BIO 320 - Lab 11 (Immunofluorescence Staining)

What is immunofluorescence staining?

Technique that allows the visualization of a protein of interest or an antigen, or lipid, or DNA in fixed/live cells or tissue sections

Describe the general process of immunofluorescence staining?

Specific fluorescent dye binds to the antigen or a specific antibody covalently conjugated with a fluorescent dye (like FITC); stained samples are examined under a confocal microscope via fluorescence microscopy

What microscope is used for fluorescence microscopy?

Confocal microscope

What is fluorescence?

Fluorescent probe (fluorophore or chromophore) is excited by specific wavelengths of light and the probe jumps to a higher energy level; immediately after activation, excited probe returns to ground state and emits radiation

When does the fluorescent probe emit radiation?

When it jumps DOWN to ground state from an excited state

What microscopic techniques are commonly used to locate fluorescent-probe-conjugated biomolecules in cells?

Conventional microscopy; confocal microscopy (both used for fluorescence microscopy)

What equation is used to calculate resolution power?

Abbe's equation

What is Abbe's equation?

Resolution limit = 0.61 times the wavelength of light divided by the refractive index (numerical aperture)

The higher the resolution limit, the higher or lower the resolving power?

LOWER

The lower the resolution limit, the higher or lower the resolving power?

HIGHER

The shorter the wavelength of light, the better or worse the resolving power?

BETTER (so the lower the resolution limit)

The longer the wavelength of light, the better or worse the resolving power?

WORSE (so the higher the resolution limit)

Units for resolution limit?

nm (nanometers)

The higher the refractive index, the better or worse the resolving power?

BETTER

The lower the refractive index, the better or worse the resolving power?

WORSE

What has the highest refractive index?

OIL (so it leads to a better resolving power)

What has the lowest refractive index?

AIR (so it leads to a worse resolving power)

What is the range for the light source for an exciting fluorescent probe?

300 nm to 600 nm

What are actin filaments? (microfilaments)

Polymers of actin that serve as a key highway system in cells

Actin filaments are known as?

Microfilaments

How are actin monomers associated with one another to form a long chain of actin fiber?

Two strands wrap around each other

Is the actin filament dynamic?

YES; sensitive to changes in chemical and mechanical environment

For immunofluorescence staining, the secondary antibody has?

FLUOROPHORE (NOT alkaline phosphatase like in the previous lab)

Dynamic actin assembly and disassembly is critical for?

Normal development; wound healing; cell migration

What is the toxin that binds to actin filaments, NOT actin monomers?

Phalloidin

Is Phalloidin a fluorophore?

NO

T/F: Phalloidin is a fluorophore.

FALSE

T/F: Phalloidin is not a fluorophore.

TRUE

How is Phalloidin involved in immunofluorescence labeling?

Chemically conjugates with a red fluorescent dye or FITC to make Rhodamine-Phalloidin or FITC-Phalloidin

Which is more harmful: FITC (Fluorescein IsoThioCyanate) or GFP?

FITC

Which is less harmful and thus used more often: FITC (Fluorescein IsoThioCyanate) or GFP?

GFP

Where do scientists isolate GFP/RFP genes from?

Aequorea victoria jellyfish

Which is shorter: excitation (absorption) wavelength or emission/fluorescence wavelength?

EXCITATION (absorption) - so it has more energy

Which is longer: excitation (absorption) wavelength or emission/fluorescence wavelength?

EMISSION/FLUORESCENCE - so it has less energy

The shorter the wavelength, the higher or lower the energy?

HIGHER

T/F: Excitation (absorption) wavelength is longer than emission/fluorescence wavelength.

FALSE

T/F: Excitation (absorption) wavelength is shorter than emission/fluorescence wavelength.

TRUE

What are some common fluorophores/chromophores used?

FITC; GFP; RFP; Rhodamine B; Acti-Stain 555; Texas Red

What fluorophore/chromophore did we use in lab?

Acti-Stain 555

How is monomeric actin made?

Free-floating ribosomes in the cytosol

What are the three different sites of yeast cells where actin molecules are recruited to?

Actin patch; actin cables (in cytoplasm); contractile ring structures (at dividing region)

F-actin (filamentous actin): polymerized or monomerized?

POLYMERIZED

Where are actin patches located?

Plasma membrane

What do actin patches at the plasma membrane play a role in?

Invagination of plasma membrane to form endocytic vesicle

How do actin filaments in actin patches help with endocytic vesicle formation?

Growth of actin filaments is associated with lengthwise growth of the vesicle

What actin binding proteins are recruited to actin patches to secure dynamic activity of actin filaments?

Abp1 (actin binding protein 1); Rvs167

What does Abp1 do? (actin binding protein 1)

STABILIZES actin filaments

What does Rvs167 do? (actin binding protein)

Causes tubulation of membrane with help of actin filaments

Inactivation of Abp1 and Rvs167 results in?

Endocytic defects

How are proteins recruited to the actin patch?

Step-wise protein recruitment; essential for proper maturation of endocytic vesicles

Which is recruited to endocytic vesicles first: Abp1 or Rvs167?

Abp1

Why is Abp1 recruited to endocytic vesicles FIRST before Rvs167?

Stabilization of actin filament (mediated by Abp1) is pivotal for establishing an endocytic scafoold that Rvs167 plays a role in inducing membrane invagination

What are cytoplasmic actin cables?

Bundles of actin filaments formed via an association between actin filaments

What do cytoplasmic actin cables do?

Serve as highway system to transport biological molecules and membrane-bound vesicles

Contractile ring structure is found where?

Dividing region between a mother and daughter cell (bud)

Contractile ring structure in yeast cells is homologous to?

Sarcomere in human muscle cells

How is cell division powered at the contractile ring structure?

Actin filaments and myosin thick filaments sliding past each other

What are actin filaments involved in collectively in yeast cells?

Endocytosis (actin patches); biomolecule transport (actin cables); cell division (contractile ring structure)

What is a sonicator?

Metal probe vibrating at a high frequency

What was formaldehyde used for?

Fixing/killing agent for immunofluorescence technology (or immunohistochemistry methods)

Upon treatment with formaldehyde, what happens to cells?

Metabolism is shut down IMMEDIATELY

Which has a stronger fluorescence: actin cables or actin patches?

ACTIN PATCHES

Which has a weaker fluorescence: actin cables or actin patches?

Actin CABLES - so longer exposures are needed

Why are patches overexposed?

For necessity

What fluorophore-tagged proteins did we use/examine?

RFP-Abp1 and GFP-Rvs167

Immunofluorescence is the labeling of _______ with fluorescent dyes.

Proteins

Immunofluorescence is often used to visualize the subcellular distribution of a __________ of interest.

Protein

Molecules absorbing the energy of electromagnetic radiation will jump to a higher energy level. When certain excited molecules return to their ground state, the _______ radiation. This phenomenon is known as?

EMIT; known as fluorescence

Fluorescent molecules are known as?

Probes?

Texas red is a fluorescent dye used in fluorescence microscopy applications. It fluoresces at a wavelength longer or shorter than its absorption wavelength?

LONGER

T/F: Fluorescent dyes absorb light at one wavelength and emit at another, longer wavelength.

TRUE

Which is lower in energy: emission state or radiation state?

EMISSION - has a longer wavelength

What do pinholes do in confocal microscopy?

Uses a pinhole to block diffuse (out-of-focus) light so you get the highest resolution out of your sample possible

Which is more powerful/better at generating high-resolution images: confocal or conventional microscopy?

Confocal

A confocal microscope differs from a typical fluorescence microscope in that?

A. Confocal microscopy scatters light off of the surface of the specimen giving striking 3-D images of the object's surface.

B. Confocal microscopy requires the sample be stained with fluorescent dyes.

C. Confocal microscopy is used to focus at different depths within the specimen, allowing a 3-D fluorescent image to be constructed.

D. B and C

E. All of the above

E?

Confocal microscopy is an optical imaging technique used to increase the optical resolution and contrast of a micrograph by using a spatial _______ to eliminate out-of-focus light in the specimen.

PINHOLE?

T/F: Microscope resolving power increases as the wavelength of radiation used to illuminate the specimen decreases.

TRUE

Monochromatic (one color) light is sometimes used to increase the resolution of the light microscope. Which color of light would you expect to give you the best resolution?

A. Red

B. Orange

C. Green

D. Blue

BLUE (has the shortest wavelength)

What two factors determine the resolving power of a light microscope? (?)

Wavelength of light; refractive index

Which of the following objectives would give you the best resolution of small objects?

A. 100x air, refractive index of 0.25

B. 100x water, refractive index of 0.65

C. 100x oil, refractive index of 1.4

D. 100x oil, refractive index of 1.25

C

Refractive index of air?

0.25

Refractive index of water?

0.65

Refractive index of oil?

1.4

The lens nearest the object to be viewed is what lens?

Objective

What does formaldehyde do in our immunofluorescence lab experiment?

Fixing/killing agent that shuts down a cell's metabolism

In order to stain actin filaments, one can use the dye called rhodamine-conjugated phalloidin. What does "rhodamine-conjugated" means?

Rhodamine (fluorphore) is chemically conjugated with phalloidin, which binds to actin polymers b/c phalloidin is NOT the fluorophore.

T/F: Phalloidin binds specifically to monomeric actin molecules.

FALSE

The size of a molecule is on the order of __________, and the size of an average cell is on the order of _________.

?

Total magnification of a 40X objective lens and 10X ocular lens?

400X

T/F: Yeast amphiphysin Rvs167 is recruited to cortical patch earlier than Abp1 is.

FALSE

Living, unstained cells and microorganisms can be observed best using?

Fluorescence microscopy with a confocal microscope.

Each actin filament is composed of?

Two strands of actin molecules wrapped together