Detailed Notes on Fluorescence Microscopy and Antibody-Based Approaches

Fluorescence Microscopy and Antibody-Based Approaches

What is Fluorescence?

• Definition: Light emitted by a substance in response to absorption of photon energy.

  • Example: rocks glowing under UV light excitation.

• Emitted light properties:

  • Longer wavelength.

  • Different color.

  • Lower energy than the excitation light.

• Persistence: Fluorescence stops when illumination ceases.

• Fluorescent molecules: Fluorophores or flus (terms used interchangeably).

• Reactions resembling fluorescence (but are not):

  • Phosphorescence: Requires light absorption, but decay and photon release are slower and persist after excitation removal (e.g., glow-in-the-dark).

  • Chemiluminescence and bioluminescence: Light released due to enzyme-substrate interaction (e.g., horseradish peroxidase and luciferase); no external light source required.

Visible Light Spectra for Microscopy

• Typically involves visible light spectra.

• Range: UV to infrared, including all colors of the rainbow.

• UV: High energy, short wavelengths.

• Infrared: Low energy, long wavelengths.

• Direction: Excitation and emission move from UV to infrared (left to right).

• Color shift: Purple excitation usually results in blue and green emission; green excitation gives yellow, orange, red, etc.

• Reason: Conservation of energy; more energy cannot be derived than what is put in.

• Jablonsky Diagram

  • Describes the fluorescence reaction.

  • An electron within an element, typically a carbon molecule, starts at its ground state.

  • A high-energy photon is absorbed, pushing the electron to a higher energy state.

  • The electron loses some energy, moving to a slightly lower energy state.

  • The remaining energy is released as a photon of a longer wavelength.

  • The electron falls back to its ground state to repeat the transition.

• Molecular Structure of Fluorophores

  • Fluorescent molecules contain aromatic rings (at least one).

  • Carbon molecules in the ring form 4 covalent bonds, but only 3 are formed in the ring, leaving a free electron.

  • Free electrons from the 6 carbon molecules form a cloud.

  • This electron cloud is exploited for fluorescence through photon absorption and emission.

• Fluorophore Usage

  • Fluorophore: Generic name for a fluorescent molecule.

  • Used to label specific areas of a cell or tissue via chemical, biological, or genetic methods.

  • Each fluorophore has unique properties based on structure.

    • Specific excitation and emission profile or spectra is one such property.

Fluorophore Excitation and Emission Spectra

• Spectrograph representation: Range of wavelengths for excitation and emission.

• Fluorescein example:

  • Excitation: 400 nm to 525 nm.

  • Emission: 480 nm to 700 nm.

  • Targeting maximum excitation and emission provides the best signal.

• Intensity Proportionality

  • Emission intensity is proportional to excitation amplitude.

    • Exciting at 40% efficiency results in 40% emission efficiency.

  • Always target 100% efficiency.

• Emission Range

  • Photons are emitted across a range of wavelengths, even at peak excitation.

• Multiple Fluorophores Considerations

  • Avoid fluorophores with overlapping excitation and emission spectra to prevent crosstalk.

• Crosstalk/Bleed Through

  • The fluorescence from one fluorophore bleeds into another's channel.

  • Results from incorrect fluorophore choice or microscope settings.

  • Correct settings improve channel separation and image quality.

• Stokes Shift

  • Definition: The difference between the wavelength maxima of excitation and emission.

  • Range: Short (few nanometers) to long (greater than 80 nm).

• Importance

  • Longer Stokes shifts are more useful because absorbed and emitted light are easier to separate using filters.

  • Very long Stokes shifts allow packing more fluorophores into samples.

  • Can illuminate multiple fluorophores with a single wavelength.

Targeting Fluorescence

• Target fluorescent molecules to specific cell parts

  • Discussed relative to what fluorescence is.

Immunofluorescence

• Pioneered by Albert Cons (1940s-50s).

• Original Use

  • Testing against pneumonia-causing bacteria.

  • Antibodies raised against bacteria were conjugated to a fluorophore (anthracine isocyanide).

  • Primary antibody bound to a specific ligand on the bacteria surface.

  • Fluorophore location indicated bacteria location.

  • Antibody alone can't be seen; a marker is needed.

• Current Immunofluorescence Method

  • Primary antibody against a specific ligand.

  • A secondary antibody with a conjugated fluorophore binds to the primary antibody.

• Reasons for Using Secondary Antibodies

  • Avoid labeling the primary antibody.

  • Commercially available secondary antibodies.

  • Allows choice of fluorophore to detect any primary antibody from a specific species.

    • E.g., If primary antibody raised in rabbit, use an anti-rabbit secondary antibody with a fluorophore.

  • Signal amplification.

    • Polyclonal secondary antibodies bind to multiple places on the primary antibody, leading to greater signal.

Immuno-fluorescence Assay

• Tertiary Antibodies

  • Possible to use a third antibody with a conjugated fluorophore.

  • Binds to multiple points on the secondary antibody.

  • Not typically used.

• Fluorophore Variety

  • Hundreds of fluorophores available across the entire wavelength spectrum (UV to far-infrared).

  • Choose fluorophore based on application.

• Standard Procedure

  • Grow cells in culture on a coverslip or in a flask.

  • Remove cells in solution and add to a clean coverslip.

• Fixation

  • Required to preserve cell structure.

  • Paraformaldehyde (PFA) covalently cross-links proteins.

  • Other fixatives: methanol, ethanol, methanol-acetone, or gluteraldehyde (reserved for specific purposes).

  • PFA preserves cell morphology, organelles, cytoskeletal structures, and subcellular localization of proteins.

• Permeabilization

  • Required because antibodies do not freely pass through the membrane.

  • Use a detergent (e.g., Triton X-100) to permeabilize cells and allow antibody access to intracellular antigens.

  • Wash with PBS.

  • If using cell surface antigens, detergent not needed.

• Blocking Buffer

  • Reduces background staining and improves sensitivity.

  • Typically BSA (alternatives: gelatin, glycine).

  • Binds to potential sites of non-specific interactions without altering the epitope.

• Primary Antibody Incubation

  • Incubate cells with unlabeled primary antibody specific for the protein/ligand of interest.

  • Approximately 1 hour at room temperature or overnight at 4°C.

  • Wash with PBS.

• Concentration

  • Titration required to determine the correct antibody concentration.

    • Too little: Not enough signal.

    • Too much: Too much background.

  • Test with serial dilutions (e.g., 1:1000, 1:500, 1:100, 1:50).

  • Antibody typically diluted in blocking buffer to reduce non-specific binding.

• Secondary Antibody Incubation

  • Incubate cells with fluorescently labeled secondary antibody for approximately 1-2 hours at room temperature in the dark.

  • Use a secondary antibody against the species in which the primary antibody was raised.

    • E.g., If primary antibody is from mouse, use an anti-mouse secondary antibody antibody with a fluorescent label.

  • Incubation times can vary.

    • Too short: Not enough signal.

    • Too long: Non-specific binding, high background.

Multiplexing Antibodies

• Can stain cells with two or more primary antibodies simultaneously to look for multiple targets.

• Considerations to prevent cross-reactivity:

  • Primary antibodies must be raised in different species (e.g., rat and rabbit, not both in rats).

    • Secondary antibodies should be raised in the same species to prevent cross reactivity.. However, it often doesn't matter as long as they are specific for only one of your primary antibodies

  • Conjugated fluorophore spectra should not overlap (e.g., green and red, not two greens).

• Examples of potential cross-reactivity:

  • Primary antibody 1 (protein 1) raised in goat; primary antibody 2 (protein 2) raised in mouse. Secondary antibody (anti-goat raised in rat) should bind to primary 1. Avoid secondary antibody (anti-mouse raised in goat) because it will cross-react with the other secondary.

  • Both primary antibodies raised in goat. Anti-goat secondary antibodies will cross-react and bind to both primary antibodies, giving a muddled signal.

  • Primary 1 and primary 2 raised in goat and mouse. Secondary antibodies (rat anti-goat and rat anti-mouse) will not cross-react because neither is anti-rat. Secondary rat anti-goat binds to primary 1, and secondary rat anti-mouse binds to primary 2.

Sample Preparation for Microscopy

• Add a mounting medium and coverslip.

• Mounting Medium

  • Viscous buffer that holds the specimen in place between the coverslip and the slide.

  • Typically contains a counterstain: a general fluorescent stain for a specific organelle (e.g., nucleus or mitochondria) to help locate the cell.

    • DNA stains: DAPI or Hoechst to stain the nucleus.

  • Contains an anti-fade agent to slow down fluorophore degradation by scavenging free radicals.

• Add a glass coverslip and seal with nail polish to prevent movement/drying out.

• Then image with a microscope.

• Multi-color Staining Example

  • Red antibody for astrocytes and green antibody for neurons, with DAPI counterstain for the nucleus.

  • Three-color image reveals tissue architecture based on staining.

Fluorescent Assay Summary

1. Grow cells in culture.

2. Fixation.

3. Permeabilization with a detergent.

4. Blocking buffer.

5. Primary antibody.

6. Secondary antibody.

7. Mount, counterstain, and coverslip.

8. Image.

Performing with Living Cells

• Immunofluorescence essays can be done with living cells in culture.

• Use primary antibody alone conjugated to a fluorophore, added to the culture media.

• Use digested antibodies, antigen-recognizing part of the antibody (fragment antigen binding).

• Generally used only for cell surface proteins because antibodies typically do not cross the membrane.

• Considerations

  • Monoclonal vs. polyclonal antibodies: personal choice.

    • Monoclonal: better specificity but lower signal.

    • Polyclonal: greater signal but more background.

  • Titration: imperative to find the best concentration for best signal with minimal background.

  • Length of antibody incubation: longer incubation improves signal but can lead to non-specific staining.

  • Antibody incompatibility when multiplexing: primary and secondary antibodies can cross-react, and fluorophore spectra can overlap, causing crosstalk.

Epitope Tags

• Use when primary antibodies are not available for a protein of interest.

• Epitope Tag

  • Short peptide sequence (8-10 amino acids) added to the N or C terminus of the protein via molecular cloning.

  • Acts as an artificial ligand recognized by a primary antibody.

  • Primary and secondary antibodies used to indirectly label the protein by labeling the epitope tag.

• Common Epitope Tags

  • Flag tags, HA tags, and Myc tags.

  • Provided by specific companies.
    Vector Usage

• Clone the protein in front of or behind the epitope tag in a vector.

• During translation, the amino acid sequence includes the protein of interest with the epitope tag attached.

• After protein folding, immunohistochemistry or immunofluorescence is used to bind specific antibodies for the epitope tag to the protein for labeling.

• Examples of Fluorescent Labeling

  • Tubulin with an HA tag assembled into microtubules is labeled with a primary antibody for the HA tag.

  • NCK1 protein with an HA tag is labeled with primary and secondary antibodies to determine its location within the cell.

Epitope Considerations

• Impact of Epitope Tag on Recipient Protein

  • Adding extra amino acids can disrupt protein folding, causing loss of activity, expression, or solubility.

  • Can disrupt subcellular localization signals, preventing correct targeting within the cell.

• N or C Terminus Addition

  • Adding the tag to the N or C terminus could disrupt the localization signal.

  • C-terminal tagging may interfere less with protein localization.

  • Generate both N-terminal and C-terminal tagged forms for comparison.

• Counterstains

  • Use counterstain to determine where a protein is located.

  • DNA stains: DAPI doesn't pass through membranes easily, whereas far easier than Hercules and stain live cells

Protein Consideration

• Consider many different fluorescent protein.

• Look into the excitation and the emission suited for your applications.

• Consider photo Stability. the less likely to be photo bleached, the better the flu for will be because it will last longer.

• The fluorophore structure can affect efficiency, Moomers don't need any Quaternary structure. They will just simply function as is, whereas a tetramer needs 4 of the sub. To actually function. So monomer is far more desirable when we're dealing with fluorescent protein

Practical Applications of GPF

• There are many constructs available for um uh for uh many fluorescent proteins that you can use to clone in your uh your protein of interest.

• GFP has an autonomous structure which means it doesn't actually require a functional protein for the GFP to be active.

• You can fuse it either at the N or C terminus without the loss of fluorescence

• Tagged proteins typically retain their activity.

• The protein has low toxicity to the host cell tissue or organism. you can splice these into the genome as tags to study the protein localizations.

• You don't need to add use immunofluorescence simple as imaging the fluorescent protein.

Microscopy and Magnification

• Allows magnification and resolution of objects invisible to the unaided eye.

• Magnification without increasing resolution is pointless.

• Goal: Improve the resolution of microscopes to see fine details.

Resolution Limitations

• Optical microscopes are diffraction limited.

  • Resolution limit is 200 nanometers, meaning objects closer than 200 nm cannot be resolved as separate entities.

• Water waves bend around objects; light waves do the same (diffraction), leading to blurring.
  Experiment

• Shining light through a small aperture

• If light was like a wave, it would diffract around the aperture and if it was a particle it would go straight to the aperture without diffracting.

• Later found out light was a particle and wave.

• Diffraction patterns are through the aperture, and we have a central spot, then multiple rings around the central spot caused by constructive and destructive interference or caused by the wave.

Eerie Disc

• The er disc is a real consequence of trying to image light moving through a microscope's lenses and apertures. Because as the light moves through, we end up with these interference patterns rather than a nice, bright, clear spot, we end up with a blurriness, which can affect the resolution of our microscope.

Objectives

• Lens is the most critical part of the microscope.

• Provides magnification and resolution.

• Resolution is derived from the numerical aperture (NA).

• NA denotes the resolving power of the lens.

• Lens Construction

  • Objective lens is complex, with multiple lenses to achieve high resolution.

NA v Resolving

• The higher the NA, the smaller the object that can be resolved.

• Laboratory microscopes typically have objectives no higher than 1.4 NA, resolving objects down to about 227 nm under ideal conditions.
  Real World Example

• Uses the NA to see the difference in the fine details. A higher NA leads to a fine detail which leads to increase sensitivity and an improved signal.

Calculation

• Do no focus too much on the details behind the formulas but understand what they mean.

• Several factors are involved in the resolution the wavelength of light, the shorter the wavelength, the better. The refractive index of the mounting and immersion media with the higher index the better the lenses can capture and focus, the light, and that enhances the resolving power.
  Essential of a Microscope

1. The objective lens's Paramount provides us with the magnification and resolution.

2. Light source provide the elimination of the sample can be mercury lamps, metal halides, LEDs and lasers

3. The Philter provides the filtration of the specific light source we require as well as filtering out the light that comes back before it goes into the imaging device, which can be as simple as looking down the eyepiece or a camera. Or a confocal detector

Fluorescent Light Source

• Light source

  • Mercury lamp, LED, metal halide, or laser.

  • Each light source emits a range of excitation wavelengths.

  • The mercury lamp can excite across the visible spectra.

  • Match the fluorophore to the light source and filter setup of the microscope.

  • Filter is needed to separate specific colours. A fluorescent setting with four blue, green, red, and for red filters.
    How Filter Cube works

1. An excitation filter allows the transmission of a specific wavelength for excitation all wavelengths apart from blue light.

2. That light goes into our diachronic mirror which allows reflection of light below a certain wavelength but transmit light above that wavelength. so it separates out the excitation and emission beams.

3. The flurophoe is then excited with photons emitted that are directed back through the objective lens where it comes into contact with the emission filter.

4. The emission filter allows for transmission of specific wavelengths corresponding to the fluorescent signal, which is emitted from our fluroor, blocking out everything . so we get a nice clean image, just from light from our fluorophore

• We're looking at green fluorescent protein which requires blue light to excite it, so we see that we have a specific excitation philtre to allow blue light to pass through.

• Light from the mirror is reflected to the object and if the object reflects it is allowed to pass through into our mission filter which has a set up blocker for blue light, but we'll allow other light past

Photo Bleaching

• Greatest challenge for fluorescent protein is the crossover bleeding.

• Consequence of that either you use the wrong settings, or the fluoride is too close so use, the correct setting in order to prevent this

• Photo bleaching is called fading as what we get when we get the photochemical altered of a dye that is permanently unable to fluoresce, so it's essentially destroyed

• Photo bleaching is for two reasons, both of which involves too much light.

  • Electrons that are kept in the excited state due to energised electron clouds in the aromatic ring

  • Fluorophores can only cycle through the excitation emission only a certain number of times before they for apart.

• Solutions:

  • Use less light

  • Close the shutters when not needed, or use a lower magnification

  • Use anti fade

Resolution

• improving the resolution relies on removing this interference pattern. if we can remove the interference pattern, we improve our resolution and achieve far better images. is the purpose for confocal my microscope

• confocal is the application between diffraction is caused by light moving through the lenses and apertures to create a disc. this is overcome by the use of lenses

• Microscope utilizes a mirror to build an image pixel by pixel. and above the image plane, we see a what's called the comfocal pinhole. is the pinhole and removes that diffraction pattern

• Depending on the depth that the laser penetrates within the sample, the resulting fluorescent light is reflected off at different angles.just be aware that thecolours do not represent, colours of the laser.

• light reflected from our plane of focus come straight to pinhole and is focused into the reader. But others, the light becomes fuzzy.

• A smaller pinhole will lead to a thinner optical section and clearer image, but image is more dimmer, because we are only looking at a thinner layer, and the oposite happens if you have a bigger pinhole

• In confocal there is a clearer focus which is not present in confocal. But we need to be very careful because we need to have a completed picture between details can often be missed.

• Multiple sections would be best

Super Resolution

• Standard limit about 20 to achieve a 20-fold increase. This is due to analysis that gets fine detail and helps determine

• The use is no real specimen is needed , but rather Post Acquisition Processing.

• Increase resolution can done by the use of diffraction grading to create an interference pattern and remove this distortion

• We also have Storm and Palm which use blinking fluoropores to localization postion.

• Uses single flashes and localizes to a a single pixel, we can build up a high resolution image.