Fluorescence Microscopy Techniques and Applications

Co-localization Analysis

• Co-localization suggests coordinated function or regulation, but doesn't confirm direct molecular interaction.

• Requires additional techniques to confirm physical interactions.

• Co-localization analysis involves observing the overlap of signals from different channels (e.g., green and red).

• Limited resolution of light microscopes prevents absolute certainty about protein interactions.

Methods for Identifying Co-localization

1. Direct Observation:

   • Simple visual assessment of signal overlap.

   • Easy method for determining if proteins or organelles overlap.

2. Scatter Plots:

   • Software-based analysis comparing ratios of two channels.

   • Plotting green intensity pixel vs. red pixel intensity.

   • A 45-degree angle indicates good co-localization, while a curved plot indicates poor co-localization.

3. Quantification:

   • Using Pearson and Mander's coefficients.

   • Pearson's coefficient close to 1 suggests co-localization.

   • Increasing trend towards quantification in research.

Visual Detection

• Merging images of different fluorophores (e.g., red and green).

• Overlapping signals appear yellow.

• Considered the easiest method for co-localization analysis.

Scatter Plot Analysis

• Plots the ratio of two channels and how they compare to each other.

• Green intensity pixel vs. red pixel intensity.

• A graph with a 45-degree angle indicates good co-localization.

• A curved graph represents poor co-localization.

Quantitative Analysis

• Determines co-localization based on quantification and provides a numerical value.

• Two main types:

  • Pearson's correlation coefficient.

  • Mander's correlation coefficient.

  • Choice depends on the specific research question.

Pearson's Correlation Coefficient

• Measures the strength of the relationship between two variables (red and green signals).

• Indicates the proportion to which both proteins overlap across the entire image.

Mander's Correlation Coefficient (M1 and M2)

• Measures the fraction of fluorescent signal A that co-localizes with fluorescent signal B, and vice versa.

• M1 represents the fraction of green signal overlapping with red.

• M2 represents the fraction of red signal overlapping with green.

• Provides two quantifiers, offering more specific information than Pearson's coefficient.

• Example:

  • Green protein present in the cytoplasm and three vacuoles.

  • Red protein present in all vacuoles but not in the cytoplasm.

  • Merged image shows co-localization in the three vacuoles.

  • Pearson's coefficient shows an overall weak relationship due to only 5% overlap.

  • Mander's coefficient shows that 5% of the green signal co-localizes with red, and 30% of the red signal co-localizes with green.

• Example:

  • Analysis of a region with EPS8 (an actin-capping protein) and actin filaments.

  • Merged image shows a white area where purple and green signals overlap.

  • Pearson's coefficient indicates a strong correlation of 0.88.

  • 91% of magenta co-localizes with green, but only 38% of green co-localizes with magenta.

Application of Co-localization in Cancer Research

• Used to identify if newly identified proteins co-localize with known tumor markers.

• Example: Upregulation of a protein in breast cancer metastasis.

  • RNA profiling identifies genes upregulated in metastatic breast tumor cells.

  • OEM protein (upregulated in metastatic breast cancer) localized in nucleoplasm and nucleoli.

  • OEM co-localizes with NPM (nucleophosphamin B23), a known tumor marker.

  • Potential therapeutic target for cancer therapies.

Förster Resonance Energy Transfer (FRET)

• Used to determine protein-protein interactions.

• Energy released by one fluorophore is captured by another and re-released.

• Requires fluorophores to be within 100 angstroms (
  $\approx$ 10 nanometers) of each other.

• Proteins must be interacting for FRET to occur.

FRET Experiment

• Uses a donor and an acceptor fluorophore (e.g., cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)).

• CFP absorbs blue light and releases cyan light.

• If YFP is within 100 Angstroms, it absorbs cyan light and releases yellow light.

• To confirm FRET, a laser is used to destroy YFP, which returns CFP to releasing cyan light.

• FRET is the transfer of excitation energy from the donor to an acceptor without the emission of a photon by the donor.

• The acceptor emits fluorescent energy instead of the donor.

• Proximity of donor and acceptor must be short (less than 10 nanometers).

• Donor's fluorescence emission spectrum must overlap with the absorption spectrum of the acceptor.

• FRET can be used to determine biosensors.

• Example: A protein tagged with CFP on one end and YFP on the other.

• Conformational changes or enzymatic reactions can bring the two proteins together, resulting in a FRET reaction.

• FRET efficiency is inversely proportional to the distance between the fluorophores.

• Can be used as a molecular ruler to measure the exact distance between two fluorophores.

Fluorescence Recovery After Photobleaching (FRAP)

• Used to study the movement of fluorophores by active transport or diffusion.

• A specific area of the cell is marked and then high-energy lasers are used to destroy all fluorescence in that area.

• The recovery of fluorescence in that area is then monitored.

• Can determine if a protein is actively transported or moves by simple diffusion.

Procedure

1. Take initial images of the cell and mark the area to be bleached.

2. Use high-energy lasers to destroy fluorescence in the marked area.

3. Continue taking images to monitor the migration of fluorescent molecules back into the bleached area.

4. Measure fluorescence intensity to determine the rate of recovery.

5. Determine if the fluorescence recovery is due to active transport or simple diffusion.

• FRAP is a live-cell experiment and cannot be performed with fixed cells.

• Used to study the mobility of molecules.

• Fluorescent molecules in a region of interest are photobleached with a high-intensity laser beam.

• Recovery is monitored over time.

• Recovery times are compared to determine how quickly proteins are trafficked.

• Example: ER labeled with KDELR-GFP, an area of ER is bleached, and the movement of the molecules back into the area is monitored.

Fluorescence In Situ Hybridization (FISH)

• Uses a fluorescently labeled oligonucleotide DNA probe that is specific to a particular region or gene.

• Probe hybridizes to double-stranded DNA.

• Wherever the DNA probe ends up, fluorescent bands appear on the chromosomes.

Applications

• Detect gene loss (e.g., deletion of a tumor suppressor gene in cancer).

• Detect gene amplification (e.g., multiple copies of an oncogene in tumors).

• Detect polysomy (e.g., determining the number of chromosomes using chromosome painting).

• Determine chromosome translocations (e.g., when an arm breaks off one chromosome and attaches to another).

• Determine the sex of a sperm.

• Phylogenetic probe to determine the phylogeny of species.

• Chromosome Painting: Allows easy sorting and identification of chromosomes.

• Can determine if an arm of a chromosome has broken off and translocated to another chromosome.

• Example: determining gene loss for mesothelioma.

• Labeling the centromeres with green and a tumor suppressor gene with red.

• In normal cells, both green and red signals are present.

• In mesothelioma tumors, the tumor suppressor gene is lost in both arms of the chromosome.

• Three-dimensional image of KRAS oncogene amplification in bile duct cancer.

Immunological Methods

• Used as a second opinion to back up microscopy findings.

• Microscopy resolution limit is about 200-250 nanometers.

• Immunological methods can make results more conclusive when protein-protein interactions cannot be visualized clearly.

Western Blot Analysis (Immunoblot Analysis)

• Detects the presence of specific proteins in tissue via antibody recognition.

• Antibodies are added to protein extracts separated by electrophoresis gel.

• Useful for detecting proteins and measuring relative levels of protein expression between tissues.

• Also used for determining the total mass of protein of interest.

Protocol For Western Blot Analysis

1. Take a biological sample (cells or tissue) and lyse it via homogenization and lysis buffer.

2. Centrifuge the crude extract to remove insoluble parts.

3. Denature the proteins via heat and detergent to disrupt their secondary and tertiary structures.

4. Add to an SDS-PAGE gel and run electrophoresis for separation by size.

5. Add a molecular weight standard ladder to determine the size of the proteins.

6. Transfer the proteins to a nitrocellulose membrane via electrophoresis.

7. Probe the membrane using a primary and secondary antibody.

8. The secondary antibody is conjugated to an enzyme (usually horseradish peroxidase).

9. Add the substrate (luminol) to produce a chemical reaction releasing light.

10. Detect the light via X-ray film to determine where the protein is within the gel.

Chemiluminescence Detection

• Luminol substrate + horseradish peroxidase + hydrogen peroxide produces an excited state, which releases light.

• Primary antibody binds to the protein of interest.

• Secondary antibody binds to the primary antibody with horseradish peroxidase conjugated to it.

• Apply to an X-ray film to detect the light from the reaction and show where your protein occurs in the gel.

Solubilize The Cell Content

1. Using lysis buffer.

2. Centrifuge to clarify soluble proteins.

3. Use heat denaturation and detergent to break down protein structures.

4. Run on an SDS-PAGE gel to separate proteins by size.

5. Transfer proteins to a nitrocellulose membrane via electrophoresis.

6. Probe using primary and secondary antibodies.

7. Detect using chemiluminescence (horseradish peroxidase) on an X-ray film.

Applications Of Western Blot Analysis

• Compare expression levels and the size of the protein of interest.

• Look at differences in normal and knockout cells.

• Look at expression differences between different tissues.

• Look at different protein isoforms.

• Look at recombinant proteins (e.g., GFP fusion protein).

Loading Control

• Normalization of protein expression levels.

• Housekeeping gene (e.g., actin, tubulin, GAPDH).

• Essential for accurate comparison of expression between tissues and cells.

Immunoprecipitation (IP) and Co-Immunoprecipitation (Co-IP)

• Microscopy is limited by resolution; IP can confirm physical protein interactions.

• Co-IP isolates a specific protein of interest and identifies any proteins bound in a complex.

• Maintains tertiary and quaternary structure until gel analysis.

• Uses Western blot to confirm isolation or Kumasi blue stain to visualize all proteins.

Immuno Precipitation Experiments:

1. Lyse cells or tissues to release contents, then centrifuge to remove insoluble components.

2. Add primary antibody specific to the protein of interest to label any associated proteins.

3. Add beads (magnetic or sepharose) coated with protein A or G, which bind to the primary antibody, allowing pelleting of proteins.

4. Pellet the protein precipitate via magnetism or centrifugation, removing non-bound proteins.

5. Remove beads via an elution buffer, leaving the protein antibody complex.

6. Run on a denaturing SDS-PAGE gel to separate proteins based on size.

7. Carry out Western blot analysis to detect proteins of interest, or excise bands from the gel for identification via mass spectrometry.

Basic Protocol Recapped:

1. Solubilize cell contents using lysis buffer and homogenize tissue.

2. Centrifuge the solution to clarify soluble proteins.

3. Add primary antibody for the protein of interest to bind and isolate the target protein and its partners.

4. Add sepharose or magnetic beads coated with protein A or G to bind to the primary antibody, allowing purification via centrifugation or magnetic attraction.

5. Use an elution buffer to separate proteins from beads, and run on a denaturing SDS-PAGE gel to separate proteins by size.

6. Carry out either a Western blot analysis or stain with Kumasi Blue.

7. Excise the bands of specific proteins for analysis via MALDI/mass spec.

Controls Required

• Loading controls are always required to validate both your positive and negative results.

• Cells and tissues can have varying levels uh of expression.

• Need a way of of normalising that across the samples to get accurate results.

Positive control

• Such as a housekeeping protein.

• If you're looking at quantification of of expression you should use a serially diluted purified protein to enable you to quantify the expression.

Negative control

• Use Beads without the primary antibody.

• Use Cell lysate, to determine if there is non-specific binding of your antibody.

Applications Of Immuno Precipitation

• Valuable tool in disease research.

• Example: Researchers were trying to determine the pathway of specific proteins involved in cancers.

• Phosphotyrosine signaling is an important intracellular communication system.

• Any changes in the activity of phosphotyrosine signals are related to many human diseases, especially in cancers.

• TKS5 protein is known to be involved in invasiveness of some cancer cells.

• TKS5 requires phosphorylation by SRC kinase, which allows for the induction of phosphotyrosine signaling.

• Using antibodies for TKS5, they found that NCK co-immunoprecipitates with TKS5.

• Deletion of SRC kinase means NCK cannot bind to TKS-5, and hence there is also no phosphotyrosine production.

• Involved in binding to TKS 5.

Use immuno precipitation and co-immuno precipitation:

• Validate suspected protein protein interactions.

• Discover novel unknown binding partners that interact with the protein of interest.

• Map binding sites involved in protein protein interaction.

• Evaluate associations of binding partner.