3.3 Fluorescence and phosphorescence in biological systems
4 min read•august 14, 2024
Fluorescence and phosphorescence are key light-emitting processes in biological systems. They occur when molecules absorb light, get excited, and then emit light at longer wavelengths. These phenomena are crucial for many biophotonic applications.
Understanding fluorescence and phosphorescence helps us study biological molecules and processes. We can use them to visualize cells, measure protein interactions, and detect various substances in living systems. This knowledge is essential for developing new imaging and sensing techniques.
Fluorescence and Phosphorescence Principles
Luminescence and Excitation
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- Fluorescence and phosphorescence are forms of luminescence, the emission of light from a substance not resulting from heat
- Fluorescence occurs when a molecule absorbs light at one wavelength and emits light at a longer wavelength after a brief interval called the fluorescence
- Phosphorescence is similar to fluorescence but with a longer emission lifetime after the initial light absorption, often continuing to emit light for some time after the source is removed
- The principles of fluorescence and phosphorescence in biology involve the excitation of electrons within molecules to higher energy states and their subsequent relaxation, emitting photons of lower energy than the absorbed light
Biological Molecules Exhibiting Fluorescence and Phosphorescence
- In biological systems, fluorescence and phosphorescence are observed in various molecules
- Proteins (green fluorescent protein, )
- Nucleic acids (ethidium bromide-stained DNA)
- Small organic compounds (, )
- The chemical structure and environment of a fluorescent or phosphorescent molecule significantly impact its spectral properties and emission characteristics
Fluorescence vs Phosphorescence Mechanisms
Excitation and Emission Processes
- Both fluorescence and phosphorescence involve the absorption of light by a molecule, causing electrons to be excited to higher energy states
- In fluorescence, the excited electron quickly returns to its ground state by emitting a photon, typically within nanoseconds of the initial excitation
- Phosphorescence involves a transition to a triplet excited state, where the electron has a parallel spin to the ground state electron, making the transition back to the ground state "forbidden" and resulting in longer emission lifetimes (microseconds to seconds)
Stokes Shift and Wavelength Differences
- Fluorescence emission generally occurs at a longer wavelength than the absorbed light due to energy loss during the brief excited state lifetime, known as the
- Phosphorescence emission also occurs at a longer wavelength than the absorbed light but with a larger Stokes shift compared to fluorescence due to the longer excited state lifetime and greater energy loss
- The Stokes shift is a key spectral characteristic that influences the application of fluorescent and phosphorescent probes
Applications of Fluorescence and Phosphorescence in Biophotonics
Microscopy and Spectroscopy Techniques
- enables the visualization of specific cellular structures, proteins, and biomolecules by labeling them with fluorescent dyes or genetically encoded fluorescent proteins
- (STED, PALM, STORM)
- Fluorescence spectroscopy allows for the quantitative analysis of fluorescent molecules, providing information on their concentration, environment, and interactions within biological systems
- spectroscopy
Molecular Interactions and Sensing
- Fluorescence () is used to study protein-protein interactions and conformational changes by measuring the energy transfer between two fluorescent molecules
- Donor-acceptor pairs (CFP-YFP, FITC-rhodamine)
- Biosensors for enzyme activity and ion concentrations
- (PLIM) exploits the longer emission lifetimes of phosphorescent probes to study biological processes with reduced background interference and improved sensitivity
- Oxygen sensing in tissues and cell cultures
- Metal ion detection ()
- Fluorescence and phosphorescence-based sensors can be used to monitor various physiological parameters in living systems
- pH sensors (, )
- Calcium indicators (, )
Factors Influencing Fluorescence and Phosphorescence
Chemical Environment and Molecular Interactions
- pH can affect the protonation state of fluorescent molecules, altering their absorption and emission spectra and
- pH-sensitive dyes (fluorescein, BCECF)
- Intracellular pH measurements
- Solvent polarity can affect the energy levels of fluorescent molecules, leading to changes in their absorption and emission spectra (solvatochromism)
- Lipophilic dyes (Nile Red, Prodan)
- Membrane fluidity and polarity studies
- Molecular interactions, such as protein binding or aggregation, can alter the fluorescence and phosphorescence properties of a molecule by changing its chemical environment or conformation
- Protein-ligand binding assays
- Amyloid fibril formation and aggregation studies
Physical Factors and Quenching
- Temperature influences the non-radiative decay processes competing with fluorescence and phosphorescence, with higher temperatures generally leading to reduced emission intensity
- Temperature-sensitive dyes (, )
- Thermal imaging and temperature mapping in biological samples
- The presence of quenchers, such as oxygen or heavy metal ions, can reduce the fluorescence and phosphorescence intensity by promoting non-radiative decay pathways
- Oxygen-sensitive probes (ruthenium complexes, )
- Fluorescence quenching-based biosensors
Spectral Characteristics of Fluorescent and Phosphorescent Materials
Absorption and Emission Spectra
- Fluorescent and phosphorescent molecules exhibit characteristic absorption and emission spectra, which provide information on their electronic structure and environment
- The absorption spectrum represents the wavelengths of light that a molecule can absorb, while the shows the wavelengths at which the molecule emits light
- Spectral overlap between the emission of one fluorescent molecule and the absorption of another is crucial for applications such as FRET and multiplexed imaging
- Förster resonance energy transfer (FRET) pairs
- Spectral unmixing in multicolor imaging
Quantum Yield and Lifetime
- The fluorescence and phosphorescence quantum yield, the ratio of emitted photons to absorbed photons, is a measure of the emission efficiency and is affected by various environmental factors
- High quantum yield dyes (rhodamines, cyanines)
- Low quantum yield probes for environmental sensing
- The fluorescence and phosphorescence lifetimes, the average time a molecule spends in the excited state before emitting a photon, can provide information on the molecular environment and interactions
- Fluorescence lifetime imaging microscopy (FLIM)
- Time-resolved fluorescence spectroscopy for protein dynamics studies
Key Terms to Review (33)
BCECF: BCECF, or 2',7'-bis(carboxyethyl)carboxyfluorescein, is a fluorescent dye used primarily to measure intracellular pH and calcium levels in biological systems. This dye is sensitive to pH changes and emits fluorescence in response, making it a valuable tool in fluorescence microscopy and flow cytometry. Its ability to provide quantitative measurements of cellular conditions allows researchers to gain insights into various physiological processes and disease states.
Confocal Microscopy: Confocal microscopy is an advanced imaging technique that allows for the visualization of samples with high resolution and contrast by using point illumination and spatial filtering. This method improves optical sectioning, enabling the collection of thin slices of a specimen, which are then reconstructed into three-dimensional images. Confocal microscopy is particularly effective in studying biological samples labeled with fluorescent markers, making it a vital tool in cellular and molecular imaging.
Cy5: Cy5 is a fluorescent dye commonly used in biological research for labeling and detecting biomolecules. Its strong fluorescence properties make it ideal for applications in imaging, flow cytometry, and various types of assays, enhancing the visibility of the target molecules. In biological systems, Cy5 is valuable for studying cellular processes and interactions due to its high quantum yield and photostability.
Emission spectrum: An emission spectrum is the spectrum of light emitted by a substance when it transitions from a higher energy state to a lower energy state, releasing photons in the process. This spectrum is unique to each element or compound and reveals information about its electronic structure, making it essential for understanding processes like fluorescence and phosphorescence in biological systems and the use of fluorescent labels and probes.
Eu-tta: Eu-tta refers to a specific type of fluorescent protein that is important in biological systems, particularly for its role in cellular imaging and tracking. This protein can emit light when excited by a specific wavelength, making it a valuable tool in research to visualize cellular processes. Understanding eu-tta is crucial for advancements in fluorescence microscopy and bioimaging techniques.
Excitation: Excitation refers to the process in which an electron in a molecule absorbs energy and moves from a lower energy level to a higher energy level. This concept is crucial in understanding how biological molecules interact with light, leading to phenomena such as fluorescence and phosphorescence, as well as their applications in various spectroscopic techniques and labeling methods.
Exogenous Fluorescence: Exogenous fluorescence refers to the phenomenon where fluorescent signals are emitted from external molecules introduced into a biological system, as opposed to those generated by the organism's own cellular components. This process is often utilized in various applications, including imaging and diagnostics, where the external fluorescent agents interact with biological structures, allowing researchers to visualize and track cellular processes more effectively.
Flavins: Flavins are a group of organic compounds derived from riboflavin (vitamin B2) that serve as essential cofactors in various biological processes, particularly in enzymatic reactions. They play a crucial role in cellular metabolism and energy production by facilitating electron transfer within the mitochondria and other cellular compartments, making them vital for processes like oxidative phosphorylation.
Fluorescence imaging: Fluorescence imaging is a powerful optical technique that utilizes the fluorescence phenomenon to visualize and analyze biological samples. By illuminating a sample with a specific wavelength of light, fluorescent molecules within the sample emit light at a longer wavelength, allowing researchers to observe cellular structures, dynamics, and interactions in real-time. This technique is crucial for studying biological processes and has applications in diagnostics, drug discovery, and basic research.
Fluorescence microscopy: Fluorescence microscopy is a powerful imaging technique that uses fluorescent dyes or proteins to visualize biological specimens at the microscopic level. This method allows researchers to observe specific cellular components and processes in real-time, providing insights into the structure and function of cells and tissues.
Fluorescent tagging: Fluorescent tagging is a technique used to label biological molecules with fluorescent dyes, enabling visualization and tracking of cellular processes under a fluorescence microscope. This method enhances the ability to study the dynamics of proteins, nucleic acids, and other biomolecules in real time, as the tagged components emit light when excited by specific wavelengths. The ability to use fluorescent tags has revolutionized research in molecular biology and biochemistry, facilitating a deeper understanding of cellular functions.
Fluorimetry: Fluorimetry is an analytical technique that measures the intensity of fluorescent light emitted by a sample after it has absorbed light or other electromagnetic radiation. This technique is widely used in various fields, especially in biology, to quantify the concentration of fluorescent molecules in a sample, providing insights into cellular processes and molecular interactions.
FRET: FRET, or Förster Resonance Energy Transfer, is a powerful phenomenon in which energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore that is in close proximity, typically within 1 to 10 nanometers. This process plays a crucial role in understanding molecular interactions and dynamics in biological systems, particularly through fluorescence and phosphorescence techniques, as well as being a key principle in the design of quantum dot-based biosensors for detecting biomolecules.
Fura-2: Fura-2 is a fluorescent calcium indicator dye that allows for the measurement of intracellular calcium levels in real-time. This compound is particularly important in studying cellular signaling processes because it exhibits a change in fluorescence when it binds to calcium ions, providing valuable insights into various physiological and pathological processes within cells.
GFP: Green fluorescent protein (GFP) is a bioluminescent protein originally found in the jellyfish Aequorea victoria, which fluoresces green when exposed to ultraviolet or blue light. This property has made GFP a valuable tool in molecular biology, allowing researchers to visualize and track biological processes in real time. Its ability to be genetically encoded makes it particularly useful for studying dynamic events in live cells and organisms.
Indo-1: Indo-1 is a fluorescent calcium indicator that is widely used in biological research to measure intracellular calcium levels. This compound exhibits fluorescence properties that are sensitive to the concentration of calcium ions, making it valuable for understanding cellular signaling and processes related to calcium dynamics in live cells. The ability of Indo-1 to emit different fluorescence intensities based on calcium binding allows researchers to visualize and quantify calcium fluctuations within various biological systems.
Intrinsic Fluorescence: Intrinsic fluorescence refers to the natural emission of light by certain biological molecules when they absorb photons and then re-emit them. This phenomenon is crucial for understanding how biological systems utilize light for various processes, such as energy transfer, cellular signaling, and detecting the presence of specific biomolecules.
Lifetime: In the context of fluorescence and phosphorescence, lifetime refers to the average duration that a molecule remains in an excited state before returning to its ground state. This time span is crucial in understanding how long a fluorescent or phosphorescent material emits light after being excited by an external energy source. The lifetime can provide insights into the molecular environment, interactions, and energy transfer processes occurring within biological systems.
NADH: NADH, or nicotinamide adenine dinucleotide (reduced form), is a crucial coenzyme involved in cellular metabolism, primarily serving as an electron carrier in the process of energy production. It plays a significant role in the transfer of electrons during biochemical reactions, particularly in cellular respiration and fermentation processes, and is essential for ATP synthesis. Understanding NADH is key to comprehending how energy is harnessed and utilized within biological systems, especially in relation to fluorescence and phosphorescence mechanisms.
Phosphorescence lifetime imaging: Phosphorescence lifetime imaging is a powerful technique used to measure the time it takes for phosphorescent materials to emit light after being excited by a light source. This method provides crucial insights into the dynamics of excited states in biological systems, allowing researchers to study interactions at the molecular level and gain a better understanding of various biological processes. The technique relies on analyzing the decay time of emitted light, which can reveal information about the local environment, such as oxygen concentration, viscosity, and molecular interactions.
Porphyrins: Porphyrins are a group of organic compounds, characterized by their large, cyclic structures that contain four nitrogen-containing pyrrole rings. These compounds are essential in various biological systems, particularly as they serve as critical components of hemoglobin and chlorophyll, playing key roles in processes like oxygen transport and photosynthesis. Their unique structural features also allow them to exhibit fluorescence, making them important in fluorescence and phosphorescence studies.
Quantum Yield: Quantum yield is a measure of the efficiency of photon emission in a given process, defined as the ratio of the number of photons emitted to the number of photons absorbed. This concept is crucial in understanding how fluorescent and phosphorescent systems behave, particularly in biological contexts where these processes are essential for imaging and sensing applications.
R. J. B. H. van der Meer: R. J. B. H. van der Meer is a prominent scientist known for his significant contributions to the fields of fluorescence and phosphorescence, particularly in relation to biological systems. His work has helped advance the understanding of how these phenomena can be utilized in various applications, including medical diagnostics and bioimaging. Van der Meer's research focuses on the mechanisms of fluorescence and phosphorescence processes at a molecular level, shedding light on their implications in biological contexts.
Resonance Energy Transfer: Resonance energy transfer is a process where energy is transferred non-radiatively between two chromophores, typically involving an excited donor molecule and an acceptor molecule that is in close proximity. This mechanism is critical in biological systems, particularly in the context of fluorescence and phosphorescence, as it helps to facilitate energy transfer necessary for processes like photosynthesis and cellular signaling.
Rhodamine B: Rhodamine B is a synthetic fluorescent dye commonly used in various biological and chemical applications, characterized by its bright pink color and strong fluorescence properties. Its ability to emit light when excited by specific wavelengths makes it a valuable tool in fluorescence microscopy, biological labeling, and as a tracer in studies of fluid dynamics and environmental monitoring.
Ruthenium Complexes: Ruthenium complexes are coordination compounds that contain ruthenium, a transition metal, bonded to various ligands. These complexes are particularly significant in biological systems due to their unique electronic properties and potential applications in photonics and biosensing, where they can act as effective fluorescent and phosphorescent probes for imaging and detecting biological molecules.
Snarf: Snarf refers to the process by which fluorescent or phosphorescent molecules absorb light energy and subsequently emit light, often utilized in various biological systems for detection and imaging purposes. This term is particularly important in understanding how certain biomolecules can indicate the presence or condition of cells, as well as their interactions with their environment through the emission of light upon excitation.
Spectrophotometry: Spectrophotometry is a quantitative measurement technique that assesses how much light a substance absorbs at different wavelengths. This method is fundamental for understanding various optical properties of materials and is essential in analyzing biological samples, where it helps characterize fluorescence and phosphorescence, as well as in the development and functioning of photodetectors that convert light into electrical signals.
Steady-state fluorescence spectroscopy: Steady-state fluorescence spectroscopy is a technique used to measure the intensity of fluorescent light emitted by a sample when it is excited by a specific wavelength of light. This method provides important information about the environment, concentration, and dynamics of fluorescent molecules in biological systems, making it a key tool for analyzing various biochemical processes and molecular interactions.
Stokes Shift: Stokes Shift refers to the difference in wavelength between the peak absorption and the peak emission of a fluorescent molecule. This phenomenon is crucial in understanding how biological systems utilize fluorescence and phosphorescence for various functions, as it allows for clear detection of emitted light after a molecule absorbs energy. The concept plays an essential role in fluorescence spectroscopy, where the shift helps distinguish between excitation and emission wavelengths, making it easier to analyze biological samples.
Super-resolution microscopy: Super-resolution microscopy is an advanced imaging technique that allows researchers to visualize biological structures at a resolution beyond the diffraction limit of conventional light microscopy, achieving details on the nanoscale. This method utilizes various innovative strategies, such as fluorescence techniques and computational algorithms, to surpass the typical limitations faced in brightfield, fluorescence, and confocal microscopy. By enhancing imaging capabilities, super-resolution microscopy plays a crucial role in cellular and molecular imaging and supports the development of cutting-edge technologies in biophotonics.
Theodor Förster: Theodor Förster was a prominent German physicist renowned for his groundbreaking work in the fields of fluorescence and energy transfer, particularly the phenomenon known as Förster resonance energy transfer (FRET). His research established fundamental principles that describe how energy can be transferred between light-absorbing molecules, playing a crucial role in understanding fluorescence and phosphorescence in biological systems. Förster's theories have led to the development of various biosensing techniques that utilize energy transfer for measuring molecular interactions and dynamics in living organisms.
Time-resolved fluorescence: Time-resolved fluorescence is a technique that measures the decay time of fluorescent light emitted by molecules after they have been excited by a light source. This method provides insights into the dynamic processes occurring within biological systems, allowing researchers to analyze molecular interactions and conformational changes with high temporal resolution.