BIO 311 Exam I: Microscopy

Basic History of Microscopes

- 1655: Robert Hooke used a microscope to see what he called "cells" named after the houses of monks

- 1674: Anthony Van L. found protozoa and bacteria

- 1838: Cell theory was developed

Neuron Cell Theories

- Reticular: Neurons exist in a vascular network and thus are not cells

- Neuronal: Neurons are in fact cells

Approximate sizes

- 100 micrometers: Plant cell that can be seen with the eye

- 10 micrometers: Animal cells and require microscope

- 1 micrometer: Mitochondria/Bacteria require microscope

- 100 nanometers: viruses and ribosomes can't be seen with LM

- 10 nanometers: proteins can't be seen with LM

- 1 nanometer: individual molecules can't be seen with LM

Resolution Limits

- The smallest possible distance that two dots can be seen as two seperate dots

- LM -> .2 micrometers

- Electron Microscope -> 2.4 angstroms

Abbe

- Created an equation that could calculate the theoretical limit of any given microscope

- Abbes equation: distance between two objects = .5 x wavelength

Practical Limit of Resolution

- Cells are bad candidate for microscopes since they are mostly water and thus lack contrast

- Organic compunds in cells absorb light, heat up easily, and move around

Super Resolution Microscopy

The one type of microscopy that doesn't obey abbe's equation

How to get contrast

- Use dyes: Fluorochromes or colorimetric

- Manipulate Light: Phase microscope or Differential Interference Contrast Microscopy

- Computer Enchancment

Bright Field Microscopy

- Oldest type of LM

- Does not Use live Cells

- Fixes cells to be observed in lifelike state

BF Microscopy Steps of Fixing cells

1) Fixation- kills cells with particular chemicals (Formaldehyde)

2) Dehydration- removal of water and replace with ethanol

3) Xyleme Replacement- replace ethanol with xylene

4) Infiltration- replace xylene with parafin (wax)

5) Microtone- Cut the sample in 10-15 micrometer slices

Very labor heavy

Alternative method for BF microscopy

Cryosections: Quickly frozen skin samples are then cut into slices

Phase Microscopy

- Makes use of living cells without fixation or dyes

- Uses light interference for contrast

Differential Interface Contrast (DIC) Microscopy or Nomarski Optics

- Used with living cels

- Creates a 3D image unlike phase microscopy

DIC/Nomarski Application

Single cell electrophysiology: Using a pipette you can measure the electric potential of the cell and monitor the flow of ions through the membrane mechanism

Darkfield Microscopy

Bright image on dark field

Polarizing Light Microscopy

- Great for specimens with highly ordered structures

- Polarized light shines upward on cell that contains moving liquid (blood vessel or other tubing)

Confocal microscopy

- Revolutionary change in microscopes

- Governed by abbe's law still

- Increases theoretical limit of resolution

Confocal microscopy components

- Monochromatic lasers

- Confocal pinholes that narrow the laser

- Point by point image scaning

- Computer to display the complied data from scan

Confocal microscopy advantages

- Less stray marks on the image

- Allows to create opitcal sections of a image

- Can use multiple dyes at once

Confocal microscopy disadvantages

- Uses flurochromes which are subject to photobleaching

Two types of Confocal microscopy

- Point scanning (previously mentioned)

- Spinning disk

Spining Disk Confocal microscopy

- Advantages: Good for living cells since it captures faster, lower laser intensity meaning less heat

Vivascope

- A type of confocal imaging system

- Purpose: Used by dermatologists for handheld skin diagnoses

- Optically sections through the skin

- Non invasive

Fluorochromes

Dyes that emit light when excited

Vital Fluorescence Microscopy

- Uses flurochromes to measure changes in cell behavior

- Dye and AM group are attached and brought into a cell. The cell separates the AM and Dye causing the dye to fluoresce

Vital Fluorescence Microscopy Applications

Can track cell behaviors:

Mitochondria activity with JC-1

- Green=monomers=low PMF=dying

- Red=J aggregate=high PMF=healthy

Alive/Dead Assay with Calcein-AM and Propidium Iodide

- Calcein: Appears green and indicates alive cell

- If a cell is dying Calcein leaves and propidium enters cell and stains the nucleus red

Calcium Concentration with Fluo3-AM

- Level of fluorescence indicates Ca concentration

Microspectrofluorometry

- Allows for a quantitative measure of fluoresnce stength

- Intensity appears as different colors

Plate reading spectrofluorometers

- Machines that can sum the fluorescent strength of a signal from a large group of cells and average out the emitted wavelength from the fluorochromes

- Gives an estimate quanitiy of fluor. strenght

Fluorescence Recovery After Photobleaching (FRAP)

- Uses photobleaching as a tool to examine the lateral fluidity of proteins

Steps:

1) A laser is targeted on one spot of the cell causing it to fluoresce

2) that spot will then become photobleached

3) If the protein in that spot is mobile then the will be filled in by other material and the photo bleaching will go away

Intracellular Injections w/ Lucifer Yellow

- Used to trace neurons in vivo

- Can indicate electrical connectivity between non neural cells too

- Must use some type of vital, fluorescent dye that is membrane insoluble and easy to diffuse

Fluorescence Immunocytochemistry

- Use of antibodies to indentfy proteins in cells

- Antigen = Protein of interest

- Epitope = Area of antigen the antibody binds to

- Antibody

Antibody Specificty vs Affinity

- Specificity: The antibody only binds to the intended protein

- Affinity: The strength of binding to the protein

Direct vs Indirect

- Direct: Antibody has a fluoro attached and lights up cell when in contact with protein

- indirect: antibody is attached to another antibody containing fluoro

- Indirect is better becase direct often sacrifices specificity

Polyclonal Antibodies

Steps:

1) Inject an antigen into a living animal

2) the creatures b-cells will make polyclonal antibodies

3) Get serum from animal containing antibodies

Monoclonal Antibodies

Steps:

1) Do the aforementioned injection and collect B cells

2) Culture the b cells and make them immortal by fusing them with myeloma cancer cell

3) The combination is a heterokayron cell that has two nuclei

4) The two nuclei fuse

5) The resulting cell can infinetly produce mAbs that are all identical

Mono vs Polyclonal

Mono is way better because it makes antibodies over and over again allowing you to pick the best once and doesn't require a living speciemen

Enzymed Linked Immunosorbent Assay (ELISA)

- Used to quantify the amount of POI

Steps: (Ex. will a drug inc protein content)

1) Have a control and experimental group

2) Isolate POI

Necrosis

Pathologic cell death

Apoptosis

- Programmed cell death

- Annexin V tages apoptic cells and results in death

GFP (green fluorescent protein)

- Originates from jellyfish

- Reporter molecule meaning that it indicates the something else occuring

- Example steps: protein expression

1) Isolate the gene of interest

2) Take the GFP sequence and turn it into a chimera gene by combining it with the GOI

3) Transfect the chimera DNA into a living cell

4) If GFP is expressed it means that GOI is also expressed

Regulated Reporting

Seeing if a gene is getting expressed (on/off)

Constituent Reporting

Always on

FRET (Forster Resonance Energy Transfer)

- Pair of fluorescent proteins with similar wavelengths where the energy is transferred

- Ability to indicate ligand-receptor in living cells

- Uses proteins in the GFP family

- Can also be used to identify phosphorylation

Autoradiography

- Ability to trace a molecule or process in a cell with a radioactive probe

Ex: Cell Division with 3H-Thymidine

1) Probe enters cells

2) Film is layered over cells

3) Spots appear on film when hit with radiation indicating s-phase occuring

FISH (Fluorescence in Situ Hybridization)

Used for detecting mRNA or Gene Sequences

Steps for mRNA detection:

1) Indentify mRNA of interest

2) Create a complimentary RNA probe with a fluorochrome

3) Binding to the RNA emits light

Steps for indentifying DNA sequence

1) Same steps but DNA must be denatured first because it is tighter

Intracellular Injection

- Single cell micropipette injection

- Electroporation

- Liposomes and nanoparticles

Single Cell micropipette injection

- Somatic cell nuclear transfer (swapping nuclei)

- Can only do one cell at a time

Electroporation

- Injects many cells at once

- Electric plates make a charge difference

- Molecules diffuse into the cells and is turned off

-Kills many cells so they cells used must be tough

Liposomes and Nanoparticles

- Several cells at once

- Never 100% effective

- Liposome: small body made of lipids containing desired molecule

- Liposome binds to the cell membrane and admits molecul and then the cell chooses to accept it

Viral transfection

Using viruses to transport genes (not fda approved)

Electronmicroscopy

-Trasmission EM

-Scanning EM

TEM

-Adheres to abbe's eq

- faster electron stream yields better resolution

- electrons travel through the specimen

- Imaging on screen

- 50-90 nanometers thin

SEM

- Scans the image from the outside

TEM Techniques

Plastic thin Sectioning

Freeze Fracture

Ultrastructural Immunocytochemistry

Plastic thin Sectioning

1) Select tissue of interest

2) Fixation

3) Dehydration

4) Infiltration of epoxy plastic

5) Ultra-thin sectioning with a ultra microtone

6) Staining with heavy metals that reflect electrons (lead as primary or uranium as negative stain)

Freeze Fracture

1) Cells are frozen then fractured with a razor blade

2) Spray with platinum and carbon to stable it

Ultrastructural Immunocytochemistry

1) uses gold particles indirectly attached to mABs

2) Allows for double labeling with different gold sizing

Laser Capture Microdissection Microscope

- Takes sections of a single cell

Atomic Force Microscope

- No lens

- A laser puts pressure on a node that outlines a sample

Two-Photon Microscope

- Thicker and deeper tissue imaging

- Uses light more efficiently leading to less photo toxicity