Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.     

Noninvasive optical imaging of staphylococcus aureus bacterial infection in living mice using a Bis-dipicolylamine-Zinc(II) affinity group conjugated to a near-infrared fluorophore

Optical imaging of bacterial infection in living animals is usually conducted with genetic reporters such as light-emitting enzymes or fluorescent proteins. However, there are many circumstances where genetic reporters are not applicable, and there is a need for exogenous synthetic probes that can selectively target bacteria. The focus of this study is a fluorescent imaging probe that is composed of a bacterial affinity group conjugated to a near-infrared dye. The affinity group is a synthetic zinc (II) coordination complex that targets the anionic surfaces of bacterial cells. The probe allows detection of Staphylococcus aureus infection (5 x 10 (7) cells) in a mouse leg infection model using whole animal near-infrared fluorescence imaging. Region of interest analysis showed that the signal ratio for infected leg to uninfected leg reaches 3.9 +/- 0.5 at 21 h postinjection of the probe. Ex vivo imaging of the organs produced a signal ratio of 8 for infected to uninfected leg. Immunohistochemical analysis confirmed that the probe targeted the bacterial cells in the infected tissue. Optimization of the imaging filter set lowered the background signal due to autofluorescence and substantially improved imaging contrast. The study shows that near-infrared molecular probes are amenable to noninvasive optical imaging of localized S. aureus infection.     

Conjugation of a new two-photon fluorophore to poly(ethylenimine) for gene delivery imaging

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.     

Primary donor photo-oxidation in photosystem I: a re-evaluation of (P700(+) - P700) Fourier transform infrared difference spectra.

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the photo-oxidation of the primary electron donor (P700) in PS I particles from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. To aid in the interpretation of the spectra, PS I particles from a site-directed mutant of C. reinhardtii, in which the axial histidine ligand (HisA676) was changed to serine, were also studied. A high-frequency (3300-2600 cm(-1)) electronic transition is observed for all PS I particles, demonstrating that P700 is dimeric. The electronic band is, however, species-dependent, indicating some differences in the electronic structure of P700 and/or P700(+) in C. reinhardtii and Synechocystis sp. 6803. For PS I particles from C. reinhardtii, substitution of HisA676 with serine has little effect on the ester carbonyl modes of the chlorophylls of P700. However, the keto carbonyl modes are considerably altered. Comparison of (P700(+) - P700) FTIR difference spectra obtained using PS I particles from the wild type (WT) and the HS(A676) mutant of C. reinhardtii indicates that the mutation primarily exerts its influence on the P700 ground state. The 13(1) keto carbonyls of the chlorophylls of P700 of the wild type absorb at similar frequencies, which has previously made these transitions difficult to resolve. However, for the HS(A676) mutant, the 13(1) keto carbonyl of chlorophyll a or chlorophyll a' of P700 on PsaB or PsaA absorbs at 1703.4 or 1694.2 cm(-1), respectively, allowing their unambiguous resolution. Upon P700(+) formation, in both PS I particles from C. reinhardtii, the higher-frequency carbonyl band upshifts by approximately 14 cm(-1) while the lower frequency carbonyl downshifts by approximately 10 cm(-1). The similarity in the spectra for WT PS I particles from C. reinhardtii and Synechocystis sp. 6803 indicates that a similar interpretation is probably valid for PS I particles from both species. The mutant results allow for an interpretation of the behavior of the 13(1) keto carbonyls of P700 that is different from previous work [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592], in which it was suggested that 13(1) keto carbonyls of P700 absorb at 1697 and 1639 cm(-1), and upshift by 21 cm(-1) upon cation formation. The interpretation of the spectra reported here is more in line with recent results from ENDOR spectroscopy and high-resolution crystallography.     

New cyanine dyes as base surrogates in PNA: forced intercalation probes (FIT-probes) for homogeneous SNP detection

Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye-PNA-conjugates, the YO-containing PNA has properties useful for homogeneous SNP detection. YO-PNA is demonstrated to signal the presence of fully complementary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single base mutations at non-stringent conditions.     

Real-time monitoring and automatic density control of large-scale microalgal cultures using near infrared (NIR) optical density sensors

Signals from near infrared (NIR) light transmittance sensors were used for both real-time monitoring of algal biomass density in growing mass cultures (200l tubular biofences), and also as feedback in a system that controlled the density of the culture by automatic injection of fresh growth medium. When operated in a semi-continuous production mode between predefined density values, diurnal growth patterns were recorded on-line that provided information on the dynamics of the microalgal cultures with respect to environmental conditions. The bioreactor system was also programmed to operate in constant biomass density mode, thereby maintaining the culture at the optimal population density (OPD), and sustaining high biomass production levels. The system has potential for operating a dynamic density set point for microalgal cultures where the optimal population density varies as a function of ambient growing conditions.     

Induced optical activity in the complex of poly(L-arginine) with azo dyes

The absorption and CD spectra of the complexes of poly(L -arginine) (PLA) and azo dyes have been measured in aqueous solution. On complexation, Blue-shifted additional absorption bands were observed. In the wide pH 4–11 range, induced CD was observed at the visible wavelengths corresponding to the blue-shifted absorption bands. The induced CD arose from the dimeric dye molecules bound to PLA in the α-helical structure. When a modified analysis of induced CE is made by the excition chirality method, the origin of the induced CD can be assigned to the dipole coupling. The PLA–dye complexes showed the counterlockwise (negative, S) chirality of the transition dipole moments of dyes.     

eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H⁺ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Identification of Mycoplasmas using a fluorophore-free microarray and infrared chemical imaging (IRCI)

A novel application of mid-infrared chemical imaging (IRCI) for the fluorophore-free detection and identification of mycoplasma species is reported for the first time. The PCR-amplified biotinylated targets hybridized to microarray probes were treated with streptavidin-gold nanoparticles followed by silver enhancement. This modification has the potential to expand the implementation of DNA microarray techniques in laboratories involved in the detection of cell substrates, other biological products, and clinical materials for the presence of mycoplasmas.     

The labelling of peptides and proteins with a new fluorophore: N-acetyl-DL-(p-N',N'-dimethylamino)phenylalanine

It was found that N-acetyl-DL-(p-N',N'-dimethylamino)phenylalanine, in the form of azlactone, can be introduced into a peptide or protein molecule as a new convenient fluorescent marker. A fluorophore of similar properties: N-acetyl-(p-amino)phenylalanine can be introduced into a peptide chain by the reaction with the azlactone of N-acetyl-(p-nitro)phenylalanine followed by reduction of nitro group to amino group. This method, however, cannot be applied to proteins.     

Monitoring nutrition in small ruminants with the aid of near infrared reflectance spectroscopy (NIRS) technology: A review

This review aims to evaluate the contribution of near infrared reflectance spectroscopy (NIRS) to monitor nutrition in small ruminants, with particular emphasis on the use of feed spectra and fecal spectra. NIRS provides satisfactory accuracy in the analysis of the chemical constituents of feeds for small ruminants, e.g., crude protein and cell wall composition, and is sometimes better than in vitro procedures for predicting in vivo digestibility and the available energy in feeds. In addition, in vitro digestibility can be accurately estimated by NIRS. The effective rumen degradability of protein could potentially be accurately predicted by NIRS, which would eliminate the need for rumen-fistulated animals. Good accuracy in the prediction of tannins has been reported for narrow, single-species applications, as well as for broad arrays of browse species. The identification of NIR segments corresponding to undigested entities has potential to help in providing spectral markers of digestibility. Fecal output can easily be evaluated, using the NIRS-aided analysis of polyethylene glycol (PEG) administered as external indigestible marker. Analysis of NIR spectra of the feces enables the accurate prediction of the chemical characteristics of the feed (dry matter digestibility and crude protein, cell wall attributes, PEG-binding tannins) in stall-fed and grazing animals, and to some extent, of the botanical composition of diets at pasture. Thus, fecal NIRS methodology holds the potential to provide nutritional diagnoses for farmers raising small ruminant.     

Cerenkov radiation energy transfer (CRET) imaging: a novel method for optical imaging of PET isotopes in biological systems

Background

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β⁺)-emitting radionuclides in small animals and humans. Upon β⁺ decay, the initial velocity of high-energy β⁺ particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.

Principal Findings

To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes ⁶⁴Cu and ¹⁸F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with ^99mTc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [¹⁸F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [¹⁸F]FDG showed CRET ratios in vivo as high as 3.5±0.3.

Conclusions

Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals.     

Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 2-aminoacridone: subpicomolar detection using an imaging system based on a cooled charge-coupled device.

Numerous monosaccharides and oligosaccharides were derivatized at their reducing end groups with the fluorophore 2-aminoacridone. The resulting fluorescent compounds were separated by PAGE using two different buffer systems. One of these, a Tris borate buffer, enabled all of the fluorescent saccharide derivatives tested to be electrophoresed and various positional isomers, anomers, and epimers could be separated. The other system consisted of a discontinuous Tris-HCl/Tris-glycine buffer and enabled the electrophoresis of acidic, but not neutral, saccharide derivatives. The acidic and neutral saccharides could thus be distinguished unequivocally. The fluorescent labeling procedure was virtually quantitative and as little as 0.63 pmol could be detected photographically when gels were illuminated by uv light. When gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrates of glycoconjugates and other naturally occurring oligosaccharides.     

Application of a full body inertial measurement system in alpine skiing: a comparison with an optical video based system

This study aims at determining the accuracy of a full body inertial measurement system in a real skiing environment in comparison with an optical video based system. Recent studies have shown the use of inertial measurement systems for the determination of kinematical parameters in alpine skiing. However, a quantitative validation of a full body inertial measurement system for the application in alpine skiing is so far not available. For the purpose of this study, a skier performed a test-run equipped with a full body inertial measurement system in combination with a DGPS. In addition, one turn of the test-run was analyzed by an optical video based system. With respect to the analyzed angles, a maximum mean difference of 4.9° was measured. No differences in the measured angles between the inertial measurement system and the combined usage with a DGPS were found. Concerning the determination of the skier's trajectory, an additional system (e.g., DGPS) must be used. As opposed to optical methods, the main advantages of the inertial measurement system are the determination of kinematical parameters without the limitation of restricted capture volume, and small time costs for the measurement preparation and data analysis.     

Regional transcranial oximetry with near infrared spectroscopy (NIRS) in comparison with measuring oxygen saturation in the jugular bulb in infants and children for monitoring cerebral oxygenation]

Using a dual channel near infrared (NIR) in vivo optical spectroscopy (INVOS) system (INVOS 3100A, Somanetics Corp. Troy, MI, USA) we investigated the relationship between jugular venous oxygen saturation (SjvO2) and regional cerebral oxygen saturation (rSO2) in 30 infants and children (mean age 4.5 years) with congenital heart disease undergoing cardiac catheterisation. The NIRS-SomaSensor (emitter and dual receiver probe) was applied at a standardised right fronto-temporal location (over the right frontal cortex) on the infant's head and covered with an adhesive flexible bandage. Using NIR light (730 and 810 nm) and two source-detector spacings (3 and 4 cm from the transmitter), percentage values of rSO2 were calculated from detected haemoglobin saturations. Simultaneously, jugular venous oxygen saturation (SjvO2) monitoring was performed via a catheter placed in the right internal jugular vein with its tip positioned in the jugular bulb, as verified by fluoroscopy. To compare the reliability of NIRS measurement characteristics, jugular venous blood was analysed for SjvO2 as a reference measure of global cerebral oxygenation, by co-oximetry (OSM3-Hemoximeter, Radiometer Copenhagen, Denmark). Other measured variables included pulse oximetry, arterial blood pressure, and venous and arterial oxygen saturations. Over a jugular venous oxygen saturation range of 31-83%, a significant positive linear correlation was found between rSO2 (NIRS measurement) and SjvO2 (jugular bulb oximetry) (r = 0.93, p < 0.001). No significant correlation was observed between rSO2 values and arterial blood saturation or pulse oximetry. The quantitative correlation between rSO2 (haemoglobin oxygenation in a small hemi-elliptical area of the brain) and reference SjvO2 measurement (method for monitoring global cerebral oxygenation) suggests that NIRS measurement with subtraction algorithm should identify predominantly intracranial saturation in the pediatric age group, and will tend to reflect global oxygenation under physiological conditions. Transcranial oximetry using dual receiving channel NIRS offers a noninvasive, real-time, reliable and practicable means of monitoring cerebral haemoglobin oxygenation changes infants and children with cyanotic and noncyanotic congenital heart disease.     

Evaluation of green forage intake and digestibility in ruminants using near infrared reflectance spectroscopy (NIRS): Developing a global calibration

The objective of this study was to evaluate the potential of near infrared reflectance spectroscopy (NIRS), applied to forage and/or faeces, to estimate the in vivo organic matter digestibility (OMD) and the organic matter voluntary intake (OMVI, g/kg metabolic weight [BW^0.75]) for a wide range of temperate forages. Two different databases, in terms of forage species and development stages were studied. The first one included two grass species and two forage mixtures for which OMD and OMVI were continuously measured during the grass-growing seasons (spring and summer). The second one contained a large set of grass and legume species and forage mixtures (142 trials) for which OMD and OMVI were measured.Forage and faeces samples were submitted to NIRS analysis and predictive calibrations were developed from forage spectra, faeces spectra, forage and faeces subtracted spectra, and faeces and forage concatenated spectra. Working on faecal spectra (alone or concatenated) enabled us to develop the best calibration equations for both OMD and OMVI estimation. The coefficient of determination (R²) was greater than 0.8. The standard error of cross validation (SECV) for OMD and OMVI was 0.021 and 4.51 g/kg BW^0.75, respectively, and the accuracy was similar to that obtained with other predictive methods. With regard to the faecal spectra (second derivative mode), the fat absorbency at wavelengths of 1730, 2310 and 2350 nm was higher when the corresponding forage was highly digestible and ingestible.In conclusion, applying NIRS to faeces is a rapid and easy analytical method that could be an interesting tool for managing grazing ruminants and optimising their performance.     

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.     

Determining skeletal muscle architecture with Laplacian simulations: a comparison with diffusion tensor imaging

Determination of skeletal muscle architecture is important for accurately modeling muscle behavior. Current methods for 3D muscle architecture determination can be costly and time-consuming, making them prohibitive for clinical or modeling applications. Computational approaches such as Laplacian flow simulations can estimate muscle fascicle orientation based on muscle shape and aponeurosis location. The accuracy of this approach is unknown, however, since it has not been validated against other standards for muscle architecture determination. In this study, muscle architectures from the Laplacian approach were compared to those determined from diffusion tensor imaging in eight adult medial gastrocnemius muscles. The datasets were subdivided into training and validation sets, and computational fluid dynamics software was used to conduct Laplacian simulations. In training sets, inputs of muscle geometry, aponeurosis location, and geometric flow guides resulted in good agreement between methods. Application of the method to validation sets showed no significant differences in pennation angle (mean difference \(0.5{^{\circ }})\) or fascicle length (mean difference 0.9 mm). Laplacian simulation was thus effective at predicting gastrocnemius muscle architectures in healthy volunteers using imaging-derived muscle shape and aponeurosis locations. This method may serve as a tool for determining muscle architecture in silico and as a complement to other approaches.