Flashcards Cell Biology (BIO 311): Exam 1

The foundation of Cell Biology

The Light Microscope

Robert Hooke

coined the term "cell" in 1665.

Antony Van Leeuwenhoek

Protozoa, Bacteria (1674)

Schleiden & Schwann

Cell Theory; regarded as the founders of cell biology.

Reticular Theory

Nerve cells were not cells; neurons are a part of a reticulum somewhat like the vascular system.

E.g. Vascular Network - Not cells.

Neuronal Theory

Neurons are really cells.

Resolving Power

Limited by the wavelength of the illuminating source.

Light Microscopes

Light resolution as we can see it is limited to 0.2 μm.

Electron Microscopes

Limit 2.4Å

Two major choices for microscopy in general:

a. Which microscope to select?

b. How to process cells/tissue?

Why aren't light microscopes capable of resolving capabilities similar to electron

microscopes?

All microscopes are considered either "diffraction limited" or "not diffraction limited" with the latter referring to "Super Resolution Microscopy."

Resolution

The capability of seeing 2 separate entities as 2 separate entities rather than a single subject.

Not the same as "enlargement"

Abbe = Abbe's Equation

Theoretical Limit Of Resolution

Dictated by the optics of the microscope, i.e. the best resolution one could have by optimizing everything.

Best possible resolution.

Can be calculated by Abbe's equation.

Practical Limit of Resolution

What you really get; it is what you really can't attain depending on other factors that influence the theoretical limit of resolution.

- Thickness, organic materials that absorb heat, lack of contrast, etc. → DECREASE RESOLUTION

- But, computer enhancement in averaging can INCREASE RESOLUTION

Cells = Poor Candidates

Mostly made of water; low density → very inherent

contrast

Section: in order to look at cells, tissue or cell has to be thick; 10 to 15 μm

Organic compounds: love to absorb radiation; generated heat, and samples will show thermal movement.

Abbe's Equation

A formula that dictates the theoretical limit of resolution for both light and electron microscopes.

d = 0.61λ/nsinθ

1800's Carl Zeiss; Abbe

Super Resolution Microscopy

NOT limited by Abbe's Equation

Dyes - 2 types

Colorimetric and Fluorochromes.

Colorimetric Dye

Absorb different wavelengths and transmit others; such as hematoxylin (nucleus) and eosin (cytoplasm) which are dyes that can be detected using the visible spectrum of light.

Fluorochromes

Fluorescent Dye → have much more utility than colorimetric probes as they can absorb certain wavelengths and emit light.

Contrast is a problem. Solutions are the following:

1. Dyes

2. Manipulating light

3. Computer Image Enhancement

Manipulating Light to fix contrast

Can manipulate the light that is going through the cells so that it generates contrast.

Both the Phase Contrast and Nomarski Optics (DIC) systems generate contrast in living cells through manipulating the light paths as they travel through the specimen. Excellent systems for viewing living cells.

Computer Image Enhancement to fix contrast

Can take the image of dyed cells and make them even better.

This is variable depending on the type of microscope, but it is used for PALM, Deconvolution Microscopy, and Super Resolution Microscopy.

Run through algorithm.

Bright-Field Microscopy

First developed by Carl Zeiss and Abbe; most commonly used one today even though it is the oldest.

Who would use Bright-Field Microscopy?

1. Pathologists

2. Cytochemists (looking for certain aspects of the cell)

3. Histologists/Histochemist → Tissue Sections

Bright-Field Microscopy - Process

1. Fixation → Kill the cells

a. Formaldehyde - function is to cross-link proteins (makes the cell wall fixed)

2. Dehydration → Removal of water

a. Remove H2O → Replace with ethanol (very labor intensive process)

3. Xylene Replacement - Replace ethanol with xylene.

4. Infiltration - replace the xylene with paraffin (liquid candle wax) then let solidify

a. Result is a solid block of tissue infiltrated in wax

5. Tissue is Cut into Sections

6. Microtome - Cuts Sections (10-15 μm)

7. Section is placed onto slide

8. Remove the paraffin using reverse of previous steps 4 → 1

What if you have a mass that needs to be diagnosed in the operating room ASAP?

Cryosections

Cryosections

Made on a cryostat; can cut frozen sections faster but the integrity of the tissue is somewhat compromised. Not as good as wax sections. Freezing substitutes for the paraffin embedding process, e.g. makes the tissue rigid.

a. Used in operating room; fast

Cryosections for MOHs Surgery

The process used by dermatologists to make sure that the margin of a skin cancer melanoma has a "clear margin" not showing cancer cells → used to treat skin cancer.

Phase Contrast Microscopy

Designed to look at living cells without fixation or dyes.

CONTRAST - Doesn't use dyes but instead uses LIGHT

INTERFERENCE

Who would use Phase Contrast Microscopy?

Used by Cell culture biologists who want to examine living cells without fixing or staining them.

Differential Interference Contrast Microscopy (DIC) - Nomarski Optics

Designed to look at living cells - no fixing, no dye.

Yields 3D-like images and can do limited optical sectioning.

Applications: Single cell electrophysiology, patch-clamp

Who would use Differential Interference Contrast Microscopy (DIC)?

Used by cell biologists who want to use living cells and see the outer surface of cells, neurobiologists.

Especially important to neurobiologists for positioning intracellular micropipettes for intracellular injection of transmembrane voltage recording as well as doing patch clamping.

Patch Clamping

Monitors ion flow through single cell membrane channels.

a. Inside-Out

b. Outside-Out

Dark Field Microscopy

Increase in contrast by having a dark field.

Can also visualize small objects in the cell with high contrast such as bacteria, microbes, mitochondria and lysosomes.

Works due to a special condenser.

Illuminates small structures with black background; less than 1 μm.

Who would use Dark Field Microscopy?

Microbiologists

Polarizing Light Microscopy

Using polarizing light to analyze "highly ordered parallel structures"

Detect small, highly ordered parallel structures in cells such as microtubules, actin/myosin.

Has a polarizer just above the light source and analyzer just above the objective lens. Both can rotate to generate contrast.

Polarizer filters light into a single plane and analyzer is used to determine if the intracellular structures rotate the plane of polarized light.

Can be used for analyzing fibrosis (scar tissue due to collagen and other extracellular matrix proteins) in organs such as the liver.

Who would use a Polarizing Light Microscope?

Neurologists and Muscle-Cell biologists

Confocal Microscopy

Revolution; Abbe's Equation

An increase by an enormous amount of the Theoretical Limit of Resolution.

Components of the Confocal Microscope.

1. Lasers - Monochromatic (consisters of the full spectrum, ~ 480 nm)

2. Confocal Pinholes - Narrows the laser beam

3. Point by point scanning of the image - looks anywhere you want within the optical cell (point by point) and takes it and sums it.

4. Computer displays the total summed image.

Advantages to Confocal Microscopy

1. Less stray image → spot by spot

2. Optical Sectioning → Can tell the microscope to take the 2D image ("2 stack") to create a 3D image of the cell.

3. Stereo Images

4. Multiple labeling (triple or quadruple) - using several dyes at once; they all blend to one color.

a. Can label 3 different structures at once.

Problem with Confocal Microscopy

Fluorochromes - Can photobleach

Photobleaching is a problem with fluorescent dyes

2 types of Confocal Microscopes

Point Scanning and Spinning Disk

Point Scanning Confocal Microscope

First generation system

that is slower but generates a bright image.

Problem with the Point Scanning Confocal Microscope

- Can photobleach

- Not good for dynamic interactions due to the time required for imaging.

- Heat is created that can negatively affect the image and practical limit of resolution. But for routine work using fixed specimens this type of confocal microscope is fine.

Spinning Disk Confocal Microscope

Second generation system that an image through several holes is simultaneously within a millisecond - good for dynamic, moving phenomena

Advantages of the Spinning Disk Confocal Microscope

1. Look at living cells

2. Faster

3. Lower laser intensity required

4. Less heat

5. Decrease in photobleaching

6. Can look at dynamic activity

Dynamic activity

Things that happen inside the cell that you wouldn't be able to see with just a confocal microscope.

Major Improvements of Confocal Microscopy over conventional Light Microscopy

- Decreases stray image by 50%

- Can optically section a cell

- Can generate stereo images

- Can use both colorimetric and fluorescent dyes

- Excellent for double or triple labeling where all two or three different colored stains can be distinguished from each other.

Vivascope

Confocal Imaging System - utilizing confocal

microscopy

- Used by dermatologists

- Handheld

- Non-invasive

- Designed for point-of-care

Fluorescence Microscopy

Fluorochromes = Fluorescent Dyes

E.g. Fluorescein

Excitation 𝝀 - 485 nm

Emitting 𝝀 - 530 nm

Vital Fluorescence Microscopy

The use of fluorochromes that can monitor specific cell functions of living cells through changes in their emission profiles.

a. JC-1

b. Calcein-AM/Propidium Iodide

c. FLUO3-AM

Qualitative: Image, microscope

Quantitative: Plate scanner "cytofluor"

Two basic methods for monitoring these dyes in cells:

Microspectrofluorometry and Plate Reading Spectrofluorometers

Mitochondria Activity using JC-1

A vital mitochondrial dye

Red = J-aggregates = HIGH PMF

Green = Monomers = LOW PMF

Calcein-AM and Propidium Iodide

"Live-Dead" assay

Calcein-AM: Can get into the cell; fills the whole

cell green.

Propidium Iodide: Nucleus is red - monitors plasma membrane integrity thus indirectly whether cells are alive (membrane intact) or dead (membrane integrity compromised)

FLUO3-AM

Can measure changes in intracellular calcium.

Microspectrofluorometry

Qualitative assessment of fluorescent probes.

1. Good for single cells or portions of cells.

2. Takes an image and alters it so you can see the changes better.

3. Cells can be genotypically identical but can react to fluorescence differently based on their phenotype.

Plate Reading Spectrofluorometers

quantitative assessment of fluorescent probes.

1. Can average the signals from thousands of cells together.

2. Generates a fluorescence intensity level value (number not an image) → NOT a microscope.

3. First one developed was Cytofluor

FRAP - Fluorescence Recovery After Photobleaching

- Can measure membrane fluidity of a particular protein in living cells in a variety of ways, most often by using a fluorescently tagged cell membrane protein.

- A defined laser beam is localized on a part of the plasma membrane where the fluorescence is photobleached.

- Next one has to redistribute itself to the bleached area (or not) this is used as an indirect method of monitoring mobility of that protein in the membrane.

i. If this is done with all cell membrane proteins, it is found that about half don't move while the other half do move (fill in the space).

TIRF - Total Internal Reflection Fluorescence Microscopy

- Designed to overcome the fact that confocal microscopes cannot easily image activities and structures near the cell surface where it is attached to the substance.

- TIRF is designed for this purpose and works by angling the excitation beam.

- Creates an "evanescent wave" that illuminates only 50 to 100 nm of the surface.

- Can't see inside, but you can see the edges.

Intracellular Injection with Lucifer Yellow

1. Tracing neurons in vivo in the ganglion.

2. Conduct electrical activity between non-neuronal cells.

3. Multiple purposes for this technique

4. Looking at neurons in situ

Ex: Ganglion cells and Epithelial Cells

5. Common dye: Lucifer Yellow injected; doesn't really affect the health of the cell for it to die; goes throughout different parts of the neuron.

6. Load micropipette with a membrane impermeable (lucifer yellow is membrane impermeable) fluorescent dye and inject into the cell.

7. Can show cell morphology or cell to cell connections.

8. Epithelial cells are connected via gap junctions.

Requirements for a Fluorescent Dye for Intracellular injection:

- Vital Dye = Can't and won't kill the cells.

- Fluorescent

- Will diffuse easily; good diffusion characteristics.

- Not lipid soluble (e.g. is contained within the cell)

Fluorescence Immunocytochemistry:

- Technique that uses antibodies to tag and visualize proteins

in cells using fluorochromes.

- Designed to locate specific molecules (proteins) in or on cells using fluorochromes that are associated with antibodies.

- Cell preparation is critical because the native binding characteristics of the antigen need to be preserved.

Fluorochrome

An alternative term for fluorescent dye.

Antigen

Substance capable of eliciting immune response.

Antigenic Determinant

Epitope - specific portion of an antigen molecule to which the antibody is capable of responding/binding.

Antibody

A molecule that can be bivalent (has two binding sites) it recognizes antigens and binds to them in the variable domain region.

Typical Structure of a Bivalent Antibody

Fab = Antigen binding site

Fc = Fluorochrome attached

Specificity

The ability of a single antibody to bind to only one antigen (target protein)

Affinity

Degree to which an antibody binds to a given antigen. High affinity means that it binds tightly (preferred) versus low affinity antibody.

How do you use antibodies to identify a protein of interest?

Direct and Indirect Techniques

Direct Technique

Cell and Protein of interest.

Uses only one antibody; antibody has fluorochrome on it; primary and only antibody used.

Indirect Technique

Uses two antibodies; add antibody that doesn't have fluorochrome attached, add secondary antibody with fluorochrome attached; preferred.

Polyclonal Antibodies

Antibodies produced by injecting animals with a specific antigen. A series of antibodies are produced responding to a variety of different sites on the antigen.

Problems with Polyclonal Antibodies

High chance of cross-reactivity & supply ends with the death of the rabbit.

Monoclonal Antibodies

Antibody generated from one B-cell from cell culture.

Hybridoma cells that produce antibodies can be cryopreserved.

Cell fusion:

a. Two cells to one cell (heterokaryon - still has two nuclei) to one cell (one cell, one nucleus)

One of the most important biologies to treat diseases such as cancer - huge market.

Fluorescence immunocytochemistry can reveal polarity.

Advantages of Monoclonal Antibodies

Much higher specificity & affinity.

Cryopreserve and thaw → immortal (stored forever as cells).

ELISA - Enzyme Linked Immunosorbent Assay

a. Not a microscope technique.

- Basically counts the number of proteins using

antibodies.

- Can quantify a target protein in cells using mAbs.

- Detects antigen concentration in a solution sample.

- Can be direct, indirect or sandwich (unique to ELISA).

- Often used to determine the relative amount of a protein present in a cell under two different conditions:

1. Colorimetric

2. Fluorescent

Apoptosis

Cell death by "suicide" - interal program that tells cells that they must die.

AKA "Programmed Cell Death"

Genetic Cell Death

Ex: Chemo, Radiation, T-cells

Necrosis

Cell death by "murder" - outside influence such as a toxin/poison (ricin) causes the cell to dye.

AKA Pathological Cell death; cells explode.

Annexin V

A stain that binds phosphatidylserine (PS) and detects one of the earliest events in apoptosis-the externalization of PS in living cells

Propidium Iodide

Stains the nucleus because the membrane is ruptured and therefore the dye can access the interiors of the cell.

Membrane IMPERMEABLE..

GFP - Green Fluorescent Protein

- Is reported as a "reporter molecule" - telling you that

something else is happening.

- Robert Tsien; a reporter molecule isolated from jellyfish (2008 Nobel Prize for GFP discovery).

- The GFP family is very colorful; can do double/triple labels with really good resolution.

GFP - Green Fluorescent Protein - How does it work?

i. Have a gene of interest

ii. Tag on GFP (add to gene of interest)

iii. You now have a chimeric gene.

iv. Expresses itself - living cells

FRET (Forster Resonance Energy Transfer)

a. Designed to indicate the proximity of two different

proteins; requires GFP-like molecules.

- Can look at ligand-protein binding.

- Requirement: has to be 1-10 nm close proximity.

- Can be used as "biosensors"

b. Used extensively in the drug discovery business to determine if a synthetic analog of a hormone can bind to its target receptor.

c. Now a "biosensor" molecule can be synthesized that can light up via FRET when it changes shape upon binding with a molecule that you want to measure.

Autoradiography

The use of radioactive probes to analyze a cellular process.

a. Relies on radioactively tagged molecules to follow or track a particular process.

b. Used less due to problems of disposal and safety.

c. Can be used to track DNA synthesis and protein trafficking.

DNA synthesis - 3H-thymidine (a DNA base)

Can be used to detect dividing cells

Protein Trafficking

35S-methionine

3H - Leucine: Amino Acid

1. Labels newly synthesized protein

2. Protein synthesis: starts at RER, then Golgi, then after 30 minutes, exits the cell.

3. Launched "protein trafficking"

Biosensors (FRET)

Protein that reveals a change in cell behavior.

Calmodulin → Changes shape when there is an increase in calcium concentration.

FISH - Fluorescence In Situ Hybridization

a. Fluorochromes

b. Relies on the fact that a complementary probe molecule can be associated with either DNA or mRNA. If DNA is to be probed it has to be destabilized through heat or some other mechanism.

c. Add a probe that now will bind to DNA or RNA due to the complementary nature of the bases.

FISH Application:

Subtyping cervical carcinomas - more than 40 types of human papilloma viruses (HPV) are known to integrate their genome in human cervical cells and cause cancer.

GMK Cells - Negative control (green monkey kidney cells)

HeLa Cells - Positive control (human cervical carcinoma cells isolated

from Henrietta Lacks)

Single Cell Micropipette Intracellular Injection

a. Using micropipettes for injection; requires generating a micropipette through which molecules or a tracer dye is injected.

b. The hole at the tip of this micropipette can be as small as the size of a mitochondria (1 micron).

c. Lucifer Yellow

d. Used for somatic cell nuclear transfer (SCNT)

e. Disadvantage: it is good for one cell at a time.

Electroporation

a. Uses a device that can generate transient holes in membranes and in many cells at the same time.

b. Allows the molecule of interest to passively diffuse into the cell.

c. Injects many cells at once; can also kill cells easily.

Liposomes and Nanoparticles

a. Can carry molecules either through direct attachment (nanoparticles) or encapsulation (liposomes) both must somehow be endocytosed by the cell.

b. Never ever 100% effective - carrier molecules of interest can end up in the lysosomes and be degraded.

c. Uses several cells at once.

d. MOST USED.

Viral Transfection

a. This is the choice of most molecular biologists doing gene transfection.

b. Can be ineffective and while able to ferry genes into the nuclear genome, viral genes can also end up in the nuclear genome.

Electron Microscopy - Two Electron Microscopes

1. Transmission Electron Microscope (TEM)

2. Scanning Electron Microscope (SEM)

Transmission Electron Microscope (TEM)

- "transmits" electrons through the specimen.

- Governed by Abbe's equation and thus is diffraction limited.

- Wavelength of electrons can be varied by changing the accelerating voltage. The faster the electrons, the shorter the wavelength, the higher the resolution.

TEM Techniques: Plastic Thin Section

1. Pick a tissue of interest.

2. Fixation → glutaraldehyde which cross links proteins - (fixes) proteins followed by osmium tetroxide (OsO4) (fixes and cross-links phospholipids).

3. Dehydration → series of steps to remove water.

4. Infiltration → epoxy as the resin; embed in plastic, not wax → need harder resin

for ultrathin sectioning.

5. Mount → Use ultramicrotome to cut and mount sections onto copper screen - float on water.

Interference colors tell you the thickness - gold, silver OK, purple = too thick.

Stain with heavy metal stains because electrons can't detect colorimetric dyes or fluorochromes → TEM is all black and white.

1. Lead - Stains membranes

2. Uranium - Counterstrain (everything else)

TEM Techniques: Freeze-fracture/etch

a. Used to look at the interior of membranes.

b. Cells are FROZEN in cryoprotectant liquid nitrogen, split with a razor blade, and then coated with a thin layer of platinum followed by carbon.

c. This created a Platinum replica → the carbon stabilized the replica but it is not electron dense.

TEM Techniques: Ultrastructural Immunocytochemistry

- Identify proteins of interest in a cell with mAbs.

- No fluorochromes - gold particles used instead.

- Can do a double label experiment with 2 different size gold particles.

TEM Techniques: Ultrastructural Autoradiography

Similar to light microscopy.