Optical Spectroscopy in Physics: Understanding Fluorescence and Jablonski Diagrams, Lab Reports of Physics

Download Optical Spectroscopy in Physics: Understanding Fluorescence and Jablonski Diagrams and more Lab Reports Physics in PDF only on Docsity! Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 1 - Lab 3: Organic Fluorophores - Reading Material References (1) Molecular Fluorescence: Principles and Applications, Bernard Valeur, Wiley-VCH, Weinheim, Germany (2002). Chapter 3: Characteristics of Fluorescence Emission. (2) Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz, Kluwer Academic / Plenum Publishers, New York, NY (1999). Chapter 3: Fluorophores. Jablonski diagram and electronic transitions The Jablonski diagram is a concise summary of the radiative and non-radiative transitions occurring between electronic states in a molecule. It comes in many forms with varying levels of complexity depending on the processes that are being considered. The figure below shows the transitions that are common to all systems. The thick, dark lines labeled S0, S1, and S2 correspond to singlet electronic states and those labeled T1 and T2 represent triplet electronic states. Vibrational levels are shown with thin lines for each state. The vertical arrows with straight lines represent radiative transitions, while the arrows with wavy lines represent non- radiative transitions. Absorption (~10-15 s) – At room temperature, the vast majority of molecules are in the lowest vibrational level of the ground state. Absorption of a photon of sufficient energy will excite the molecule from the ground state (S0) to an excited state (S1, S2, etc.). Generally, the excitation will be to a higher vibrational level of the excited state. We will focus mainly on photo- excitation, but it also possible to excite molecules via chemical reactions (chemiluminescence), thermal excitation (incandescence), and biological processes (bioluminescence). IC: internal conversion ISC: inter- system crossing vibrational relaxation Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 2 - Vibrational relaxation (~10-12 s) – This occurs within a given electronic state. When a molecule reaches a higher vibrational level of an electronic state, it will quickly relax to the lowest vibrational level of that state. The energy is released as heat to the surrounding solvent molecules. Internal conversion (IC, ~10-13 to 10-9 s) – This is a transition between two electronic states with the same energy (horizontal transition) and the same spin multiplicity (i.e. singlet-singlet or triplet-triplet). The rate will depend on the initial and final states of the transition. We will consider two cases, summarized in the following examples. (1) A molecule is excited to a higher vibrational level of the state S2. Vibrational relaxation occurs quickly to the lowest vibrational level of S2, then it undergoes internal conversion from S2 to S1, followed by vibrational relaxation to the lowest vibrational level of S1. The overall rate is ~10-13 to 10-11 s. This process explains why most transitions from excited to ground states occur from the lowest vibrational level of the first excited state S1 regardless of the initial excitation. (2) The molecule can then transit from the lowest vibrational level of S1 to a higher vibrational level of S0. This would be followed by rapid relaxation to the lowest vibrational level of the ground state. This mechanism is one of the non-radiative transitions that can occur during de-excitation. The time-scale for S1 to S0 internal conversion is ~10-11 to 10-9 s. The difference in rates for these processes is related to the greater energy difference between the initial and final states (i.e. S0-S1 energy difference is greater than S1-S2). Fluorescence (~10-10 to 10-8 s) – This, of course, is the radiative transition we are most interested in. It generally occurs from the lowest vibrational level of the first excited state S1 to a higher vibrational level of the ground state S0 (exceptions, though rare, do exist). Intersystem crossing (ISC, ~10-10 to 10-4 s) – This is a transition between states of different multiplicity (i.e. singlet-triplet or triplet-singlet), but the same energy (horizontal transition). It requires an interaction leading to a change of electronic spin. This can be achieved through interactions with other molecules or through spin-orbit coupling within the molecule (e.g. the heavy atom effect). For isolated or shielded molecules, it can lead to phosphorescence emission. For non-protected molecules in solution, it generally leads to non-radiative de-excitation because phosphorescence is a very slow process (~10-6 to 102 s), leaving many opportunities for non- radiative decay. Other processes – The above processes are relevant to almost any situation. In addition, there are other de-excitation processes that are dependent upon the local environment (e.g. interactions with other solute molecules). These include processes such as proton transfer, electron transfer, dynamic quenching, resonance energy transfer, photo-induced chemical reactions, structural changes, and excimer and exciplex formation. Later in the semester, we will cover the processes of dynamic quenching and resonance energy transfer in detail. Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 5 - Fluorophore properties Fluorescence probes come in myriad forms with a diverse array of properties and functionalities. Their tremendous diversity is what makes fluorescence spectroscopy such a rich and powerful field of study. When selecting the appropriate fluorophore for a specific application, many factors must be considered. The following table lists some of the important properties and gives examples of their relevance: property relevance absorption spectrum type of light sources available emission spectrum detector range, interference from autofluorescence Stokes shift separation of absorption and emission extinction coefficient efficiency of absorption quantum yield signal-to-noise ratio lifetime time scale of the process studied photostability # of photons collected before photobleaching, signal degradation polarization anisotropy of the probe solvent interactions polarity, pH, viscosity, temperature, pressure solute interactions energy transfer, specific binding, dynamic quenching Types of fluorophores There are many possible ways in which to group similar fluorophores into distinct classes. The following list is based mainly on their applications (e.g. protein labeling), but a few are based on similar fluorescence properties (e.g. long-lifetime dyes). (1) Intrinsic or natural fluorophores: Fluorescent compounds are found in many living systems. These include the fluorescent amino acids tryptophan, tyrosine, and phenylalanine found in proteins, as well as enzyme cofactors such as NADH, FAD, and riboflavin found in cells and tissues. While intrinsic fluorophores can be used to study cellular dynamics and protein structure, more often they serve as an unwanted background “autofluorescence” that must be separated from the desired signal. (2) Covalent protein-labeling fluorophores: These fluorophores are designed with reactive groups for labeling proteins with a fluorescent tag, allowing us to monitor their behavior. A wide variety of reactive derivatives are available. For example, sulfhydryl groups can be labeled through reaction with iodoacetamides or malemides and amine groups through reaction with isothiocyanates, N-hydroxysuccinimides, or sulfonyl chlorides. (3) Non-covalent protein-labeling fluorophores: These fluorophores have non- covalent interactions with proteins that often lead to a change in fluorescence Fluorescein-Isothiocyanate Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 6 - properties upon binding. For example, 1-anilinonaphthalene-8-sulfonic acid (ANS) is known to preferentially bind to hydrophobic pockets in proteins. In solution, ANS has a very weak greenish fluorescence, but after binding, it acquires a bright blue fluorescence. (4) DNA probes: There are many fluorophores that spontaneously bind to DNA and display enhanced emission upon binding. For example, ethidium bromide is only weakly fluorescent in water with a lifetime around 1.7 ns, but upon binding to double-stranded DNA, its fluorescence intensity increases 30-fold with the lifetime increasing to about 20 ns. Other types of DNA probes are designed specifically to bind (intercalate) within the major or minor grooves of double-stranded DNA or to bind preferentially to certain base sequences. (5) Chemical sensing probes: This class of fluorophores is used to quantify the amount of a given substance, often times ions like Na+, Ca2+, Cl-, and Mg2+. Binding of the ions to the fluorophore may cause such measurable effects as spectral shifts or changes in fluorescence intensity. For example, the probe Calcium Green shows about a 10-fold increase in intensity upon binding of Ca2+. (6) Environmental probes: This class includes many fluorophores that are sensitive to changes in solvent conditions such as pH, polarity, viscosity, or temperature. For example, this week we will study the molecule Prodan, for which the emission wavelength (and Stokes shift) is highly sensitive to the solvent polarity. (7) Membrane probes: This class of fluorophores is used for labeling membranes. For example, hydrophobic probes such as DPH preferentially bind to the non-polar regions of membranes, where they are highly fluorescent, while those remaining in the aqueous phase have minimal fluorescence. More hydrophilic probes like fluorescein or Texas Red can be forced to localize to the membrane by attaching long acyl chains to them. There are also membrane-specific probes with fluorescence properties that depend on the electric potential across the membrane. (8) Red and near-IR dyes: Long-wavelength dyes are of interest largely because they minimize problems with autofluorescence background in cells and tissues. This is true because autofluorescence is strongest in the UV and blue and generally decreases as the wavelength is increased. This class of dyes is also excitable with simpler (i.e. cheaper) laser sources such as laser diodes. The cyanine dyes Cy-3, Cy-5, and Cy-7 are examples of long-wavelength dyes that have received extensive use in DNA sequencing. PRODAN Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Liu - 7 - (9) Long-lifetime dyes: Most organic fluorophores (with only a few exceptions) have lifetimes between about 1 and 10 ns. This severely limits the range of dynamic processes that can be studied using these dyes. Fortunately, there are a few types of organometallic fluorophores that have much longer lifetimes. Perhaps the most commonly employed are the lanthanides Eu3+ and Tb3+, which are fluorescent in aqueous solutions and have lifetimes around 0.5 to 3 ms. Unfortunately, they also have very low extinction coefficients (less than 10 L/mol-cm), so they must be chelated with organic ligands that absorb light efficiently and transfer the energy to the metal ion. (10) Fluorescent proteins (visible): There are several classes of proteins that have intrinsic fluorescence at longer wavelengths than that discussed above for the aromatic amino acids. The most common and widely used of these are the green fluorescent proteins (GFPs). GFP is derived from a bioluminescent jellyfish and contains a highly fluorescent chromophore surrounded by a protective β-barrel structure. The GFPs are particularly useful because the chromophore forms spontaneously (from a 3 amino acid sequence) during protein folding and does not require any enzymes for catalysis. Thus, the gene for GFP is often inserted directly into other genes to synthesize proteins with a GFP molecule already attached. A range of colors are available by making specific mutations. GFP chromophore