Computer-operated microspectrofluorimetry to identify formaldehyde-induced fluorophores of biogenic monoamines and precursor substances in models and tissue sections

By means of a histochemical reaction using formaldehyde vapour (Falck and Owman 1965), biogenic monoamines and precursor substances, i.e., L-DOPA, dopamine, noradrenaline, adrenaline, 5-hydroxytryptophan and 5-hydroxytryptamine, may be converted into fluorophores with specific spectral characteristics, i.e., the emission spectrum, excitation spectrum and fading curve. The registration and correction of the spectral properties and changes induced by acidification with hydrochloric acid vapour or treatment with ammonia vapour, of these formaldehyde-induced fluorophores, are performed by an automated microspectrofluorimeter, developed by modification of a Leitz MPV 2 system. This work deals with the instrumental configuration and certain methodological features in order to identify the fluorogenic biogenic monoamines and precursor substances in models and tissue sections. Registrations of excitation peak values, for the first time extended to a wavelength range from 240-460 nm, are discussed, which enable the calculation of peak ratio values 410/260, 380/320, 320/260 or 385/315, suitable as identification parameters for formaldehyde-induced fluorophores of biogenic monoamines and precursor amino acids.     

Microspectrofluorimetric analysis of the formaldehyde-induced fluorophores of 5-hydroxytryptamine and dopamine in intrapulmonary neuroepithelial bodies after administration ofl-hydroxytryptophan andl-DOPA

Summary To demonstrate the intracellular store of 5-hydroxytryptamine and dopamine in pulmonary neuroepithelial bodies of the neonatal rabbit after treatment with the corresponding amino-acid precursorsl-5-hydroxytryptophan orl-3,4-dihydroxyphenylalanine, formaldehyde-induced flourescence in combination with microspectrofluorimetric analysis has been used. Emission spectra and excitation spectra in an extended wavelength range from 240 to 460 nm, the displacement of excitation peaks after exposure to hydrochloric acid vapour, and calculation of peak ratio values 410/260, 380/320, 320/260 for phenylethylamine fluorophores and 385/315 for indolylethylamine fluorphores were performed. Thus, the presence of 5-hydroxytryptamine without occurrence of 5-hydroxytryptophan was demonstrated in pulmonary neuroepithelial bodies after administration of the corresponding biological precursor, while dopamine combined with 5-hydroxytryptamine were clearly revealed after administration ofl-3,4-dihydroxyphenylalanine. The rate of photodecomposition always corroborated these findings.Dedicated to Prefessor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Differentiation of the formaldehyde-induced fluorescent products of noradrenaline and dopamine by microspectrofluorometry

Synopsis The influence of concentration of dopamine and noradrenaline on the spectral characteristics of their formaldehyde-induced fluorophore, together with the influence of duration of reaction with formaldehyde, has been investigated in a model system. No substantial differences between fluorescence spectra of the amine fluorophores were observed. Accordingly, the influence of hydrochloric acid and ammonia vapours on the fluorophores was investigated. A shift to shorter wavelengths in the excitation maximum of each fluorophore was observed after a brief exposure to hydrochloric acid vapour; more prolonged exposure resulted in a pronounced shift back to longer wavelengths in the case of noradrenaline but no substantial change was observed with dopamine. Brief exposure to hydrochloric acid vapour resulted in a substantial increase in the rate of fading of the noradrenaline fluorophore on exposure to exciting radiation. It is suggested that this offers a convenient way of differentiating the amines.     

The distribution and endocrine nature of the abdominal paraganglia of adult man

The paraganglia of adult man were studied using the formaldehyde-induced fluorescence (FIF) method for histochemical characterization of biogenic monoamines. Microspectrofluorimetry was used to record the emission spectra and fluorescence intensities of the paraganglionic cells. The study of samples from six patients showed that well vascularized paraganglia were widely distributed throughout the retroperitoneal spaces. The paraganglia exhibited strong FIF with the spectral characteristics of monamines. Treatment with HC1 caused an increase in the fluorescence intensity of the paraganglia and a simultaneous shift of the emission maximum from 480--495 nm. This change suggests the presence of high concentrations of tryptophyl-containing peptides and is not due to monoamines. The possibility of a dual endocrine function for the paraganglia is discussed.     

Factors influencing the fluorescence spectra of the formaldehyde-induced reaction product of 5-hydroxytryptamine

Summary Fluorescence excitation and emission spectra of the formaldehyde-induced fluorophore of 5-hydroxytryptamine in a Sephadex model have been examined following exposure to hydrochloric acid or ammonia vapour. Exposure to hydrochloric acid vapour produced excitation spectra with broad maxima centred around 400 nm, whilst exposure to ammonia vapour intensified the maximum normally seen at approximately 450 nm relative to that seen at 400 nm. The emission maximum was generally broad and poorly defined following exposure to hydrochloric acid vapour; exposure to ammonia vapour had little effect on its location. Exposure of the formaldehyde-induced fluorophore in models containing 5-hydroxytryptamine to 300 nm irradiation caused a substantial shift in the position of the emission maximum; a concomitant increase in the fluorescence intensity was also observed. When the fluorescence present in duodenal enterochromaffin cells was examined after similar treatments, a number of differences in the response of the fluorophore were noted.     

The histochemical demonstration of catecholamines and tryptamines by acid- and aldehyde-induced fluorescence: microspectrofluorometric characterization of the fluorophores in models

Synopsis Fluorescence excitation and emission spectra are reported of the fluorophores derived from various catecholamines, tryptamines and related substances treated with vapours of acetaldehyde, glutaraldehyde and acetic acid. In most cases, the excitation maxima were at about 400–410 nm, and emission maxima at about 530 nm (catecholamines) or 560 nm (tryptamines). The microspectrofluorometric differentiation of individual arylethylamines by the relative height of excitation peaks at about 400 and 330 nm after treatment with acetic acid vapour is discussed.     

Luminescence excitation spectra reveal low-lying excited states in stacked adenine bases

Luminescence emission, polarization, and excitation spectra of polyadenylic acid (poly(A)) have been studied in room-temperature aqueous solution (pH 8). The temperature dependence of the luminescence of poly(A) at two different excitation wavelengths in the range 10-70 degrees C has also been studied and compared with that of adenosine 5'-monophosphate (AMP). It has been found that the luminescence excitation spectrum and the polarization curve of poly(A) solution reveal a low-intensity electronic transition at about 320 nm which is red-shifted by approximately 0.9 eV from the maximum of the absorption spectrum at 260 nm. A model of two luminescent stacked forms is suggested. The difference in the ground state levels of these two stacked forms obtained from the temperature dependencies of the emissions is insignificant ( approximately 1 kcal/mol). This means a lowering of the excited state of the stacked form with the 320 nm/420 nm absorption/emission bands by approximately 0.9 eV as compared to the main form with the 260 nm/400 nm absorption/emission bands. The low-lying excited states suggest the stronger electronic coupling of the bases in a certain stacked form. It is proposed that such clusters of the stacked bases could provide the wire-like conductivity in the short segments of DNA.     

Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine.

A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines. On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.     

Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde

Summary The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas glutationedopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480–485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilibrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.     

Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

We report on a novel technology for multicolor gene and chromosome detection as well as for three-dimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumor-bearing mice was performed.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine

Summary A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.This work was supported by grants from the Jalmari and Rauha Ahokas Foundation and the J.K. Paasikivi Foundation     

Discovery of signaling effect of UV-B/C light in the extended UV-A/blue-type action spectra for step-down and step-up photophobic responses in the unicellular flagellate algaEuglena gracilis

Summary Cultures of unicellular algal flagellateEuglena gracilis grown in different conditions were subjected to action spectroscopy for step-down and step-up photophobic responses, respectively. The spectral region was extended into the UV-B/C as well as in the UV-A and visible regions with the Okazaki Large Spectrograph as the monochromatic light source. The photophobic responses of the cells were measured with an individual-cell assay method with the aid of a computerized video motion analyzer. In the UV-A and visible regions, the shapes of the action spectra were the so-called UV-A/blue type. In the newly studied UV-B/C region, new action peaks were found at 270 nm for the step-down response and at 280 nm for the step-up one. The absorption spectrum of flavin adenine dinucleotide (FAD) appeared to fit the action spectrum for the step-up response, whereas the shape of the step-down action spectrum, which has a UV-A peak (at 370 nm) higher than the blue peak (at 450 nm), appeared to be mimicked by the absorption spectrum of a mixed solution of 6-biopterin and FAD. These observations might also account for the fact that the UV-B/C peak wavelength at 270 nm of the action spectrum for the step-down response is shorter by 10 nm than the action spectrum for the step-up response at 280 nm.Abbreviations FAD flavin adenine dinucleotide - FWHM spectral full width at half maximum - NIBB National Institute for Basic Biology - OLS Okazaki Large Spectrograph - PFB paraflagellar body - UV-A ultraviolet light of spectral region between 320 and 400 nm - UV-B/C ultraviolet light of spectral region between 190 and 320 nm     

Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines

Summary Glyoxylic acid vapour is a most powerful reagent for the fluorescence histochemical visualization of biogenic monoamines. In the present investigation the mechanisms of fluorophore formation in the glyoxylic acid reaction has been studied in detail for tryptamine in histochemical models and in freeze-dried tissue, utilizing microspectrofluorometric, Chromatographic, and mass spectrometric techniques in combination with isotope measurements.The glyoxylic acid-tryptamine reaction proceeds through an initial Pictet-Spengler type cyclization to 1,2,3,4-tetrahydro--carboline-1-carboxylic acid, followed by two alternative fluorophore forming reactions yielding 3,4-dihydro--carboline, or the 2-carboxymethyl-3,4-dihydro--carbolinium and 2-methyl-3,4-dihydro--carbolinium salts, which are all strongly fluorescent. It is shown that the yield of fluorophores is considerably higher in the glyoxylic acid vapour reaction than in the formaldehyde vapour reaction of the standard Falck-Hillarp method, and that this higher efficiency of glyoxylic acid is due to the most favourable catalysing properties of the carboxylic group of the glyoxylic acid molecule.     

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.     

Ultrastructural,fluorescence microscopic and microfluorimetric study of the innervation of the axial complex in the sea urchin,Sphaerechinus granularis (Lam.)

Summary In juxtaposition with the contractile epithelia of the axial complex of the sea urchin, Sphaerechinus granularis, several types of nerve fibers with different vesicle populations were determined. Nerve terminals, filled with clear vesicles and dense core vesicles, form synaptoid neuromuscular junctions. Close to the somatocoelic epithelia of the axial and terminal sinus septa, numerous axon profiles form a nerve plexus. Among the epithelial cells covering the plexus, two types of nerve cells can be distinguished which presumably produce neurosecretory and aminergic granules, respectively. Monoamine fluorescence (formaldehyde-induced fluorescence, Falck-Hillarp technique) was analyzed microspectrofluorimetrically. The emission spectrum of the fluorophores occurring in the present material shows a maximum at 475 nm and is characteristic of catecholamines; the excitation maximum at 380 nm after formaldehyde treatment is typical of catecholamines at low pH only. Since the peak ratio (370:320 nm) does not change after HCl-vapor treatment, the fluorophores are likely to be indicative of dopamine.     

Spectral Properties of Single Gold Nanoparticles in Close Proximity to Biological Fluorophores Excited by 2-Photon Excitation

Metallic nanoparticles (NPs) are able to modify the excitation and emission rates (plasmonic enhancement) of fluorescent molecules in their close proximity. In this work, we measured the emission spectra of 20 nm Gold Nanoparticles (AuNPs) fixed on a glass surface submerged in a solution of different fluorophores using a spectral camera and 2-photon excitation. While on the glass surface, we observed the presence in the emission at least 3 components: i) second harmonic signal (SHG), ii) a broad emission from AuNPS and iii) fluorescence arising from fluorophores nearby. When on the glass surface, we found that the 3 spectral components have different relative intensities when the incident direction of linear polarization was changed indicating different physical origins for these components. Then we measured by fluctuation correlation spectroscopy (FCS) the scattering and fluorescence signal of the particles alone and in a solution of 100 nM EGFP using the spectral camera or measuring the scattering and fluorescence from the particles. We observed occasional fluorescence bursts when in the suspension we added fluorescent proteins. The spectrum of these burst was devoid of the SHG and of the broad emission in contrast to the signal collected from the gold nanoparticles on the glass surface. Instead we found that the spectrum during the burst corresponded closely to the spectrum of the fluorescent protein. An additional control was obtained by measuring the cross-correlation between the reflection from the particles and the fluorescence arising from EGFP both excited at 488 nm. We found a very weak cross-correlation between the AuNPs and the fluorescence confirming that the burst originate from a few particles with a fluorescence signal.     

Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.     

Action spectra for polarotropism and phototropism in protonemata of the fern Adiantum capillus-veneris

The action spectrum for polarotropism was determined, using the Okazaki large spectrograph, by brief irradiation with light between 260 nm and 850 nm in single-celled protonemata of the fern Adiantum capillus-veneris L., which had been cultured for 6 days in red light and then in the dark for 15 h. The action spectrum had a peak at around 680 nm. This effect was nullified by subsequent irradiaton with far-red light, and typical red/far-red reversibility was observed, indicating the involvement of phytochrome. Polarized ultraviolet or blue light had no effect on the direction of apical growth. The action spectrum for phototropism was also determined in the red light region by means of brief microbeam irradiation of a flank of the subapical region of the protonema. This spectrum showed a peak at 662 nm which was consistent with the absorption peak of phytochrome, but not with the peak of the action spectrum for polarotropism.