OAT Biol 8: Microscopy

What is microscopy?

• What is the point?

• Microscopy aims to magnify the image of a given specimen

• Preparative techniques are used to ease viewing and interpretation of the specimen

Fixation

• Meaning

• What is the point of that

Fixation: “Sticking” or “securing” cells to a slide, preserving them in as close to a lifelike state as possible

Staining

• Def

• What is the point of this

• What does it do the specimen

• Application of stains or dyes to a specimen to add color and contrast

  • Different structures may become more easily distinguished

  • Staining often kills the specimen

Optical Microscopy Def

Cells are viewed directly

• Thin specimen are illuminated with a light source, while the lenses magnify the resulting image

• Living cells CAN be viewed using this method

Types of Optical Microscopes (3 types)

Stereo Microscope

Compound Microscope

Bright Field Microscope

Stereo Microscope

Def

Low magnification is used to view the surface of a specimen

• 3d

Compound Microscope

• Positives

• Negatives

• DEF

Multiple lenses are used to provide variable, adjustable magnification

• Viewing of simple, one-cell thick samples requiring fixation and staining

• Poor image contrast

Bright Field Microscope

Compound microscope with a bright light

Phase Contrast Micrscopes

View thin samples of live, unstained cells

• High image contrast

• Type of compound microscope

• Does NOT require fixation

Fluorescent Microscope

View live cells that are tagged with fluorophores

• Cellular components are visible with this method

Fluorophores

Fluorophores: Fluorescent chemical markers used to “tag” target structures

Immunoflorescence Microscopy

• Fluorophores are used to identify the location of target proteins

FRAP (fluorescence recovery after photobleaching)

• Quantitatively measures biomolecule movement in a live cell

Protocol:

• Baseline fluorescence is measured, and an area of the sample is photobleached

• Over time, photobleached molecules are replaced by unbleached molecules via diffusion

• Gradually, the area will recover fluorescence based on cell dynamic

Electron Microscopy

• How it works

• Mortality

• What do you need to treat the specimen with

Cells are viewed indirectly

• Electrons are fired at the sample

• The electrons bounce off of the sample, causing them to pass through a magnetic field

• Smaller objects can be viewed, however, specimen must be stained with a metal coating and killed

SEM (Scanning Electron Microscope)

• Def

• Type of sample

(SEM): High resolution 3D images of the surface of a dehydrated sample

Transmission electron microscope (TEM):

• Def

• Type of sample

High resolution 2D images of the internal structures of a sample

Bacterial Growth Curve

• Def

• What does the it look like

• What are the phases of it

A graphic depiction of the phasic growth and death of bacteria from culturing

What are the four phases of the Bacterial Growth curve?

• Lag

• Exponential (log)

• Stationary Phase

• Death Phase

Bacterial Growth curve: Lag Phase

Lag phase: Bacterial proliferation is stagnant as cells initially adapt to new environment (culture medium)

• Growth rate = death rate

Bacterial Growth curve: Exponential (log) Phase

A period of exponential cell growth

• Growth rate > death rate

Bacterial Growth curve: Stationary Phase

A second period of stagnant proliferation

• Growth rate = death rate

• Total population is much higher than in lag phase

Bacterial Growth curve: Death Phase

Rapid decline in bacterial population size

• Death rate > growth rate

• Caused by lack of resources after log and stationary phases

Differential Centrifugation

• What compenents are created?

• What is the point of it?

• What does it seperate?

• What does it allow for furthermore?

• Separating solutes in a given solvent based on size and density

• Test tubes filled are subjected to several rounds of centrifugation, with each round increasing centrifugal forces

• Each step selectively pellets components within a specific size or density range

• Pellet: The components which have sedimented to the bottom of the tube

• Allows for the fractionation of cell organelles and macromolecules

List these from dense to least dense:

• Er and Golgi Fragments

• Mitochondria, Chloroplasts and Lysosomes

• Nucleus

• Ribosomes and large macromolecules.

From MOST dense to LEAST dense (correct order)

1. Nucleus

2. Mitochondria, chloroplasts, and lysosomes

3. ER and Golgi fragments

4. Ribosomes and large macromolecules

Karyotyping

• Def

• Uses

• What phase do we undergo this technique?

• Observing chromosomes under a microscope to identify potential chromosomal abnormalities

Ex: Trisomy 21 / Down syndrome

• Observation occurs during metaphase

CRISPR (if u dont know this i would be a little upset)

• Uses

• Def

• What does it do specifically?

● Technology allowing the editing of specific genomic regions

● Target sequences may be inserted or deleted

● Uses: Gene therapy

DNA fingerprinting

• Uses

• What does it do?

• What innate technology does it use?

• An identification technique which relies on the inherent uniqueness of each human genome

  • Specific regions of noncoding DNA are fragmented with restriction enzymes and analyzed

  • Uses: Paternity testing and forensic identification

DNA Sequencing

• Def

• Example of DNA Sequencing

• Determining the order of nucleotides in a given DNA sequence

  • EX: Sanger Sequencing

Sanger sequencing:

• Sanger sequencing: Uses PCR to make DNA copies out of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs)

• ddNTPs lack the 3’ OH necessary to create phosphodiester bonds, ending elongation once they are inserted

• Using both dNTPs and ddNTPs allows for variety in fragment lengths, which enables the sequence to be determined

PCR (polymerase chain reaction)

• 3 steps

• def

• An automated process that allows for the creation of 1+ billion of copies of a DNA fragment

3 Steps:

• Denaturation

• Annealing

• Elongation

PCR: Denaturation

• What is the temp

• Denaturation (94-95° C): Intense heating separates the DNA double strands into single strands

PCR: Annealing

• Temp

• Def

Primer annealing (55° C): The sequences are cooled, allowing DNA primers

to hybridize with the single strands

PCR: Elongation

• Elongation (72° C): Moderate temperature elevation encourages Taq polymerase activity, adding nucleotides to the 3’ ends of the strands

Bacterial Cloning

• General Def

(eukaryotic gene product cloned using prokaryotic cells)

Process of Bacterial Cloning

• Eukaryotic gene products are cloned using prokaryotic cells

Process:

• Processed eukaryotic mRNA is converted to cDNA (copy DNA) using reverse transcriptases

• cDNA is incorporated into a plasmid using restriction enzymes and DNA ligase

• Plasmid vector is taken up by competent bacterial cells via transformation

• Process continued:

• Gene of interest is located using antibiotic resistance (antibiotic resistance gene is attached to target gene) and color change methods

Uses: Medicine production

Gene Therapy

• Def

• How does it work

• Types of it

• What does it treat?

• Medical treatment where target genes are inserted into patient cells

• Viruses are the preferred vector

• High transduction rate (most efficient)

• Potential to trigger an immune response

• Non-viral vectors do not cause immune responses but are less efficient

Examples include CRISPR and naked plasmid DNA

Uses: Treatment of diseases with clear genetic involvement

Enzyme Linked Immunosorbent Assay (ELISA)

• Purpose

• Def

• What is it used for

• Determines if a person possesses a specific antigen

• Antibodies are placed on a microtiter plate with a sample from the individual

If the individual possesses the antigens of interest a color change will occur

Uses: Disease diagnosis (HIV)

Pulse Chase Experiments:

● Observing protein movement through a cell

● Pulse phase: Amino acids are radioactively labeled and then incorporated into proteins

● Chase phase: Prevention of radioactively labeled protein production

● Radioactive proteins are tracked using simple staining

● Uses: Study gene expression and protein fates (synthesis, movement, degradation) within a cell

Pulse Phase (Pulse Chase Experiment)

● Pulse phase: Amino acids are radioactively labeled and then incorporated into proteins

Chase Phase (Pulse Chase Experiment)

● Chase phase: Prevention of radioactively labeled protein production

Gel Electrophoresis (another obvious one)

• Use

• How does it undergo it’s use mechanically

• Charges top and bottom

• What travels the furthest

● A technique to separate DNA fragments or proteins by size and relative charge

● Samples are placed into wells within an agarose gel

● The gel is situated within an electric field

Top: negative cathode

Bottom: positive anode

Smaller and more negatively charged fragments travel further from the top of the gel

Uses: Genotyping, fingerprinting, diagnostic testing

SDS Page

• Used in protein gel electrophoresis

• Sodium dodecyl sulfate (SDS): A strong detergent which denatures proteins and gives them a negative charge

• Denatured proteins are placed into polyacrylamide gel wells

• Protein fragments are separated out based on density and size for analysis

• Uses: Assessing protein size and purity

Exonucleases and Endonucleases

• Def

• Nucleotide cleaving enzymes differing in their target location

Exonucleases

• Where does it excise

• What does that produce then?

● Exonucleases: Cleave nucleotides from the ends of a polynucleotide chain

• Can only produce sticky ends

Endonuclease:

Cleave nucleotides from the inside of a polynucleotide chain

• Can produce sticky ends or blunt ends

Restriction Enzymes

• Where do they affect the subject

• What is the subjet

• Restriction enzymes: Special endonucleases that mostly cut DNA at palindromic sequences

Palindromic Sequences

Palindromic sequences: Short regions where the sequence reads the same in the 5’-3’ on both strands

Recombinant DNA

DNA produced when DNA fragments from different sources are joined together at blunt and/or sticky ends

Blunt ends

Blunt ends: DNA fragments without any unpaired nucleotides

Sticky Ends

DNA fragments with single-chain overhanging segments (unpaired nucleotides)

Southern Blotting

• A technique used to identify fragments of a known DNA sequence within a large population of DNA

• Electrophoresed DNA is separated into single strands

• The strands are then identified via complementary DNA probes

Northern Blotting

• Identify known fragments of RNA using an RNA probe

• Conducted in a similar fashion to the Southern Blot but using RNA

Western Blotting

• Quantifies the amount of a target protein in a sample

• Uses SDS-PAGE

• Proteins are treated with primary antibodies, which bind to the target protein, and secondary antibodies, which bind to both an indicator and the primary antibody

TYPES OF BLOTTING

SNOW DROP

Genomics

• The study of all genes present in an organism’s genome and how they interacts

Gene Annotation

• Gene annotation: The process of identifying the location of genes and coding regions within a genome and determining each of their functions

• Requires a genomic library and DNA microarray

Genomic Library

● Store the DNA of an organism’s genome

● DNA fragments are incorporated into plasmids

● These fragments can be screened for using antibiotic resistance and color changing techniques

● They can they be cloned by bacterial cloning

DNA Microarrays

• Def

• Process

• Contain thousands of DNA probes

• Bind to complementary DNA fragments, allowing researchers to see which genes are expressed

Protocol:

• Isolate a cell and remove the mRNA before exposing it to reverse transcriptase in order to produce cDNA

• Hybridize the cDNA with DNA probes and examine the microarrays for fluorescence. The microarray can now be compared with the sequenced genome

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