Two-photon Fluorescence Cross-correlation Spectroscopy Research Articles

Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime Analysis.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions suchas protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures suchas lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guideof how to set up such a tool.

Lipoplexes for Gene Delivery Characterized by Fluorescence Correlation Spectroscopy

Liposomes composed of cationic lipids have been extensively investigated as drug delivery systems [1] and carrier for gene therapy and regenerative medicine as an alternative to viral approaches [2]. One exemplary regenerative medicine concept is the use of induced pluripotent stem cells (iPS) reprogrammed through the introduction of pluripotency genes such as OCT4, Sox2, KLF4 and c-Myc [3]. In this work, we study the complexation of adequate liposome formulations with these four genes through Fluorescence Cross-Correlation Spectroscopy (FCCS). [4]Liposomes stained with the carbocyanine dye 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD) and fluorescently labelled plasmids carrying the OCT4, Sox2, KLF4 and c-Myc genes are characterized via single photon FCCS studies with a confocal microscope (LSM780, Zeiss, Oberkochen, Germany). Using CX-rhodamine and fluorescein as fluorescent labelling for the genes, we create two complementary wavelength windows to the far red wavelength window necessary to observe DiD fluorescence. Thereby, we create the possibility to monitor liposome loading with more than one gene.[1] Allen, T. M. & Cullis, P. R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev. 65, 36-48 (2013).[2] Niidome, T. & Huang, L. Gene Therapy Progress and Prospects: Nonviral vectors. Gene Ther. 9, 1647-1652 (2002).[3] Takahashi, K. & Yamanaka, S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126, 663-676 (2006).[4] Merkle, D., Lees-Miller, S. P. & Cramb, D. T. Structure and Dynamics of Lipoplex Formation Examined Using Two-Photon Fluorescence Cross-Correlation Spectroscopy. Biochemistry 43, 7263-7272 (2004).

Analysis of DNA-Dependent Protein Kinase-Mediated DNA End Joining by Two-Photon Fluorescence Cross-Correlation Spectroscopy

Nonhomologous end joining (NHEJ) is the primary mechanism by which mammalian cells repair DNA double-strand breaks (DSBs). Proteins known to play a role in NHEJ include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the Ku 70/Ku 80 heterodimer (Ku), XRCC4, and DNA ligase IV. One of the main roles of the DNA-PKcs-Ku complex is to bring the ends of the DSB together in a process termed synapsis, prior to end joining. Synapsis results in the autophosphorylation of DNA-PKcs, which is required to make the DNA ends available for ligation. Here, we describe a novel assay using two-photon fluorescence cross-correlation spectroscopy that allows for the analysis of DNA synapsis and end joining in solution using purified proteins. We demonstrate that although autophosphorylation-defective DNA-PKcs does not support DNA ligase-mediated DNA end joining, like wild-type (WT) DNA-PKcs, it is capable of Ku-dependent DNA synapsis in solution. Moreover, we show that, in the presence of Ku, both WT DNA-PKcs and autophosphorylation-defective DNA-PKcs promote the formation of multiple, large multi-DNA complexes in solution, suggesting that, rather than align two opposing DNA ends, multiple DNA-PK molecules may serve to bring multiple DNA ends into the NHEJ complex.

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.

Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry.

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated, and autophosphorylated forms of the enzyme. Key binding parameters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.

Structure and dynamics of lipoplex formation examined using two-photon fluorescence cross-correlation spectroscopy.

The conditions required to form transfectable lipoplexes have been extensively studied [Zuhorn, I. S., and Hoekstra, D. (2002) J. Membr. Biol. 189, 167-179]. However, to date, experiments have not addressed either the order of events of lipoplex formation in solution or the maximum number of DNA molecules per vesicle in stable single-vesicle lipoplexes. In this study, we have employed two-photon excitation fluorescence correlation spectroscopy (TPE-FCS) and two-photon fluorescence cross-correlation spectroscopy (TPE-XCS) to examine both fluorescence-labeled DNA and cationic vesicle structure and dynamics simultaneously. The dependence of large aggregated lipoplex formation on DNA-to-cationic lipid charge ratio was determined, as was the maximum number of 40 bp double-stranded DNA oligonucleotides able to bind to a single vesicle.

Detection of specific DNA sequences using dual-color two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a biomedical research tool. FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. They can also serve as a diagnostic, detecting particular molecular species with high sensitivity and specificity. We here demonstrate that dual-color FCS measurements can be applied to detect and quantify the concentration of specific non-fluorescent molecular species without requiring any modifications to the molecule of interest. We demonstrate this capability by applying dual-color two-photon fluorescence cross-correlation spectroscopy to detect single stranded gamma tubulin DNA in solution with high sensitivity. This quantification is independent of molecular size, and the methods introduced can be extended to measurements in complex environments such as within living cells.

Intracellular calmodulin availability accessed with two-photon cross-correlation.

The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca(2+) signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca(2+)/CaM-dependent protein kinase II is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca(2+)/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca(2+)/CaM-dependent protein kinase II in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein-protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.