Browsing by Subject "fluorescence"

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  • Kuha, Jonna; Järvinen, Marko; Salmi, Pauliina; Karjalainen, Juha (Springer Link, 2020)
    Hydrobiologia 847 21 (2020)
    Organic matter (OM) other than living phytoplankton is known to affect fluorometric in situ assessments of chlorophyll in lakes. For this reason, calibrating fluorometric measurements for OM error is important. In this study, chlorophyll (Chl) fluores cence was measured in situ in multiple Finnish lakes using two sondes equipped with Chl fluorometers (ex.470/em.650–700 nm). OM absorbance (A420) was measured from water samples, and one of the two sondes was also equipped with in situ fluorometer for OM (ex.350/em.430 nm). The sonde with Chl and OM fluorometers was also deployed continuously on an automated water quality monitoring station on Lake Konnevesi. For data from multiple lakes, inclusion of water colour estimates into the calibration model improved the predictability of Chl assessments markedly. When OM absorbance or in situ OM fluorescence was used in the calibration model, predictability between the in situ Chl and laboratory Chl a assessments was also enhanced. However, correction was not superior to the one done with the water colour estimate. Our results demonstrated that correction with water colour assessments or in situ measurements of OM fluorescence offers practical means to overcome the variation due to OM when assessing Chl in humic lakes in situ.
  • Rautaniemi, Kaisa; Vuorimaa-Laukkanen, Elina; Strachan, Clare J.; Laaksonen, Timo (2018)
    Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and gamma- and alpha-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 degrees C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.
  • Rojalin, Tatu (Helsingfors universitet, 2015)
    Raman spectroscopy is based on vibrations that occur between the atoms of a compound. The overall structural energy is derived from the electronical energy as well as vibrational, rotational and translational energy. In Raman spectroscopy the vibrational and rotational energies are essential. Usually the excitation energy used in Raman spectroscopy can be either in the region of visible light or NIR. The sample absorbs the energy and energy is also scattered back to all possible directions. Elastic scattering is called the Rayleigh scattering. In that case the back-scattered photons have an equal energy as the original excitation energy. However, some of the scattering happens inelastically and it forms the basis of Raman-phenomena. If the detected photons have smaller energy than the original, it is called the Stokes scattering. If the energy is bigger, it is anti-Stokes scattering. Raman is typically very rare and weak phenomenon. The spectral features in Raman spectra consist of the intensities and energies of the back scattered photons. Raman spectroscopy provides very accurate and detailed structural information on the molecule. It is basically a label-free technique with minimal need for sample preparation and the measurements can also be carried out e.g. through container walls. Further, Raman is quite insensitive to hydrous samples and it is suitable to solutions and biological assessments. However, there are some drawbacks that are formed by the luminescence phenomena i.e. fluorescence. Strong fluorescent backgrounds can mask the relevant Raman features in spectra because Raman and fluorescence are competetive processes. For instance many drug molecules have such structures that they cause strong fluorescence. It is also one of the reasons that pharmaceutical applications and measurements have been partly limited due to this problem. There are applications to improve and enhance a Raman signal. For example resonance phenomena and SERS are favored. To solve the fluorescence-related problems there are also means; one can change the laser wavelenght, photobleach the sample or apply different kinds of data manipulation techniques to the spectral data achieved. There are drawbacks with these methods. They can be slow, complex, damage the samples and still insufficient fluorescence suppression is a problem. In this study a novel time-gated CMOS-SPAD detection technique is applied to non-fluorescent and fluorescent drug measurements. The new detection system has a programmable on-chip delay time and it is synchronized with a picosecond pulsed laser. The scattered photons can be measured in the time scale when they are simultaneously measured in traditional energy and intensity wise. Raman scattering occurs in the timescale of sub-picoseconds while the fluorescence phenomena happen typically in the order of nanoseconds. This time difference can be exploited effectively to suppress the fluorescence. In the literature review of this study the basis of vibrational spectroscopy is introduced - especially Raman spectroscopy. The techniques related, as well as the novel time-resolved technique are covered. Further, different kinds of applications in the field of Raman spectroscopy are reviewed, mainly pharmaceutics-related and biologically relevant applications. In the experimental work the focus was to compare a continuous-wave 785 nm laser setup coupled with the CCD-detector to the pulsed picosecond 523 nm laser coupled with the CMOS-SPAD-detector. The measurements were performed on different kinds of drugs, both non-fluorescent and fluorescent. The aim was to obtain information on the effectiveness of CMOS-SPAD-technique on fluorescence suppression for solid drugs and solutions. Secondary goals were to collect knowledge on the similarities and differences between the Raman setups used for solution measurements, to optimize and discuss the key elements of setups for solids and solutions and to show preliminarily the applicability of the CMOS-SPAD-system on fluorescent drug's solutions as well as find out the requirements related to quantitative assessments using Raman spectroscopy. In drug research there is also constant need for reliable in vitro cell assays. The assessments made in this study may prove useful to the future applications e.g. measurements with living cells. An effective fluorescence suppression was achieved to strong fluorescent backgrounds using the novel time-resolved CMOS-SPAD-detection system coupled with the pulsed picosecond 532 nm laser. The setup is potentially a convenient tool to overcome many fluorescence-related limitations of Raman spectroscopy for laboratory and process analytical technology (PAT) use in the pharmaceutical setting. The results achieved encourage to consider that with careful calibration and method validation there is potential for quantitative analysis, biopharmaceutical and biological applications e.g. in vitro cell studies where most Raman techniques suffer from strong fluorescence backgrounds. Other potential fields for future applications can be also considered.
  • Juvonen, Risto O.; Rauhamäki, Sanna; Kortet, Sami; Niinivehmas, Sanna; Troberg, Johanna; Petsalo, Aleksanteri; Huuskonen, Juhani; Raunio, Hannu; Finel, Moshe; Pentikäinen, Olli T. (2018)
    Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.
  • Karasev, M. M.; Stepanenko, O. V.; Rumyantsev, K. A.; Turoverov, K. K.; Verkhusha, V. V. (2019)
    High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (650-900 nm) open a possibility for in vivo imaging of biological processes at the micro-and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes-natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
  • Zhang, Zhen; Kumar, Shiv; Bagnich, Sergey; Spuling, Eduard; Hundemer, Fabian; Nieger, Martin; Hassan, Zahid; Köhler, Anna; Zysman-Colman, Eli; Bräse, Stefan (2020)
    Six luminophores bearing an OBO-fused benzo[fg]tetracene core as an electron acceptor were designed and synthesized. The molecular structures of three molecules (PXZ-OBO, 5PXZ-OBO, 5DMAC-OBO) were determined by single crystal X-ray diffraction studies and revealed significant torsion between the donor moieties and the OBO acceptor with dihedral angles between 75.5 and 86.2 degrees. Photophysical studies demonstrate that blue and deep blue emission can be realized with photoluminescence maxima (lambda(PL)) ranging from 415 to 480 nm in mCP films. The emission energy is modulated by simply varying the strength of the donor heterocycle, the number of donors, and their position relative to the acceptor. Although the DMAC derivatives show negligible delayed emission because of their large singlet-triplet excited state energy difference, Delta E-ST, PXZ-based molecules, especially PXZ-OBO with an experimental Delta E(ST)of 0.25 eV, demonstrate delayed emission in blend mCP films at room temperature, which suggests triplet exciton harvesting occurs in these samples, potentially by thermally activated delayed fluorescence.
  • Filpponen, Ilari; Sadeghifar, Hasan; Argyropoulos, Dimitris S. (2011)
    In this communication a method for the creation of fluorescent cellulose nanoparticles using click chemistry and subsequent photodimerization of the installed side-chains is demonstrated. In the first step, the primary hydroxyl groups on the surface of the CNCs were converted to carboxylic acids by using TEMPO-mediated hypohalite oxidation. The alkyne groups, essential for the click reaction, were introduced into the surface of TEMPO-oxidized CNCs via carbodiimide-mediated formation of an amide linkage between monomers carrying an amine functionality and carboxylic acid groups on the surface of the TEMPO-oxidized CNCs. Finally, the reaction of surface-modified TEMPO-oxidized cellulose nanocrystals and azido-bearing coumarin and anthracene monomers were carried out by means of a click chemistry, i.e., Copper(I))-catalyzed Azide-Alkyne Cycloaddition (CuAAC) to produce highly photo-responsive and fluorescent cellulose nanoparticles. Most significantly, the installed coumarin and/or anthracene side-chains were shown to undergo UV-induced [2+2] and [4+4] cycloaddition reactions, bringing and locking the cellulose nanocrystals together. This effort paves the way towards creating, cellulosic photo responsive nano-arrays with the potential of photo reversibility since these reactions are known to be reversible at varying wavelengths.
  • Fowler, Michael; Duhamel, Jean; Qiu, Xing Ping; Korchagina, Evgeniya; Winnik, Francoise M. (2018)
    Aqueous solutions of a series of monodisperse poly(N-isopropylacrylamide)s end-labeled with n-butyl-1-pyrene at one or both chain ends (Py-n-PNIPAMs with n=1 or 2) were studied by turbidimetry, light scattering, and fluorescence. For a given polymer concentration and heating rate, the cloud point (T-c) of an aqueous Py-n-PNIPAM solution, determined by turbidimetry, was found to increase with the number-average molecular weight (M-n) of the polymer. The steady-state fluorescence spectra and time-resolved fluorescence decays of Py-n-PNIPAM aqueous solutions were analyzed and all parameters retrieved from these analyses were found to be affected as the solution temperature passed through T-c, the solution cloud point, and T-m, the temperature where dehydration of PNIPAM occurred. The trends obtained by fluorescence to characterize the aqueous Py-n-PNIPAM solutions as a function of temperature were found to be consistent with the model proposed for telechelic PNIPAM by Koga et al. in 2006. (c) 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018, 56, 308-318