HeLa cells from Henrietta Lacks; who do human cell lines belong to?

Cell senescence

Cells eventually cease division

Immortal cell lines

Tumor lines & stem cells

Creating immortal cell lines

Transforming cells with genes that induce continual cell division

Forming hybrid cells by fusing them with an existing tumor line, such as a hybridoma used to synthesize antibodies

Key parameters of microscopy

1. Magnification

2. Contrast

3. Resolution

Resolution

The ability to distinguish details

Resolution limits

• Naked eye: 0.2 mm

• Light microscope: 0.3 um

• Super-resolution fluorescence microscope: 20 nm

• Electron microscope: 0.2 nm

Types of light microscopy

1. Brightfield

2. Dark field

3. Phase contrast

4. Differential interference contrast (DIC)

Physical requirement for specimens in light microscopy

Specimens must be thin enough for light to move through (up to ~50 um thick, about one cell layer) Multicellular tissues require thin sections (Histology)

Preparing multicellular tissue for histology

1. Fix specimen to preserve structures

2. Embed in a matrix

3. Slice and produce thin sections

Light traveling through a light microscope

Light moves through a condenser lens and then up to the objective lens

Brightfield microscopy

Thin sections of tissue are usually stained to add contrast; commonly used in medicine

Dark Field microscopy

Light is shone at an oblique so that direct light does not reach the objective lens (creating a dark background) Light hitting the specimen is scattered and reaches the objective so only the specimen is lit

Phase Contrast/DIC microscopy

White light hits the specimen and wavelengths move out of phase based on the depth of the specimen, making depth visible to the eye. DIC specifically compares out of phase wavelengths with a reference wavelength that is in phase

Fluorophore

A fluorescence marker/dye used to visualize individual components of a cell. It undergoes excitation when hit with a specific wavelength of light, absorbs energy, and emits energy at a lower level (generating a higher wavelength) A filter blocks unwanted wavelengths not emitted

Fluorescent in Situ Hybridization (FISH)

A technique where a fluorophore attached to a specific cell component, such as DNA or RNA, to visualize it

Preparing DNA for FISH

• Denature DNA into single strand

• Form a label that is complementary in sequence

• Attach a fluorophore directly to the DNA, or attach an antigen to the DNA that is then recognized by a fluorophore-bound antibody/protein

Immunofluorescence

A technique using antibodies tagged with a fluorescent dye to visualize specific target molecules within a sample

Antibody

A Y-shaped protein made by B-cells that has two identical antigen-binding sites

Epitope

The specific part/region of an antigen to which an antibody binds. An antigen can have multiple epitopes

Polyclonal Antibody

A collection of different antibodies that bind to different epitopes on the same antigen

1. Inject an animal (e.g., rabbit) with an antigen.

2. Collect blood after an immune response occurs.

3. Separate and isolate the mixture of antibodies that bind to the target antigen.

Monoclonal Antibody

A identical set of antibodies produced by a single clone of cells that binds to one specific epitope on an antigen

1. Inject an animal (e.g., mouse) with an antigen

2. Harvest immune (B) cells

3. Fuse B-cells with myeloma cells to form hybridomas

4. Propagate desired clones to produce highly specific, consistent antibodies

Immunofluorescence staining

1. Fixation: Immobilize/kill the cell

2. Primary Antibody: Add an antibody engineered to bind specifically to the target antigen

3. Secondary Antibody: Add a fluorophore-conjugated antibody that binds to the primary antibody (species-specific, e.g., anti-mouse)

Gene tagging with a fluorophore

Fuses a gene of interest with a fluorophore gene (e.g., GFP) and transforms it into the organism. Protein expresses tagged in live cells, allowing real-time in vivo tracking

FRET (Förster Resonance Energy Transfer)

Detecting protein-protein interactions. When two labeled proteins interact in close proximity, energy transfers (resonance) between their fluorophores

FRAP (Fluorescence Recovery After Photobleaching)

Measuring the mobility/movement of molecules across a membrane or within a cell

1. Bleach a specific region of a fluorescently labeled cell using high-intensity UV light

2. Monitor the time it takes for unbleached fluorescent molecules to diffuse back into the area (recovery)

Deconvolution Microscopy

A computational technique where software uses mathematical algorithms to remove out-of-focus blur/light, sharpening the final image at the plane of focus

Confocal Microscopy

A technique that uses a pinhole to eliminate out-of-focus light and scans a focal plane to take precise optical sections/images of a specimen

Electron microscopy

Uses electrons to produce a wavelength of 0.004 nm

Transmission Electron Microscopy (TEM)

1. Fix specimen and stain with electron-dense material

2. Make ultrathin sections

3. Bombard specimen with electrons (electrons scatter on the specimen)

4. Detect electrons to produce a high-resolution image

Immunogold labeling in TEM immunocytochemistry

A technique where antibodies conjugated to gold particles are applied to cells so specific molecules can be visualized under TEM

Cryo-Electron Microscopy

It fixes specimens by rapid freezing rather than harsh chemicals, maintaining the sample's natural structure. The entire process is conducted under freezing conditions

Scanning Electron Microscopy (SEM)

To visualize the 3D surface of a specimen (no thin sectioning required)