EYES-Fluorescence Techniques

Molecules Review: Advanced Fluorescence Microscopy Techniques

Introduction

• This review focuses on techniques like FRAP, FLIP, FLAP, FRET, and FLIM that exploit fluorescence properties.

• These techniques utilize how fluorochromes excite and emit light, get damaged during excitation, or undergo non-radiative decay.

Fluorescence

The Physical Phenomenon

• Fluorescence is a photoluminescent process where a substance absorbs and re-emits light.

• Photoluminescence includes fluorescence and phosphorescence, generated through physical, mechanical, or chemical excitation.

• Fluorescent probes/dyes are called 'fluorochromes'; 'fluorophore' refers to fluorochromes bound to biological macromolecules (nucleic acids, lipids, proteins).

• Fluorochromes include organic molecules, inorganic ions (e.g., lanthanides), fluorescent proteins (e.g., GFP), and atoms (e.g., gaseous mercury).

• Quantum dots are used for bio-imaging and theragnostics (combination of diagnostics and therapeutics).

• Fluorescence involves photon emission at a longer wavelength, as shown in the Jabłoński diagram.

• When light hits a fluorescent sample, atoms/molecules absorb a quantum of light, exciting a valence electron from the ground state (GS0) to a higher energy level (ESn).

• This is a fast, femtosecond process requiring energy $ΔE = E<em>{ESn} - E</em>{GS0}$.

• Energy of photons is expressed by Planck’s law: $E = h \cdot \frac{c}{\lambda} = h \cdot ν$ where:

  • E is energy (J).

  • h is Planck's constant (J.s).

  • ν is frequency (s$^{-1}$).

  • λ is wavelength (m).

  • c is the speed of light (m.s$^{-1}$).

• Numerous allowed transitions populate vibrational energy levels of excited states, forming the absorption spectrum.

• Electrons quickly relax to the lowest excited sublevel in picoseconds via non-radiative conversions, releasing heat.

• Emission spectra are usually independent of excitation wavelength due to rapid relaxation (Kasha’s rule).

• Absorption and emission transitions often involve the same energy levels, leading to near mirror image spectra, though exceptions exist.

• External conversion depletes the excited state.

• Intersystem crossing (singlet to triplet state) is slow and spin-forbidden but can occur in molecules with heavy atoms.

• Lanthanide ions show delayed fluorescence (luminescence) with micro/millisecond lifetimes due to intra-configurational f-f transitions, distinguishable from organic fluorochromes which are generally in the nanosecond range.

• Electron returns to ground state (GS0), emitting a photon with longer wavelength.

• Photon emission (π→π or π→n transition) from singlet states occurs between $10^{-9}$ and $10^{-6}$ seconds (fluorescence).

• Emitted light is collected through the same objective lens used for excitation (epi-fluorescence microscopy).

• Fluorochromes undergo repetitive cycles of excitation/emission until destruction or covalent modification occurs.

• Transition from triplet state (ET1) to ground state (GS0) is slower (phosphorescence), with emission over longer periods.

• Fluorescence emission occurs at longer wavelengths (lower energy) than excitation light (Stokes shift).

• Anti-Stokes shifts (emission wavelength < excitation wavelength) occur during photon upconversion or two-photon excitation.

• Fluorochromes have characteristic excitation and emission spectra dependent on their vibronic configuration.

Overview of Fluorescence Characteristics

• Essential fluorescence characteristics include lifetime, quantum yield, quenching, photobleaching, and energy transfer.

• These properties can be artifacts or used to solve scientific questions (e.g., FRAP for diffusion, FRET for molecular interactions).

• Quantum yield (Ф) determines fluorochrome brightness: Φ = \frac{\text{# photons emitted}}{\text{# photons absorbed}} = \frac{Γ}{Γ + k_{nr}}, where:

  • Γ is the rate constant of emission.

  • $k_{nr}$ is the sum of all non-radiative decay processes.

  • $k<em>{nr}$ encompasses intersystem crossing ($k</em>{isc}$), internal conversion ($k<em>{ic}$), predissociation ($k</em>{pd}$), dissociation ($k<em>{d}$), and external conversion ($k</em>{ec}$)

• Quantum yield is sensitive to solvent polarization, pH, fluorochrome concentration, and presence of molecular oxygen.

• Comparative method by Williams et al. is used for recording Ф.

• Fluorescence lifetime (τ) is the average time an electron spends in the excited state before returning to the ground state: $τ = \frac{1}{Γ + k_{nr}}$.

• Fluorescence intensity decay (It) over time (t) following excitation: $I(t) = I_0 \cdot \exp(-\frac{t}{τ})$ where I0 is the initial intensity.

• During lifetime, fluorochrome may undergo conformational changes, diffuse, or interact with molecules, enabling lifetime measurements to probe such actions.

• Anisotropy refers to varying properties along different axes; fluorescence polarization measures fluorochrome rotation.

• Quenching reduces quantum yield or lifetime through interaction with a quencher.

  • Dynamic quenching: collision of quencher and excited fluorochrome, decreasing lifetime and intensity.

  • Static quenching: direct interaction forming a non-fluorescent ground state complex.

  • Self-quenching (concentration quenching): close proximity of identical molecules.

  • Color-quenching: emitted photons absorbed by a strongly colored component.

• Fluorescence intermittency/"blinking": fluorochrome alternates between fluorescent and dark states.

• Autofluorescence: fluorescence from cellular components (flavins, NADH, elastin, collagen).

  • Strategies to avoid autofluorescence: precise filtering, probes fluorescing outside the autofluorescence window (near-infrared), time-resolved techniques, upconverting fluorochromes.

  • Autofluorescence window approximately encompasses the range from 350 to 600 nm.

• Photobleaching: ability to repetitively enter excitation/emission cycles is interrupted.

  • Reactions of triplet state with molecular oxygen cause transformation to a reactive singlet state i.e. singlet oxygen, formation of superoxide anion radical and other reactive oxygen species

• Resonance energy transfer: excited state energy transferred from a donor fluorochrome to an acceptor chromophore via dipole-dipole coupling (Förster).

• Charge-transfer complexes: nanosecond short-lived dimers (excimer/exciplex) show red-shifted emission.

Fluorescence Microscopy

General Concepts

• First fluorescence microscopes developed in early 20th century.

• Oskar Heimstädt (1911) developed the first working fluorescence microscope.

• Epi-fluorescence microscope (1929) by Philipp Ellinger and August Hirt improved signal-to-noise ratio.

• Max Haitingen introduced 'fluorochrome' for fluorescent stains.

• Albert Coons (early 1940s) labeled antibodies with fluorescent dyes.

• Lasers (1960s) provided high spatial and temporal coherence.

• Green fluorescent protein (GFP) by Tsien, Chalfie, and Shimomura (1990s) enabled live-cell imaging.

• GFP is a 238 amino acid protein that shows bright green fluorescence and was first isolated from the jellyfish Aequorea victoria.

• Wild-type GFP consists of a -barrel structure in which the essential chromophoric moiety, an amino acid triplet of Ser65, Tyr66, and Gly67, lies at the centre.

• Scientists modified GFP to expand color spectrum, improve photostability, and enhance quantum yield.

• Fluorescent proteins (FPs) identified from other species, like Zoanthus (ZsYellow) and Discosoma (DsRed), have expanded the color palette.

• Quantum dots (QDs) are inorganic semiconducting nanoparticles with core-shell configuration.

• QDs size determines their fluorescent properties.

• QDs have broad absorption spectra and relatively long lifetimes.

• Fluorescence microscopy is standard for live cell imaging.

• Advantages include spatio-temporal resolution and less destructiveness than other techniques.

• Selectivity and contrast enhancements are achieved through labeling and maximizing collection of emitted light while minimizing collection of excitation light.

• Fine cellular structures and single-molecules can be made visible.

• Three basic components are present in every light microscope: an illumination source, a magnifying lens, and an image acquisition device.

• Wide field fluorescence microscope uses a high power lamp (mercury or xenon source), which excites the fluorochromes in the fluorescently labeled sample and induces fluorescence emittance.

• Confocal laser scanning microscope using photomultiplier tube.

• Excitation filter narrows wavelength range to excite fluorochromes efficiently, while emission filter blocks excitation light from reaching the detector.

• Lasers are commonly utilized in confocal and multiphoton laser scanning microscopy to point illuminate the sample.

Resolution in Fluorescence Microscopy

• Optical resolving power determines detail observed, influenced by physical factors and instrument limitations.

• Diffraction changes incidence light direction when light hits an object

• Numerical aperture (N.A.) defined by Ernst Abbe determines objective’s light capturing capacity: $N.A. = n \cdot sin(α)$, where:

  • n is the refractive index of the medium between the object and the objective

  • α is half the objective opening angle

• Immersion oil increases N.A. and resolution.

• Resolution is the smallest distance between two points discriminated as separate points.

• Contrast is the difference between maximum and minimum intensity between two objects.

• PSF (point spread function) characterizes the system; Airy pattern is intensity distribution of PSF in the focal plane.

• Resolution: $d = \frac{λ}{2NA} = \frac{λ}{2(n \cdot sin α)}$

• Rayleigh criterion: two points are resolved when the first minimum of one Airy disc is aligned with the central maximum of the second Airy disc.

• Ernst Abbe described that the smallest resolvable distance between two points using a conventional light microscope cannot be smaller than half the wavelength of the imaging light.

Confocal Laser Scanning Microscopy (CLSM)

• CLSM combines high-resolution optical imaging with depth selectivity.

• Laser beam focuses on the sample and collects emitted photons pixel-by-pixel with a PMT.

• Acquires well-focused images from various depths through "optical sectioning."

• Pinhole aperture before the detector prevents out-of-focus light from reaching the detector.

• Abrogation of interference of out-of-focus and stray light with the in-focus signal cause a considerable increase in resolving power.

• Focal point in the sample and the pinhole lie in conjugate planes, as shown in Figure 9B, and this optical arrangement of the focal points is called ‘confocal’.

• Optical sections of the specimen can be obtained as well as 3D structure reconstruction (stack of individual sections).

• Non-linear behavior: sensitive to the square of the light intensity.

Multiphoton Fluorescence Microscopy

• Uses focused laser beams, point-by-point scanning, and optical sectioning.

• Multiple photon excitation (MPE) involves simultaneous absorption of multiple photons by fluorochromes.

• Femtosecond mode-locked pulsed lasers are required.

• Two-photon excitation is a non-linear process that increases with the second power of the excitation light intensity.

• Excitation is restricted to a small volume in the focal plane of the specimen (no need for a pinhole).

• Sectioning capabilities are caused on the excitation side and therefore there is no need for a pinhole to block fluorescence from out-of-focus locations.

• The process is extremely low and results in a reduced overall photobleaching.

• Deep red or infra-red excitation light can be used in TPE, which penetrates much deeper into the specimen.

• Two-photon autofluorescence microscopy (2PAM) assesses the disease states in tissues based on changes in morphological, spectral, and lifetime parameters.

• Second-harmonic imaging microscopy (SHIM) is based on the nonlinear optical effect of second- harmonic generation (SHG).

• Multiphoton fluorescence microscopy offers reduced photobleaching, low photo-toxicity, higher penetration depths, and higher spatial resolution.

Photobleaching-based Techniques for Assessing Cellular Dynamics

Fluorescence Recovery after Photobleaching (FRAP)

• Developed by Axelrod et al. in the 1970s.

• Measure protein mobility in living cells by measuring the rate of fluorescence recovery at a previously bleached site.

• Use of micro- injection or permeabilization techniques can disrupt cell and cellular homeostasis.

• FRAP is generally suitable to study and investigate:

  • Protein/molecule movement and diffusion (diffusional speed).

  • Compartmentalization and connections between intracellular compartments.

  • The speed of protein/molecule exchange between compartments (exchange speed).

  • Binding characteristics between proteins.

• Immobilization of proteins that bind to large structures

• Basic Principles: Fluorescent molecules are irreversibly photobleached in a small area, and diffusion of non-bleached molecules into the bleached area is recorded.

• Mobile fraction (Mf): fraction of fluorescent molecules that can participate in this exchange.

• Immobile fraction (If) fraction that cannot exchange.

• The speed of recovery to half the plateau intensity (I∞) is called ‘half maximum’ or ‘half life’ (τ½).

• Determination of the half life and immobile/mobile fractions via curve fitting of the experimental data points using a simple exponential equation: $I(t) = M_f \Big(1 - e^{-\frac{t}{τ}}\Big)$ where t is time.

  • From this is extracted:

    • Mobile fraction = Mf

    • Immobile fraction (IMf ) = 1– Mf

    • Half life: $τ_{\frac{1}{2}} = -ln(0.5)τ$

• Information of diffusion of fluorochrome.

• Different profiles of temporal fluorescence intensity plot provide information about protein mobility and indicate whether it should be classified as high, intermediate or immobile (Figures 13B–D).

Practical Aspects and CLSM-Specific Considerations

• Images are processed to generate a kinetic plot of photobleaching by displaying the temporal fluorescence changes in the bleached region of the cell.

• Background bleaching can be corrected for mathematically and can be minimized ab initio

• Applying line scans instead of 2D scans.

• Using fluorescent probes that are less susceptible to photobleaching.

• Decreasing the laser power or the pixel resolution by zooming out or using faster scans.

• In a quantitative FRAP experiments the crispness of the image itself is often less important and therefore the spatial resolution is sacrificed for the benefit of maximizing the temporal resolution.

• The ideal fluorescent probe for use in photobleaching studies should be highly fluorescent (high quantum yield), but only moderately susceptible to photobleaching.

• Applying the following correction can result in more suitable results: $I(t) = M<em>f \Big(1 - e^{-\frac{t}{τ}}\Big) + Be^{-\frac{t}{τ</em>2}}((y_0)$

Inverse FRAP (iFRAP) in Cell Biology

• Modified version of FRAP method.

• Initially developed to study the mobility of molecules in small areas of the nuclei and their exchange with the surrounding nucleoplasm.

• Entire population of fluorochromes in the cell is bleached, except the accumulated fluorochromes in a small part of the organelle.

• Particularly useful to study the residency time of molecules in small organelles.

• The main limitations of iFRAP lies in the long time needed to photobleach the entire cell.

• Therefore, iFRAP is mostly useful for analyzing the dissociation kinetics of molecules bound to an immobile intracellular structure.

• Exemplified the work of dynamics mRNAs at speckles

Summary of Steps to Perform in FRAP Experiments:

1. Definition of the cell region to be bleached (ROI).

2. Acquisition of control images to measure intensity before bleaching.

3. Brief illumination of the bleach region with very high laser intensity. Ideally, the bleaching event should be ultra-short followed by subsequent image acquisition without time delay.

4. Recording the progress of fluorescence recovery in the bleached area with high temporal resolution.

5. Changes in intensity in the bleached region represent the sum of all movement of the fluorescent molecules, whether passive (e.g., diffusion or active e.g., transport).

6. The regeneration time (half-recovery period) is a measure for the speed of protein movement.

Potential Complications and Pitfalls

1. Living cells often move during an experiment.

2. The total amount of excitable fluorochromes reduces overtime.

3. When bleaching a region in a three-dimensional sample.

4. In some instances, the final FRAP results are determined by the size of ROI.

5. With presence of lower levels of flurochromes, a higher intensity is needed to obtain sufficient signals.

6. When bleached and fluorescent molecules are exchanged with compartments distant from the bleached region.

7. Fluorochrome intermittency (blinking) reversible photobleaching may cause flawed FRAP results.

8. Photo-induced cross-linking may occur

9. Repeating FRAP on the same sport constitutes an important control to exclude differences in FRAp results due to photo-damage.

Fluorescence Loss in Photobleaching (FLIP)

The Basic Principles of FLIP:

• Complementary technique to FRAP.

• Used to reveal the connectivity between different compartments in the cell of the mobility of a molecule within the whole compartment.

• Repetitive bleaching of the same region so thereby preventing recovery of that particular region.

• The repetitive bleaching occurs adjacent to the unbleached ROI.

• Loss in fluorescence defines the mobile fraction of the fluorescently labeled protein.

• Incomplete loss in fluorescence defines the immobile fraction

• FLIP is a direct method for studying exchange of molecules between two compartments (e.g., compartments that are separated by lipid bilayers).

• FLIP experiments are very useful to demonstrate the connectivity and flux between different regions of the cell.

• Continuity of cellular structures, such as the Golgi Apparatus, the endoplasmic reticulum, and the protein between the Nucleus and cytoplasm and the Nucleolus, has been studied using FLIP.

• FLIP is often used in conjunction with FRAP experiments to obtain combined information regarding active or passive transport.

Summary of Steps to Perform in FLIP Experiments:

1. Definition of the cell region to be bleached (ROI).

2. Acquisition of control images to measure the intensity before bleaching.

3. Brief repeated illuminations of the bleach region, with very high laser intensity.

4. Recording the progress of fluorescence decay and adjacent non-bleached areas, with high temporal resolution.

5. Changes in the intensity of the non-bleached region represent some of all movement of fluorescent molecules, whether passive (e.g., diffusion) or active (transport).

6. The decay time is a measure of the speed of protein movement.

Fluorescence Localization after Photobleaching (FLAP) and Photo-Activation Methods

• Tracking all the labeled molecules is impossible for the FRAP and FLIP methods.

• Tracking of labeled proteins possible by Spotaio-Temporal Resolution

• Detection and tracking of sub-populations that move rapidly and has short residence times.

• Major advantage over conventional methods.

• FLAP is a technique, one label molecule of interest carries two fluorescent labels, one label is locally bleached, and the second remains intact and used as a reference label

• Absolute flap signal: subtracting the bleached signal from the unbleached one, allowed the tracking of the labeled molecule

• Relative Flap image can also be calculated to show the full ablated fraction of molecules which is then used in Pixel images.

• Photo-activation a fluorescent labeled from often fluorescent protein is irreversible activated from a low fluorescent (dark) state to a bright fluorescent, and the irradiated sample is light by a specific wavelength intensity for a particular duration.

• This photo conversion also follows different rules, is a procedure of pre-mRNA from speckles.

• Photo-conversion or switching following different principles.

• Photo-convertible proteins Likeaids are irreversibly converted from green to red fluorescent with a pulse of ultraviolet light. Unlike photo-activation, the Fluorochrome changes it's fluorescent color.

Energy Transfer Methods for Inter- and Intra-Molecular Interaction Measurements

Förster Resonance Energy Transfer (FRET)

• FRET uses dipole-dipole range coupling from an excited donor fluorochrome to another molecule or acceptor.

• Can be used to determine molecular interaction/molecular proximity (10 nm ) beyond resolution limits of the classical light microscope.

• Only works when there is a close proximity between the two molecules.

• FRET efficiency (EFRET):

  • Depends on distance, spectral overlap, and dipole orientation.

  • Scales with inverse 6th power law: $E<em>{FRET} = \frac{R</em>0^6}{R_0^6 + r^6}$

  • R0: Förster radius (50% FRET efficiency).

  • Effective range: 3–8 nm.

• Förster Distance depends on spectra overlap and the molecular orienation

  • $R<em>0 = 0.211 \Big( K^2n^{-4}Q</em>DD\Big)^{\frac{1}{6}}$

  • Q - Quantum Yield

  • J - Spectral Overlap Integral between donor + acceptor.

  • K - is dipole orientation function

FRET Couples

• Organic fluorochromes offer advantages over FPs (red-emitting dyes, higher photon counts, higher extinction coefficients, emission ranges outside autofluorescence window).

• ATTO dyes show excellent photostability and brightness.

• Lanthanide-based chelates (e.g., Eu(III), Tb(III), Sm(III)) have long fluorescence lifetimes and as such are ideal donors for time-resolved FRET measurements.

• First truly effective fluorescent protein FRET pair, devoid of problems such as poor photophysical properties and ineffective overlap integrals, consisted of CFP as the donor and YFP as the acceptor [.

• Wide range of genetically modified forms of FPs makes it possible to list a number of different FRET-pairs based on applications that are being resolved

• FRET theory is based on the assumption In a FRET couple, only a single donor and a single acceptor are present with very weak coupling.

Applications of FRET in Cell Biology

• FRET studies can lead to myriads of biological processes such as

  • Protein-protein interactions

  • Signal Transduction

  • Enzyme Activation

  • Calcium Signaling

  • Nucleic Acid Stuidies

  • Characterization of Gene Expression and Real-Time PCR assays

• FRET-biosensors utilize conformational change in a sensory domain to change FRET signal.

• There are three basic approaches

  • Interaction of the labeled proteins results in a FRET signal

  • Proteolysis of an intramolecular Labeled by molecules leatads to seperation of Donor and Acceptor

  • The labeled by molecule or intermolecularly in which ligand of substrate stimulation occurs leading to FRET increase.

• Other intermolecular FRET biosensors consisted of a CFP/YFP FRET pair to detect direct intermolecular integrin interactions

• Some advanced biosenesors is called “computational multiplexing” with are currently in use.

Approaches to FRET imaging

• Live-cell FRET microscopy combined with various techniques (anisotropy measurements, , and FLIM and super-resolution microscopy) to determine FRET.

Basic Priciples: Donor and Acceptor Photobleaching

• Donor Photobleaching involves measuring the bleaching rate of the donor

• Fluorochromes with longer lifetimes higher bleaching rates.

• Since the photobleaching time varies inversely with fluorescence, reduced photobleaching rate can be used to calculate FRET.

• The best way is to see the same specimen as it controls.

• Calculate the FRET efficiently use that: $E = 1 - I(Da)/I(D)$

  • Advantages: Relatively Straightforward to operate

    • Carried out on any light Fluorescence source.

    • Requires Only A single specimen preparation.

  • Disadvantages: that are some issues with Photo-damage in life cells

Summary of Steps for Acceptor-Photobleaching

1. Choose an appropriate FRET couple to perform their experiments..

2. Acquire images of the donor in the presence of the acceptor and of the acceptor. At a low laser intensity before bleaching.

3. Draw a Roi within the image, the bleaching area and the part in which the fret efficiency will be calculated

4. ZOOM IN on the Roi and photo bleach the exceptor with high laser intensity..

5. Zoom out to the original magnification and re-record the donor (ID) acceptor images (post-bleach)

6. Utilizing and algorithm that corrects for SBT is other unwanted artifacts, the fret signal can be consolidated.

7. Used cross correlation with Allen and images that are measured

8. Calculate the FRET efficiencies

Sensitized Emission

• Requires high quality, suitable, and specific filters with appropriate set of controls.

• This image channels the Fret signal to readily calculates from it

• The FRET is used with correction factors:

  • The youvan method- to correct the Fred signal and calculate it according to: Fc = F donor exceptor corrs

• Advantages : Sensitized Emissions and fret Measurements Can Be Performed on a widefield microscope And Confocal microscopeWith the respective dichloic and filter sets.

• Sensitized Emission FRET Measurements: reduce the risk of cross talk and other unwanted effects to a minimum, is a further development of the method and is named. SpECTRAL Imaging

Fluorescence Lifetime Imaging Microscopy (FLIM)

• Fluorescence and fluorochromes lifetime are mapped within images of living cells

• Is affected by energy transfer therefore essential

• Fret efficiency is calculated according to a formula: E = 1 - Tao (Da) / Taoh (D)

• Has been used for application of protein to protein and dynamics measurements with hgh temporal specificity.

Polarization Anisotropy Imaging

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• The polarization is defined as the intensity corrected difference as a function to the X citation polarization.

• The approach can be used with high sigal and it's well suited for application is high content screening.

Homo-FRET versus Hetero-FRET

• Hetero energy transfer only works between with those things are different this case and energy transfer occurs irreversible the donor to the receptor

• Homo energy transfer is almost the same as as hetero energy transfer where the donor transfers this is excited state energy the identical in closed position accepts it

Advances in Protein-Interaction Methods

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