ABSTRACT
Two-photon photodynamic therapy is a promising therapeutic method which requires the development of sensitizers with efficient two-photon absorption and singlet-oxygen generation. Reported here are two new diketopyrrolopyrrole-porphyrin conjugates as robust two-photon absorbing dyes with high two-photon absorption cross-sections within the therapeutic window. Furthermore, for the first time the singlet-oxygen generation efficiency of diketopyrrolopyrrole-containing systems is investigated. A preliminary study on cell culture showed efficient two-photon induced phototoxicity.
Subject(s)
Coloring Agents/chemistry , Photosensitizing Agents/chemistry , Porphyrins/chemistry , Pyrroles/chemistry , Singlet Oxygen/chemistry , Cell Survival/drug effects , Coloring Agents/pharmacology , HEK293 Cells , HeLa Cells , Humans , Photochemotherapy , Photons , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Pyrroles/pharmacologyABSTRACT
Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature.
ABSTRACT
Two-photon excited microscopy has evolved as a routine technique for long-term cellular and in vivo imaging and is now available in most optical microscopy facilities. Classical dyes and fluorescent proteins, developed for epifluorescence or confocal microscopy, are used but unfortunately present a low efficiency upon two-photon excitation inducing the need of high excitation power (over 20 mW). To reduce this excitation power, new dyes need to be developed, allowing really low two-photon excitation power in the milliwatt or sub-milliwatt range. We report here the conception, synthesis, and physicochemical and photophysical properties of new functionalized diketopyrrolopyrrole (DPP) derivatives acting as fluorescent tags for biomolecules. They present high two-photon absorption cross-sections and bright luminescence around 600 nm. These two-photon optimized fluorophores were bioconjugated to HIV-I Tat (44-61), and their cellular localization was observed by two-photon excited microscopy using sub-milliwatt laser excitation power.
Subject(s)
Fluorescent Dyes/chemistry , Photons , Pyrroles/chemistry , Fluorescent Dyes/chemical synthesis , Gene Products, tat/chemistry , HIV-1/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence , Molecular Structure , Pyrroles/chemical synthesisABSTRACT
The idea of using light to unleash biologically active compounds from inert precursors (uncaging) was introduced over 30 years ago. Recent efforts prompted the development of photoremovable protecting groups that have increased photochemical efficiencies for one- and two-photon excitation to allow more sophisticated applications. This requires characterization of one- and two-photon photochemical efficiencies of the uncaging processes.The present chapter focuses on the characterization of one-photon quantum yields and two-photon cross-sections.
Subject(s)
Photolysis , Photons , Azo Compounds/chemistry , Azo Compounds/radiation effects , Buffers , Chromatography, High Pressure Liquid , Light , Quantum Theory , Reference Standards , Spectrum Analysis/methods , Spectrum Analysis/standardsABSTRACT
The design, preparation and characterisation of a library of malachite green (MG) derivatives for two-photon RNA labelling is described. Some of these MG derivatives exhibit an increased affinity for an MG-aptamer, as well as improved two-photon sensitivity when compared to the classical malachite green chloride. The underlying mechanisms and potential benefits for in vivo RNA visualisation are discussed.
Subject(s)
RNA/analysis , Rosaniline Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Humans , Kinetics , Photons , RNA/chemistry , RNA/metabolism , Rosaniline Dyes/chemical synthesis , Rosaniline Dyes/toxicity , Spectrometry, Fluorescence/methodsABSTRACT
Different photoremovable protecting groups in the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl series with a donor-acceptor biphenyl backbone, known to display excellent two-photon absorption cross-sections, were investigated in order to develop efficient two-photon sensitive photoremovable protecting groups. The 2-(o-nitrophenyl)propyl series was a more versatile platform to increase the two-photon sensitivity of photoremovable protecting groups, leading to the p-alkoxy and p-bisalkylamino-4-nitro-[1,1'-biphenyl]-3-yl)propyl derivatives: PENB and EANBP respectively. Those two photoremovable protecting groups are to date the best caging groups for two-photon excitation at 800 and 740 nm respectively, offering attracting perspectives in chemical biology.
Subject(s)
Biphenyl Compounds/chemistry , Nitrobenzenes/chemistry , Photons , Molecular Structure , PhotolysisABSTRACT
Transforming synaptic input into action potential output is a fundamental function of neurons. The pattern of action potential output from principal cells of the mammalian hippocampus encodes spatial and nonspatial information, but the cellular and circuit mechanisms by which neurons transform their synaptic input into a given output are unknown. Using a combination of optical activation and cell type-specific pharmacogenetic silencing in vitro, we found that dendritic inhibition is the primary regulator of input-output transformations in mouse hippocampal CA1 pyramidal cells, and acts by gating the dendritic electrogenesis driving burst spiking. Dendrite-targeting interneurons are themselves modulated by interneurons targeting pyramidal cell somata, providing a synaptic substrate for tuning pyramidal cell output through interactions in the local inhibitory network. These results provide evidence for a division of labor in cortical circuits, where distinct computational functions are implemented by subtypes of local inhibitory neurons.
Subject(s)
Interneurons/cytology , Neural Inhibition/physiology , Synapses/physiology , Animals , Biophysics , Cerebral Cortex/cytology , Channelrhodopsins , DNA-Binding Proteins/genetics , Dependovirus/genetics , Electric Stimulation , Excitatory Amino Acid Antagonists/pharmacology , GABA Antagonists/pharmacology , Glutamate Decarboxylase/genetics , Glutamic Acid/pharmacology , In Vitro Techniques , Interneurons/physiology , Ligand-Gated Ion Channels/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Neurological , Mutation/genetics , Neural Inhibition/drug effects , Neural Inhibition/genetics , Parvalbumins/genetics , Patch-Clamp Techniques , Photic Stimulation , Pyramidal Cells/cytology , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Pyridazines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Glycine/genetics , Synapses/genetics , Synaptic Transmission , Transcription Factors/genetics , Transduction, Genetic , Valine/analogs & derivatives , Valine/pharmacology , gamma-Aminobutyric Acid/pharmacologySubject(s)
Biphenyl Compounds/chemistry , Neurotransmitter Agents/chemistry , gamma-Aminobutyric Acid/chemistry , Animals , Brain Chemistry , Neurotransmitter Agents/metabolism , Patch-Clamp Techniques , Photochemical Processes , Photolysis , Photons , Pyramidal Cells/chemistry , Pyramidal Cells/metabolism , Rats , Spectrophotometry, Ultraviolet/methods , gamma-Aminobutyric Acid/metabolismABSTRACT
Boron-containing two-photon-absorbing fluorophores have been prepared as new bifunctional molecules, potentially useful in two-photon excited microscopy (TPEM) and boron neutron capture therapy. They are based on a one-dimensional conjugated system containing a p-carborane entity at one end of the molecule and various electron-donating groups containing oxygen or nitrogen atoms at the other end. We investigated their one- and two-photon photophysical properties. They showed efficient fluorescence in an organic solvent, as well as in water for two of them, allowing microscopy on cell cultures. High two-photon absorption cross sections were determined in the 700-900 nm range. TPEM images were obtained with these new p-carborane-containing fluorophores, with laser intensities in the submilliwatt range.
Subject(s)
Biosensing Techniques/methods , Boranes/chemical synthesis , Fluorescent Dyes/chemical synthesis , Boranes/metabolism , Boron Neutron Capture Therapy , Female , Fluorescence , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Lasers , Light , Microscopy, Fluorescence, Multiphoton , Photochemical Processes/radiation effects , PhotonsABSTRACT
A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.
Subject(s)
DNA/analysis , Fluorescent Dyes/chemistry , Microscopy, Fluorescence, Multiphoton/methods , Nitriles/chemistry , Photons , Animals , Cattle , Fluorescent Dyes/chemical synthesis , Nitriles/chemical synthesisABSTRACT
Total synthesis and photophysical properties of PENB-DDAO, a photoactivatable 1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO) derivative of a far-red emitting fluorophore, are described. The photoremovable group of the DDAO phenolic function comprises a donor/acceptor biphenyl platform which allows an efficient (> or = 95%) and rapid (< 15 micros time-range) release of the fluorescent signal and displays remarkable two-photon uncaging cross sections (delta(a) x Phi(u) = 3.7 GM at 740 nm). PENB-DDAO is cell permeable as demonstrated by the triggering of cytoplasmic red fluorescent signal in HeLa cells after one-photon irradiation (lambda(exc) around 360 nm) or by the generation of a red fluorescent signal in a delineated area of a single cell after two-photon photoactivation (lambda(exc) = 770 nm).
Subject(s)
Acridines/analysis , Fluorescent Dyes/analysis , Microscopy, Fluorescence/methods , Acridines/chemical synthesis , Acridines/metabolism , Cell Membrane Permeability , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Models, Molecular , PhotochemistryABSTRACT
New boron containing two-photon absorbing fluorophores have been prepared. Centered on a pyrazabole central core, various conjugated systems and end groups were investigated to modulate their physicochemical properties (alkoxy, diphenylamino, and boron dipyromethene groups). One and two-photon photophysical characterizations were performed, showing efficient fluorescence in organic solvents. High two-photon absorption cross sections were determined in the 500-800 nm range. Two-photon excited microscopy images were also obtained with these new boron containing fluorescent bioprobes with laser intensities in the milliwatt range.
Subject(s)
Biosensing Techniques/methods , Boron/chemistry , Fluorescent Dyes/chemistry , Photons , Spectrometry, Fluorescence/methods , Absorption , Color , Fluorescence , Photochemistry/methodsABSTRACT
Light-responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light-triggered chemical tools, specially how two-photon excitation, "caged" fluorophores, and the photoregulation of protein activities in combination with time-resolved x-ray techniques should break new grounds in the understanding of dynamic biological processes.
Subject(s)
Engineering , Glutamates/chemistry , Photolysis , Photons , Glutamates/chemical synthesis , Molecular StructureABSTRACT
A pi-extended [2-(2-nitrophenyl)propoxy]carbonyl (NPPOC) derivative has been prepared as an efficient UV and near-IR photolabile protecting group for glutamate. This glutamate cage compound exhibits efficient photorelease upon one-photon excitation (epsilonPhi=990 M(-1) cm(-1) at 315 nm). In addition, it also shows efficient photorelease in activation of glutamate receptors in electrophysiological recordings. Combined with a high two-photon uncaging cross-section (deltaPhi=0.45 GM at 800 nm), its overall properties make this new cage-3-(2-propyl)-4'-methoxy-4-nitrobiphenyl (PMNB)-for glutamate a very promising tool for two-photon neuronal studies.
Subject(s)
Glutamic Acid/chemistry , Photons , Electrophysiology , Glutamic Acid/chemical synthesis , Hippocampus/metabolism , Molecular Structure , Patch-Clamp Techniques , Photochemistry , Spectrophotometry , Tissue Culture TechniquesABSTRACT
Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.
Subject(s)
Brain/cytology , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Photons , Animals , Cell Line , Cytoplasm/metabolism , Diffusion , Fluorescence Recovery After Photobleaching , Fluorescent Dyes/toxicity , Humans , Mice , Mice, Nude , Microscopy, Fluorescence, MultiphotonABSTRACT
We report herein the molecular engineering of an efficient two-photon absorbing (TPA) chromophore based on a donor-donor bis-stilbenyl entity to allow conjugation with biologically relevant molecules. The dye has been functionalized using an isothiocyanate moiety to conjugate it with the amine functions of poly(ethylenimine) (PEI), which is a cationic polymer commonly used for nonviral gene delivery. Upon conjugation, the basic architecture and photophysical properties of the active TPA chromophore remain unchanged. At the usual N/P ratio (ratio of the PEI positive charges to the DNA negative charges) of 10 used for transfection, the transfection efficiency and cytotoxicity of the labeled PEI/DNA complexes were found to be comparable to those of the unlabeled PEI/DNA complexes. Moreover, when used in combination with unlabeled PEI (at a ratio of 1 labeled PEI to 3 unlabeled PEI), the labeled PEI does not affect the size of the complexes with DNA. The labeled PEI was successfully used in two-photon fluorescence correlation spectroscopy measurements, showing that at N/P = 10 most PEI molecules are free and the diffusion coefficient of the complexes is consistent with the 360 nm size measured by quasielastic light scattering. Finally, two-photon images of the labeled PEI/DNA complexes confirmed that the complexes enter into the cytoplasm of HeLa cells by endocytosis and hardly escape from the endosomes. As a consequence, the functionalized TPA chromophore appears to be an adequate tool to label the numerous polyamines used in nonviral gene delivery and characterize their complexes with DNA in two-photon applications.
Subject(s)
Fluorenes/chemistry , Fluorescent Dyes/chemistry , Gene Transfer Techniques , Phenyl Ethers/chemistry , Photons , Polyethyleneimine/chemistry , DNA/chemistry , Endocytosis , Fluorenes/chemical synthesis , Fluorescent Dyes/chemical synthesis , HeLa Cells , Humans , Isothiocyanates/chemistry , Microscopy, Fluorescence , Phenyl Ethers/chemical synthesis , Spectrum Analysis , TransfectionABSTRACT
We report here the synthesis and characterization of a new type of non-ionic blue fluorescent water-soluble chromophores specifically designed for two-photon absorption microscopy. The water solubility is induced by introduction of short oligo(ethylene glycol) monomethyl ether moieties. This work has led to low molecular weight dyes with efficient two-photon absorption cross sections and high fluorescence quantum yield in organic solvents as well as in aqueous solutions.
Subject(s)
Biphenyl Compounds/chemistry , Biphenyl Compounds/chemical synthesis , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Stilbenes/chemistry , Stilbenes/chemical synthesis , Molecular Structure , Molecular Weight , Photons , Quantum Theory , Sensitivity and Specificity , Solubility , Spectrometry, Fluorescence , Water/chemistryABSTRACT
The thermotropic polymorphism of a series of guanidinium alkanesulfonates (6 < or = n < or = 18) was investigated by optical microscopy and differential scanning calorimetry. Hydrogen bonding was analyzed by infrared spectroscopy. Molecular volumes were measured by dilatometry. The structure of the crystal, smectic A, and ordered smectic phases observed were studied by X-ray diffraction and utilized to prove that the supramolecular arrangement of the molecules in the crystal survives in the smectic phases at high temperature.
