Journal articles: 'Two-photon excitation fluorescence of proteins'

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Diaspro, Alberto, Giuseppe Chirico, and Maddalena Collini. "Two-photon fluorescence excitation and related techniques in biological microscopy." Quarterly Reviews of Biophysics 38, no. 2 (May 2005): 97–166. http://dx.doi.org/10.1017/s0033583505004129.

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1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.

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Paul, Uchenna P., Li, Milton L. Lee, and Paul B. Farnsworth. "Compact Detector for Proteins Based on Two-Photon Excitation of Native Fluorescence." Analytical Chemistry 77, no. 11 (June 2005): 3690–93. http://dx.doi.org/10.1021/ac048161z.

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Wan, H., C. Soeller, D. R. Garrod, C. Robinson, and M. B. Cannell. "Quantitative Immunocytochemistry of Proteins Using 2- Photon Microscopy and Digital Image Analysis." Microscopy and Microanalysis 4, S2 (July 1998): 416–17. http://dx.doi.org/10.1017/s1431927600022200.

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The two photon microscope provides optical sectioning of fluorescent specimens with a resolution comparable to that obtained in confocal microscopy (see refs 2,3). However, the excited volume in 2-photon microscopy is limited to the focal volume (unlike conventional fluorescence microscopy where excitation occurs throughout the specimen). This means that photodamage is limited to the plane of section being examined. Thus, the light emitted from each point in the specimen depends on the amount of fluorochrome present without the problem of prior illumination (of other planes within the specimen) reducing the photon yield so a better signal to noise ratio can be obtained when examination of multiple image planes is needed. Since 2-photon excitation spectra are wide and chromatic aberration is eliminated (because the emitted light does not have to be focused on a pinhole), it is possible to excite several fluorochromes simultaneously and map their positions with high accuracy.

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Botchway, Stanley W., Ignasi Barba, Randolf Jordan, Rebecca Harmston, Peter M. Haggie, Simon-Peter Williams, Alexandra M. Fulton, Anthony W. Parker, and Kevin M. Brindle. "A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging." Biochemical Journal 390, no. 3 (September 5, 2005): 787–90. http://dx.doi.org/10.1042/bj20050648.

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A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

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Kierdaszuk, Borys, Ignacy Gryczynski, Anna Modrak-Wojcik, Agnieszka Bzowska, David Shugar, and Joseph R. Lakowicz. "FLUORESCENCE OF TYROSINE AND TRYPTOPHAN IN PROTEINS USING ONE- AND TWO-PHOTON EXCITATION." Photochemistry and Photobiology 61, no. 4 (April 1995): 319–24. http://dx.doi.org/10.1111/j.1751-1097.1995.tb08615.x.

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Chirico, G., A. Diaspro, F. Cannone, M. Collini, S. Bologna, V. Pellegrini, and F. Beltram. "Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins Induced by Two-Photon Excitation." ChemPhysChem 6, no. 2 (February 11, 2005): 328–35. http://dx.doi.org/10.1002/cphc.200400318.

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Wang, Ke, Tzu-Ming Liu, Juwell Wu, Nicholas G. Horton, Charles P. Lin, and Chris Xu. "Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy." Biomedical Optics Express 3, no. 9 (July 31, 2012): 1972. http://dx.doi.org/10.1364/boe.3.001972.

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Cannell, M. B., and C. Soeller. "Optical Sectioning in Fluorescence Microscopy by Confocal and 2-Photon Molecular Excitation Techniques." Microscopy Today 5, no. 8 (October 1997): 12–15. http://dx.doi.org/10.1017/s1551929500056741.

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Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labeled materials (such as intracellular ions and proteins) in both fixed and living cells, However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane, This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 µm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source.

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Cannell, M. B., and C. Soeller. "High Resolution Imaging Using Confocal and 2-photon Molecular Excitation Microscopy." Microscopy Today 8, no. 5 (June 2000): 20–26. http://dx.doi.org/10.1017/s1551929500065196.

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Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labelled materials (such as intracellular ions and proteins) in both fixed and living cells. However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane. This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 μm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source. This converts the microscope from an imaging system to a point detector and images are produced by scanning the illuminating and detecting point over the specimen to build an image (in much the same way that a television set produces an image). The basic idea behind the confocal system is shown in Figure 1, and it should be noted that light from points outside the focal plane is defocused at the pinhole and so does not pass through the pinhole efficiently.

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Tao, Wen, Michael Rubart, Jennifer Ryan, Xiao Xiao, Chunping Qiao, Takashi Hato, Michael W. Davidson, Kenneth W. Dunn, and Richard N. Day. "A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy." American Journal of Physiology-Cell Physiology 309, no. 11 (December 1, 2015): C724—C735. http://dx.doi.org/10.1152/ajpcell.00182.2015.

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The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

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Olivini, Francesca, Sabrina Beretta, and Giuseppe Chirico. "Two-Photon Fluorescence Polarization Anisotropy Decay on Highly Diluted Solutions by Phase Fluorometry." Applied Spectroscopy 55, no. 3 (March 2001): 311–17. http://dx.doi.org/10.1366/0003702011951713.

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We report here the phase-sensitive measurements of two-photon fluorescence polarization anisotropy (FPA) on highly diluted solutions. We first describe the characterization of the response of the two-photon microscope to the light polarization and its test by means of measurements of the FPA on rhodamine 6G as a function of the viscosity. Further, we report the study of the FPA at high dilutions on a globular protein, the beta-lactoglobulin B (BLG) labeled with Alexa 532. The number of molecules (from 0.4 to 17 molecules per excitation volume) is measured by means of fluorescence correlation spectroscopy (FCS). The average rotational and translational diffusion coefficients measured with the FPA and FCS methods are in good agreement with the protein size and do not show a substantial dependence on the protein concentration, despite the very low signal (≌3 times the background) observed for highly diluted solutions.

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WASYLEWSKI, Zygmunt, Henryk KOLOCZEK, Alicja WASNIOWSKA, and Krystyna SIZOWSKA. "Red-edge excitation fluorescence measurements of several two-tryptophan-containing proteins." European Journal of Biochemistry 206, no. 1 (May 1992): 235–42. http://dx.doi.org/10.1111/j.1432-1033.1992.tb16921.x.

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Stapelfeldt, Henrik, and Leif H. Skibsted. "Modification of β–lactoglobulin by aliphatic aldehydes in aqueous solution." Journal of Dairy Research 61, no. 2 (May 1994): 209–19. http://dx.doi.org/10.1017/s0022029900028223.

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SummaryEach of the secondary lipid oxidation products pentanal, hexanal and heptanal was found to react with β–lactoglobulin (β–lg) in a two-phase model System (aqueous phosphate buffer–1-octanol) yielding fluorescent condensation products (emission maximum, 410 nm; excitation maximum, 350 nm). Protein polymers were detected by size-exclusion HPLC, and the rate of reaction paralleled the formation of fluorescent products, with the reactivity being pentanal > hexanal > heptanal. Simultaneously, the reaction also changed the intrinsic fluorescence of β–lg, and in particular pentanal reduced the intensity of tryptophan fluorescence (emission maximum, 332 nm; excitation maximum, 288 nm) by 30%. These findings are discussed with reference to the effect of peroxidizing lipids on the physical properties of whey proteins and the use of protein fluorescence (induced by the reaction with aldehydes) as marker for the oxidative status of milk and whey protein products.

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Roorda, Robert D., Tobias M. Hohl, Ricardo Toledo-Crow, and Gero Miesenböck. "Video-Rate Nonlinear Microscopy of Neuronal Membrane Dynamics With Genetically Encoded Probes." Journal of Neurophysiology 92, no. 1 (July 2004): 609–21. http://dx.doi.org/10.1152/jn.00087.2004.

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Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.

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Mazo-Vargas, Anyimilehidi, Heungwon Park, Mert Aydin, and Nicolas E. Buchler. "Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy." Molecular Biology of the Cell 25, no. 22 (November 5, 2014): 3699–708. http://dx.doi.org/10.1091/mbc.e14-07-1187.

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Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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Bal, Ufuk, Volker Andresen, Brenda Baggett, and Urs Utzinger. "Intravital Confocal and Two-Photon Imaging of Dual-Color Cells and Extracellular Matrix Mimics." Microscopy and Microanalysis 19, no. 1 (February 2013): 201–12. http://dx.doi.org/10.1017/s1431927612014080.

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AbstractWe report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

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Zhang, Mingshu, Zhifei Fu, and Pingyong Xu. "Extending the spatiotemporal resolution of super-resolution microscopies using photomodulatable fluorescent proteins." Journal of Innovative Optical Health Sciences 09, no. 03 (May 2016): 1630009. http://dx.doi.org/10.1142/s1793545816300093.

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In the past two decades, various super-resolution (SR) microscopy techniques have been developed to break the diffraction limit using subdiffraction excitation to spatially modulate the fluorescence emission. Photomodulatable fluorescent proteins (FPs) can be activated by light of specific wavelengths to produce either stochastic or patterned subdiffraction excitation, resulting in improved optical resolution. In this review, we focus on the recently developed photomodulatable FPs or commonly used SR microscopies and discuss the concepts and strategies for optimizing and selecting the biochemical and photophysical properties of PMFPs to improve the spatiotemporal resolution of SR techniques, especially time-lapse live-cell SR techniques.

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Lakowicz, J. R., I. Gryczynski, L. Tolosa, J. D. Dattelbaum, F. N. Castellano, L. Li, and G. Rao. "Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes." Acta Physica Polonica A 95, no. 1 (January 1999): 179–96. http://dx.doi.org/10.12693/aphyspola.95.179.

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Ruehr, M. L., and M. Bond. "Analisis of PKA Binding to A-Kinase Anchoring Proteins Using Fluorescence Resonance Energy Transfer." Microscopy and Microanalysis 5, S2 (August 1999): 508–9. http://dx.doi.org/10.1017/s1431927600015865.

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The cAMP dependent-protein kinase (PKA) signaling pathway plays a key role in the sympathetic regulation of muscle contraction in adult cardiac myocytes. This pathway may be regulated via compartmentalization of its components, whereby A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular areas to give each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid, and the regulatory subunit of PKA (R).Fluorescence resonance energy transfer (FRET) was used to monitor binding events between the type II regulatory subunit of PKA (RE) and Ht31 or Ht31P, a mutated form of Ht31 which does not bind RII. Each protein was fused to a derivative of the green fluorescent protein (GFP) so that the excitation-emission spectra of the two fluorescent proteins overlap.

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Kohler, R. H., P. Schwille, W. W. Webb, and M. R. Hanson. "Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy." Journal of Cell Science 113, no. 22 (November 15, 2000): 3921–30. http://dx.doi.org/10.1242/jcs.113.22.3921.

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Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

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Carillo, Maria Antonietta, Mathieu Bennet, and Damien Faivre. "Interaction of Proteins Associated with the Magnetosome Assembly in Magnetotactic Bacteria As Revealed by Two-Hybrid Two-Photon Excitation Fluorescence Lifetime Imaging Microscopy Förster Resonance Energy Transfer." Journal of Physical Chemistry B 117, no. 47 (November 11, 2013): 14642–48. http://dx.doi.org/10.1021/jp4086987.

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Lee, Jiung-De, Ping-Chun Huang, Yi-Cheng Lin, Lung-Sen Kao, Chien-Chang Huang, Fu-Jen Kao, Chung-Chih Lin, and De-Ming Yang. "In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions." Microscopy and Microanalysis 14, no. 6 (November 6, 2008): 507–18. http://dx.doi.org/10.1017/s1431927608080628.

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AbstractThe high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)–labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)–labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (τpeak) and the left side of the half-peak width (τ1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

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Gautam, Saurabh, and Munishwar N. Gupta. "Solid state fluorescence of proteins in high throughput mode and its applications." F1000Research 2 (March 25, 2019): 82. http://dx.doi.org/10.12688/f1000research.2-82.v2.

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Direct comparison between fluorescence spectra of a sample in solution and solid state form is valuable to monitor the changes in protein structure when it is “dried” or immobilized on a solid surface (for biocatalysis or sensor applications). We describe here a simple method for recording fluorescence emission spectra of protein powders without using any dedicated accessory for solid samples in a high-throughput format. The 96-well plate used in our studies, was coated black from all the sides and the excitation and emission paths are identical and are from the top of the well. These two features minimize scatter and provide fairly noise free spectra. Even then the fluorescence intensity may be dependent upon many factors such as the extent of protein aggregation, morphology and sizes of the protein particles. Hence, (changes in) λmax emission may be a more reliable metric in the case of fluorescence spectra of proteins in the solid state. However, any large changes in the intensity could indicate changes in the microenvironment of the fluorophore. The fluorescence emission spectra were blue-shifted (4 to 9 nm), showed an increase in the intensity for different proteins studied upon lyophilization, and were similar to what has been reported by others using available commercial accessories for solid state samples. After validating that our method worked just as well as the dedicated accessories, we applied the method to compare the fluorescence emission spectra of α-chymotrypsin in solution, precipitated form, and the lyophilized powder form. We further examined the fluorescence emission spectra of green fluorescent protein (GFP) in solution and solid form. We also analyzed fluorescence resonance energy transfer (FRET) between tryptophan (Trp57) and the cyclic chromophore of GFP. These findings pointed towards the change in the microenvironment around the cyclic chromophore in GFP upon lyophilization.

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Nguyen, Trinh T., and David T. Cramb. "Elucidation of the mechanism and energy barrier for anesthetic triggered membrane fusion in model membranes." Canadian Journal of Chemistry 97, no. 6 (June 2019): 474–82. http://dx.doi.org/10.1139/cjc-2018-0405.

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Membrane fusion is vital for cellular function and is generally mediated via fusogenic proteins and peptides. The mechanistic details and subsequently the transition state dynamics of membrane fusion will be dependent on the type of the fusogenic agent. We have previously established the potential of general anesthetics as a new class of fusion triggering agents in model membranes. We employed two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to report on vesicle association kinetics and steady-state fluorescence dequenching assays to monitor lipid mixing kinetics. Using halothane to trigger fusion in 110 nm diameter dioleoylphosphatidylcholine (DOPC) liposomes, we found that lipid rearrangement towards the formation of the fusion stalk was rate limiting. The activation barrier for halothane induced membrane fusion in 110 nm vesicles was found to be ∼40 kJ mol−1. We calculated the enthalpy and entropy of the transition state to be ∼40 kJ mol−1 and ∼180 J mol−1 K−1, respectively. We have found that the addition of halothane effectively lowers the energy barrier for membrane fusion in less curved vesicles largely due to entropic advantages.

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Batista, A., C. Loureiro, J. Domingues, J. S. Silva, and A. M. Morgado. "Corneal Metabolic State Assessment by Fluorescence Lifetime Imaging Microscopy." Microscopy and Microanalysis 19, S4 (August 2013): 7–8. http://dx.doi.org/10.1017/s1431927613000652.

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A long time objective of ophthalmologists is to diagnose corneal cells dysfunction prior to its pathologic expression. With this motivation, we are currently developing a new instrument for in vivo metabolic imaging of corneal tissues.Metabolic alterations are known to be the first sign of several corneal pathologies and can be assessed through non-invasive monitoring of metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The quantification of the relative proportions between free and protein-bound NADH and FAD can be achieved using fluorescence lifetime-resolved methods. This approach has already been applied in age-related macular degeneration, diabetic retinopathy and epithelial cancer.FAD and NADH imaging can be performed by one-photon excitation (1PE) and two-photon excitation (2PE) techniques. The latest has the advantage of allowing simultaneous excitation of both metabolic co-factors. However, there are still safety concerns when considering in vivo ocular studies in humans using 2PE.Due to these concerns we used, as a first approach, a 1PE system for evaluating the feasibility of corneal FAD imaging. The use of FAD has advantages over NADH. It can be excited over longer excitation wavelengths, is more resistant to photo-bleaching and is located exclusively in the mitochondrial space.A PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan) was used to monitor FAD autofluorescence. The instrument uses a 440 nm pulsed diode laser (330 ps) running at a pulse rate of 40 MHz. The instrument was modified by us to allow the acquisition of both fluorescence lifetime and reflectance images and to enhance scattered light rejection.Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH, Berlin, Germany). The fluorescence decay times were obtained after applying a non-linear least square fit to the decay data and the goodness of fit was evaluated by the analysis of the residuals and the chi-squared (χ2).We have acquired fluorescence lifetime images of ex vivo healthy Wistar rat corneas (Fig.1) using two different instrument setups: 1- using the emission filters provided by the manufacturer; 2- placing extra emission filters to fully reject the scattered excitation light. In both setups, FAD fluorescence data presented a bi-exponential decay with a short (protein-bound FAD) and a longer (free FAD) lifetime component.While both setups provide FAD fluorescence decays, only the second retrieves valid metabolic information. We obtained two lifetime components, one of 0.118 (0.028) ns and another of 2.11 (0.16) ns, with a relative contributions of 39.4 (2.2) and 60.6 (2.2), respectively. These values are in accordance with the literature.Corneal layer discrimination is possible based on morphologic characteristics. However, the fluorescence lifetime images do not provide morphological detail (Fig.1), possibly because FAD is only present in the mitochondria. These organelles are small and tend to accumulate around the nuclei.So, we modified the instrument’s optical setup to allow the acquisition of both fluorescence lifetime images and reflectance images. Figure 2 shows an example of the corneal epithelial layer.The image resolution and depth penetration are still not ideal. Since the assessment of corneal endothelial layer metabolic function is also within our goals, we are currently implementing further modifications to improve both the instrument’s resolution and depth penetration.The characterization of FAD fluorescence lifetime in unhealthy corneas is important to detect corneal dysfunctions prior to its pathologic expression. Therefore, we intend to study metabolic altered Wistar rat corneas. The alterations will be induced by potassium cyanide, which is a reversible inhibitor of the fourth complex of the mitochondrial electron transport chain.Financial support received from the Fundação para a Ciência e a Tecnologia under the research projects PTDC/SAU-BEB/104183/2008 and PTDC/SAU-ENB/122128/2010.

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Gallo, Eugenio, Sophia Wienbar, Avin C. Snyder, Kalin V. Vasilev, Bruce A. Armitage, and Jonathan W. Jarvik. "A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families." Protein & Peptide Letters 21, no. 12 (November 5, 2014): 1289–94. http://dx.doi.org/10.2174/0929866521666140616121800.

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Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.

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McGuinness, Colin D., Kulwinder Sagoo, David McLoskey, and David J. S. Birch. "Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295nm light-emitting diode and time-correlated single-photon counting." Applied Physics Letters 86, no. 26 (June 27, 2005): 261911. http://dx.doi.org/10.1063/1.1984088.

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Takai, Akira, Masahiro Nakano, Kenta Saito, Remi Haruno, Tomonobu M. Watanabe, Tatsuya Ohyanagi, Takashi Jin, Yasushi Okada, and Takeharu Nagai. "Expanded palette of Nano-lanterns for real-time multicolor luminescence imaging." Proceedings of the National Academy of Sciences 112, no. 14 (March 23, 2015): 4352–56. http://dx.doi.org/10.1073/pnas.1418468112.

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Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ∼20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca2+ dynamics in different cellular compartments in a single cell.

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Dramićanin, Tatjana, Lea Lenhardt, Ivana Zeković, and Miroslav D. Dramićanin. "Biophysical characterization of human breast tissues by photoluminescence excitation-emission spectroscopy." Journal of Research in Physics 36, no. 1 (January 1, 2012): 53–62. http://dx.doi.org/10.2478/v10242-012-0013-z.

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Abstract Fluorescence excitation-emission spectroscopy was used to investigate specimens of normal and malignant human breast tissues. Measurements were performed in two spectral regions: in the excitation range from 335nm to 400nm and emission range from 430nm to 625 nm, and in the excitation range from 400nm to 470nm and emission range from 500nm to 640 nm. It was found that fluorescence spectra are composed mainly of the emissions of extracellular proteins and that the differences in the intensity of their emissions reveal the changes in the tissue structure and morphology. These differences could be best observed in the emission spectra excited with 370 nm, 425nm and 455nm radiation. Statistical analysis revealed several spectral subregions that exhibited extremely significant statistical difference between normal and malignant breast tissues. The origin of these differences was elaborated, and prospects for optical diagnostics of breast cancer was discussed.

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Morgan, M. Thomas, Daisy Bourassa, Shefali Harankhedkar, Adam M. McCallum, Stephanie A. Zlatic, Jenifer S. Calvo, Gabriele Meloni, Victor Faundez, and Christoph J. Fahrni. "Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells." Proceedings of the National Academy of Sciences 116, no. 25 (June 3, 2019): 12167–72. http://dx.doi.org/10.1073/pnas.1900172116.

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Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

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Tarver, Crissy L., and Marc Pusey. "A low-cost method for visible fluorescence imaging." Acta Crystallographica Section F Structural Biology Communications 73, no. 12 (November 10, 2017): 657–63. http://dx.doi.org/10.1107/s2053230x17015941.

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A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

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Zhang, Xi, Mingshu Zhang, Dong Li, Wenting He, Jianxin Peng, Eric Betzig, and Pingyong Xu. "Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy." Proceedings of the National Academy of Sciences 113, no. 37 (August 25, 2016): 10364–69. http://dx.doi.org/10.1073/pnas.1611038113.

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Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent “on” to “off” state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm2) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

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Kosuge, Kotaro, Ryutaro Tokutsu, Eunchul Kim, Seiji Akimoto, Makio Yokono, Yoshifumi Ueno, and Jun Minagawa. "LHCSR1-dependent fluorescence quenching is mediated by excitation energy transfer from LHCII to photosystem I in Chlamydomonas reinhardtii." Proceedings of the National Academy of Sciences 115, no. 14 (March 19, 2018): 3722–27. http://dx.doi.org/10.1073/pnas.1720574115.

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Photosynthetic organisms are frequently exposed to light intensities that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O2, which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosynthetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii, the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipation of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii, we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light-harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1.

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Li, Huali, Huaina Yu, and Tongsheng Chen. "Partial Acceptor Photobleaching-Based Quantitative FRET Method Completely Overcoming Emission Spectral Crosstalks." Microscopy and Microanalysis 18, no. 5 (October 2012): 1021–29. http://dx.doi.org/10.1017/s1431927612001110.

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AbstractBased on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

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Wang, Run, and Frank V. Bright. "Comparison between Covalent Attachment and Physisorption of 2-(p-Toluidinyl)naphthalene-6-Sulfonate (TNS) to Proteins." Applied Spectroscopy 47, no. 6 (June 1993): 800–806. http://dx.doi.org/10.1366/0003702934066929.

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Previously, we studied the physisorption of 2-( p-toluidinyl)naphthalene-6-sulfonate (TNS) to the hydrophobic sites or cavities of bovine serum albumin (BSA) and determined the effects of acrylamide (quencher) and urea (denaturant) on the complex using steady-state and dynamic fluorescence spectroscopy. In this paper, we extend this work to bovine β-lactoglobulin (β-Lg) and immunoglobulin G (IgG), and compare the complexes to the covalently attached TNS. These results show that the hydrophobic sites of different proteins can be probed by TNS with the use of steady-state and time-resolved fluorescence spectroscopy. The TNS binding site in β-Lg is the most hydrophobic in comparison to BSA and IgG. The excited-state decay kinetics for the TNS/protein complexes is best described by a distribution (Gaussian) plus a discrete decay model. TNS/protein adducts (covalent attachment of TNS), whereby the TNS sulfonate is converted to a sulfonamido group, strongly affects the probe photophysics. For example, a red shift is observed in the emission and excitation spectra due to covalent attachment. Acrylamide quenches the fluorescence of TNS/protein complexes, but does not quench 2-( p-toluidinyl)naphthalene-6-(N-propyl)-sulfonamide(TNS-PA) or the covalent TNS/protein adducts. Furthermore, the fluorescence decay kinetics of the covalent TNS/protein adducts is always best described by a double discrete decay model. In all cases studied, added urea does not affect the recovered lifetime values but does decrease the pre-exponential factor for the longer lifetime component. This result suggests a single-step urea denaturation mechanism involving two protein states—folded (native) and unfolded (denatured).

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Khan, Faez Iqbal, Fakhrul Hassan, Razique Anwer, Feng Juan, and Dakun Lai. "Comparative Analysis of Bacteriophytochrome Agp2 and Its Engineered Photoactivatable NIR Fluorescent Proteins PAiRFP1 and PAiRFP2." Biomolecules 10, no. 9 (September 7, 2020): 1286. http://dx.doi.org/10.3390/biom10091286.

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Two photoactivatable near infrared fluorescent proteins (NIR FPs) named “PAiRFP1” and “PAiRFP2” are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAiRFP2 were more deviated than Agp2 from its native conformation and the hydrophobic regions that were buried in PAiRFP1 and PAiRFP2 core exposed to solvent molecules. The eigenvalues and the trace of covariance matrix were found to be high for PAiRFP1 (597.90 nm2) and PAiRFP2 (726.74 nm2) when compared with Agp2 (535.79 nm2). It was also found that PAiRFP1 has more sharp Gibbs free energy global minima than Agp2 and PAiRFP2. This comparative analysis will help to gain deeper understanding on the structural changes during the evolution of photoactivatable NIR FPs. Further work can be carried out by combining PCR-based directed mutagenesis and spectroscopic methods to provide strategies for the rational designing of these PAiRFPs.

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Xu, Huacheng, Pinjing He, Guanzhao Wang, and Liming Shao. "Three-dimensional excitation emission matrix fluorescence spectroscopy and gel-permeating chromatography to characterize extracellular polymeric substances in aerobic granulation." Water Science and Technology 61, no. 11 (June 1, 2010): 2931–42. http://dx.doi.org/10.2166/wst.2010.197.

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Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy and gel-permeating chromatography (GPC) were employed to characterize the extracellular polymeric substances (EPS) in aerobic granulation. EPS matrix in this study was stratified into four fractions: (1) supernatant, (2) slime, (3) loosely bound EPS (LB-EPS), and (4) tightly bound EPS (TB-EPS). The results showed that the dissolved organic carbon was mainly distributed in TB-EPS fraction, and increased with increasing the operating time. The supernatant, slime, and LB-EPS fractions exhibited four fluorescence peaks, an autochthonous signature, unimodal MW distribution and lower molecular weight (MW) (3 < log [MW]<5), whereas the TB-EPS fraction only had two peaks, an allochthonous signature, multiple peaks and higher MW (5 < log [MW]<7). It was deemed that the formation of aerobic granules was correlated with the accumulation of proteins in the TB-EPS fraction. EEM spectroscopy and GPC profiles could be used as appropriate and effective methods to characterize the EPS in aerobic granulation from a micro-view level.

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Periasamy, Ammasi, and Richard N. Day. "PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins." Microscopy and Microanalysis 3, S2 (August 1997): 133–34. http://dx.doi.org/10.1017/s1431927600007558.

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The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

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So, Peter T. C., Chen Y. Dong, Barry R. Masters, and Keith M. Berland. "Two-Photon Excitation Fluorescence Microscopy." Annual Review of Biomedical Engineering 2, no. 1 (August 2000): 399–429. http://dx.doi.org/10.1146/annurev.bioeng.2.1.399.

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Hänninen, Pekka, Jori Soukka, and Juhani T. Soini. "Two-photon Excitation Fluorescence Bioassays." Annals of the New York Academy of Sciences 1130, no. 1 (May 2008): 320–26. http://dx.doi.org/10.1196/annals.1430.040.

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Pape, Jasmin K., Till Stephan, Francisco Balzarotti, Rebecca Büchner, Felix Lange, Dietmar Riedel, Stefan Jakobs, and Stefan W. Hell. "Multicolor 3D MINFLUX nanoscopy of mitochondrial MICOS proteins." Proceedings of the National Academy of Sciences 117, no. 34 (August 11, 2020): 20607–14. http://dx.doi.org/10.1073/pnas.2009364117.

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The mitochondrial contact site and cristae organizing system (MICOS) is a multisubunit protein complex that is essential for the proper architecture of the mitochondrial inner membrane. MICOS plays a key role in establishing and maintaining crista junctions, tubular or slit-like structures that connect the cristae membrane with the inner boundary membrane, thereby ensuring a contiguous inner membrane. MICOS is enriched at crista junctions, but the detailed distribution of its subunits around crista junctions is unclear because such small length scales are inaccessible with established fluorescence microscopy. By targeting individually activated fluorophores with an excitation beam featuring a central zero-intensity point, the nanoscopy method called MINFLUX delivers single-digit nanometer-scale three-dimensional (3D) resolution and localization precision. We employed MINFLUX nanoscopy to investigate the submitochondrial localization of the core MICOS subunit Mic60 in relation to two other MICOS proteins, Mic10 and Mic19. We demonstrate that dual-color 3D MINFLUX nanoscopy is applicable to the imaging of organellar substructures, yielding a 3D localization precision of ∼5 nm in human mitochondria. This isotropic precision facilitated the development of an analysis framework that assigns localization clouds to individual molecules, thus eliminating a source of bias when drawing quantitative conclusions from single-molecule localization microscopy data. MINFLUX recordings of Mic60 indicate ringlike arrangements of multiple molecules with a diameter of 40 to 50 nm, suggesting that Mic60 surrounds individual crista junctions. Statistical analysis of dual-color MINFLUX images demonstrates that Mic19 is generally in close proximity to Mic60, whereas the spatial coordination of Mic10 with Mic60 is less regular, suggesting structural heterogeneity of MICOS.

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Casals, C., E. Miguel, and J. Perez-Gil. "Tryptophan fluorescence study on the interaction of pulmonary surfactant protein A with phospholipid vesicles." Biochemical Journal 296, no. 3 (December 15, 1993): 585–93. http://dx.doi.org/10.1042/bj2960585.

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The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A, shielding was only observed on interaction with DPPC. From the intrinsic fluorescence measurements as well as from the quenching experiments, we concluded that the interaction of some phospholipid vesicles with SP-A produces a conformational change on the protein molecule and that the interaction of SP-A with DPPC is stronger than with other phospholipids. This interaction appeared to be independent of Ca2+ ions. Physiological ionic strength was found to be required for the interaction of SP-A with negatively charged vesicles of either DPPG or DPPC/DPPG (7:3, w/w). Intrinsic fluorescence of SP-A was sensitive to the physical state of the DPPC vesicles. The increase in intrinsic fluorescence of SP-A in the presence of DPPC vesicles was much stronger when the vesicles were in the gel state than when they were in the liquid-crystalline state. The effect produced by SP-A on the lipid vesicles was also dependent on temperature. The aggregation of DPPC, DPPC/DPPG (7:3, w/w) or dimyristoyl phosphatidylglycerol (DMPG) was many times higher below the phase-transition temperature of the corresponding phospholipids. These results strongly indicate that the interaction of SP-A with phospholipid vesicles requires the lipids to be in the gel phase.

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Wei, Yani, Luhui Wang, Yingying Zhang, and Yafei Dong. "An Enzyme- and Label-Free Fluorescence Aptasensor for Detection of Thrombin Based on Graphene Oxide and G-Quadruplex." Sensors 19, no. 20 (October 12, 2019): 4424. http://dx.doi.org/10.3390/s19204424.

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An enzyme- and label-free aptamer-based assay is described for the determination of thrombin. A DNA strand (S) consisting of two parts was designed, where the first (Sa) is the thrombin-binding aptamer and the second (Se) is a G-quadruplex. In the absence of thrombin, Sa is readily adsorbed by graphene oxide (GO), which has a preference for ss-DNA rather than for ds-DNA. Upon the addition of the N-methyl-mesoporphyrin IX (NMM), its fluorescence (with excitation/emission at 399/610 nm) is quenched by GO. In contrast, in the presence of thrombin, the aptamer will bind thrombin, and thus, be separated from GO. As a result, fluorescence will be enhanced. The increase is linear in the 0.37 µM to 50 µM thrombin concentration range, and the detection limit is 0.37 nM. The method is highly selective over other proteins, cost-effective, and simple. In our perception, it represents a universal detection scheme that may be applied to other targets according to the proper choice of the aptamer sequence and formation of a suitable aptamer-target pair.

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Lakowicz, Joseph R., Ignacy Gryczynski, Henryk Malak, and Zygmunt Gryczynski. "Two-Color Two-Photon Excitation of Fluorescence." Photochemistry and Photobiology 64, no. 4 (October 1996): 632–35. http://dx.doi.org/10.1111/j.1751-1097.1996.tb03116.x.

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XU, Q., K. SHI, S. YIN, and Z. LIU. "Chromatic two-photon excitation fluorescence imaging." Journal of Microscopy 235, no. 1 (July 2009): 79–83. http://dx.doi.org/10.1111/j.1365-2818.2009.03183.x.

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Kessler, Manfred A., Andreas Meinitzer, Walter Petek, and Otto S. Wolfbeis. "Microalbuminuria and borderline-increased albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay." Clinical Chemistry 43, no. 6 (June 1, 1997): 996–1002. http://dx.doi.org/10.1093/clinchem/43.6.996.

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Abstract We report a new automated fluorescence assay for determination of albumin in urine. The dye Albumin Blue 580 specifically binds to albumin with exhibition of strong red fluorescence. The albumin concentration is calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. Two Cobas Fara programs cover working ranges of 2–200 and 1–50 mg/L with detection limits of 1.4 and 0.4 mg/L, respectively. Within-run CVs (n = 10) ranged from 1.7% (189 mg/L) to 8.9% (7.2 mg/L) for 2–200 mg/L and from 2.9% (43.3 mg/L) to 5.7% (2.3 mg/L) for the 1–50 mg/L range. A test of urine samples (n = 100) submitted to routine analysis gave results that agreed well with those by the Behring nephelometric assay: AB 580 = 0.922 (± 0.010) BNA + 4.16 (± 0.78). No interference was detected from other urine components, including several proteins and 46 drugs. The high specificity and sensitivity make the method ideal for determination of microalbuminuria. In addition, the method is fast, inexpensive, and well-suited for clinical laboratory application and thus may be used instead of immunoassays.

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Slimani, Amel, Delphine Tardivo, Ivan V. Panayotov, Bernard Levallois, Csilla Gergely, Frederic Cuisinier, Hervé Tassery, Thierry Cloitre, and Elodie Terrer. "Multiphoton Microscopy for Caries Detection with ICDAS Classification." Caries Research 52, no. 5 (2018): 359–66. http://dx.doi.org/10.1159/000486428.

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Dentin carious lesion is a dynamic process that involves demineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the caries process using the collagen two-photon excitation fluorescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries Detection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG intensities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Terrer et al.: J Dent Res 2016; 96: 574–579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and the ICDAS scale. Comparison of the efficiency of nonlinear optical signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical application. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.

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Feng, Lin, Yanqing An, Jianzhong Xu, and Shichang Kang. "Characteristics and sources of dissolved organic matter in a glacier in the northern Tibetan Plateau: differences between different snow categories." Annals of Glaciology 59, no. 77 (October 1, 2018): 31–40. http://dx.doi.org/10.1017/aog.2018.20.

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AbstractDissolved organic matter (DOM) in mountain glaciers is an important source of carbon for downstream aquatic systems, and its impact is expected to increase due to the increased melting rate of glaciers. We present a comprehensive study of Laohugou glacier no. 12 (LHG) at the northern edge of the Tibetan Plateau to characterize the DOM composition and sources by analyzing surface fresh snow, granular ice samples, and snow pit samples which covered a whole year cycle of 2014/15. Excitation–emission matrix fluorescence spectroscopy analysis of the DOM with parallel factor analysis (EEM-PARAFAC) identified four components, including a microbially humic-like component (C1), two protein-like components (C2 and C3) and a terrestrial humic-like component (C4). The use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) showed that DOM from all these samples was dominated by CHO and CHON molecular formulas, mainly corresponding to lipids and aliphatic/proteins compounds, reflecting the presence of significant amounts of microbially derived and/or deposited biogenic DOM. The molecular compositions of DOM showed more CHON compounds in granular ice than in fresh snow, likely suggesting newly formed DOM from microbes during snowmelting.

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HÄNNINEN, P. E., E. SOINI, and S. W. HELL. "Continuous wave excitation two-photon fluorescence microscopy." Journal of Microscopy 176, no. 3 (December 1994): 222–25. http://dx.doi.org/10.1111/j.1365-2818.1994.tb03518.x.

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Liu Li-Xin, Qu Jun-Le, Lin Zi-Yang, Chen Dan-Ni, Xu Gai-Xia, Hu Tao, Guo Bao-Ping, and Niu Han-Ben. "Time-resolved two-photon excitation fluorescence spectroscopy." Acta Physica Sinica 55, no. 12 (2006): 6281. http://dx.doi.org/10.7498/aps.55.6281.

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Journal articles on the topic 'Two-photon excitation fluorescence of proteins'

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