Two-photon microscopy of deep intravital tissues and its merits in clinical research.

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity.

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as "reactive-tracers" for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.

A modified Ce/Mg-BCIP-NBT formazan/indigoblue technique for demonstration of non-specific alkaline phosphatase activity.

The wide ranged structurally variability of formazans and their accessibility for auxiliary additives as redoxmediators or metals provide an easy tunable chromogenic visualization technique. We present here an improved nitro blue tetrazolium (NBT) 5-bromo-4-chloro-3-indolyl phosphate (BCIP) method which is superior to the classical McGadey's procedure regarding proper precipitation and localization as well as sensitivity. Different metal additives as well as the overall reaction course modifying additives (redox mediators, chelating additives, buffer) were optimized.

New fluorescent method for the histochemical detection of dipeptidyl peptidase IV using glycyl-l-prolyl-2-anthraquinonyl hydrazide as substrate.

Glycyl-4-prolyl-2-anthraquinonylhydrazide (Gly-Pro-2-AH) was synthesized and used as a new fluorogenic substrate for the histochemical detection of dipeptidyl peptidase IV activity (DPP IV). The enzymatic hydrolysis liberates 2-anthraquinonyl hydrazine (2-AH). Further on, the primary reaction product reacts with an aromatic aldehyde to give an insoluble hydrazone. The final reaction product fluoresces orange-red when exited by green light (lambda(exc)=520-580 nm) and marks sites of enzymatic activity by an intensive fluorescence. This fluorescent method permits highly sensitive enzyme detection and causes only very low background tissue fluorescence. Thus enzyme locations in the capillary bed endothelium properly and sensitively stained, which has not been achieved by now. The new method is used successfully to demonstrate the enzyme in cryotome tissue sections from several rat organs.

New fluorescent method for the histochemical detection of tripeptidyl peptidase I using glycyl-l-prolyl-l-met-2-anthraquinonyl hydrazide as substrate.

A new substrate for tripeptidyl peptidase I (TPP I; E.C.3.4.14.9)-Gly-L-Pro-L-Met-2-anthraquinonyl hydrazide (Gly-Pro-Met-2-AH) is synthesized and used for the fluorescent histochemical detection of the enzyme. The enzyme liberates low soluble 2-anthraquinonylhydrazine, which-couples quickly with 3-nitrobenzaldehyde (3-NBA) yielding a highly fluorescent water-insoluble hydrazone--3-nitrobenzylidene-2-anthraquinonylhydrazone. The latter compound is localized precisely at sites of enzymatic activity and marks them with a very bright and stable orange-red fluorescence after excitation with conventional monochromatic andlaser green light (lambda(exc)=520-580 nm). The new technique is used successfully for the visualization of the enzyme in tissue sections of different rat organs - and represent the first fluorescent histochemical method for that peptidase.

Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

The expression and localization of somatostatin receptors in dorsal root ganglion neurons of normal and monoarthritic rats.

Somatostatin has antinociceptive effects by acting on somatostatin (sst) receptors in primary afferent neurons. Five sst receptor subtypes (sst(1-5)) have been identified. In the present study we assessed the expression and localization of the sst receptor subtypes in lumbar dorsal root ganglia of normal rats and of rats with unilateral antigen-induced arthritis (AIA) in the knee joint. We used polymerase chain reaction (PCR) of material from dorsal root ganglia and immunohistochemistry in dorsal root ganglion paraffin sections. PCR data show that sst(1), sst(2(a)), sst(2(b)), sst(3), and sst(4) receptors are expressed in lumbar dorsal root ganglia of the rat. The sst(5) receptor was expressed in a few samples. Available antibodies revealed sst(2(a)) and sst(2(b)) receptor-like immunoreactivity in the vast majority of neurons, and sst(4) receptor-like immunoreactivity in about 40% of the dorsal root ganglion neurons and in some satellite cells. Real time PCR at 3, 10 and 21 days after induction of AIA did not reveal changes in receptor expression. Immunohistochemistry showed that a similar high proportion of neuronal profiles expressed sst(2(b)) receptor-like IR in control and AIA rats, but the proportion of neuronal profiles with sst(2(a)) receptor-like IR was significantly lower in acute and chronic AIA rats than in control rats. Although the proportion of neuronal profiles with sst(4) receptor-like IR was significantly higher at 21 days than at 3 days values at 3 or at 21 days were not significantly different from control. These data show that the majority of dorsal root ganglion neurons exhibit somatostatin receptor-like IR thus suggesting a high potential for inhibition by somatostatin. The reduction in the proportion of neuronal profiles with sst(2(a)) immunoreactivity suggests that inhibition of neuronal activity by somatostatin is reduced during painful arthritis.

Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study.

OBJECTIVE: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. METHODS: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin-eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). RESULTS: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. CONCLUSION: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.

Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves.

The multidisciplinary research of tissue engineering utilizes biodegradable or decellularized scaffolds with autologous cell seeding. Objective of this study was to investigate the impact of different decellularization protocols on extracellular matrix integrity of xenogeneic tissue by means of multiphoton femtosecond laser scanning microscopy, biochemical and histological analysis. Pulmonary valves were dissected from porcine hearts and placed in a solution of trypsin-EDTA and incubated at 37 degrees C for either 5, 8, or 24 h, followed by a 24 h PBS washing. Native and decellularized valves were processed for histology, DNA, cell proliferation, matrix proteins and biomechanical testing. Multiphoton NIR laser microscopy has been applied for high-resolution 3D imaging of collagen and elastin. Distinct differences in several ECM components following decellularization time were observed. Total GAG contents decreased in a time-dependent manner, with o-sulfated GAGs being more susceptible to degradation than n-sulfated GAGs. Efficiency of insoluble collagen extraction increased proportionally with decellularization time, suggesting ECM-integrity may be compromised with prolonged incubation. Biomechanical testing revealed a gradual weakening of mechanical strength with increased decellularization time. The enzymatic decellularization process of heart valves revealed a time-dependent loss of cells, ECM components and biomechanical strength. In order to avoid any immune response a thorough decellularization of 24 h remains mandatory.

Recent advances in catalytic peroxidase histochemistry.

Immunoassays have developed to become an important analytical tool in life sciences for detection of endogenous and exogenous targets. Among the most important enzyme labels horseradish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (GAL) is HRP the smallest enzyme and plays nowadays an outstanding role. The oldest substrates are chromogens widely applied for localization of sites of peroxidase (PO) activity in histochemistry as well as for colorimetric applications. They are represented by a diversity of aromatic amines and phenols. Encouraged by development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels represent the most sensitive and worthwhile detection tools to date. In contrast to chromogens fluorescent labels for detection PO activity are confined only to a few substrates developed more recently. These substrates are mostly applied in histochemistry at a short time scale due to their frequently high solubility. At the long time scale sole exception is so far the tyramine based fluorochome deposition technique (more general: catalytic reporter deposition, CARD). Despite quite different staining behavior both fluorometric and product deposition related principles are based on 4-hydroxy phenylalkyl substrates. The following article reviews basic principles of peroxidatic substrate degradation processes including chromogenic and fluorescent approaches with emphasis on recent advances in development of chromogens and fluorogens for application in histology. As a result of systematic efforts towards the design of substrates, the range of classical precipitating chromogens as well as fluorescent techniques could be complimented by novel highly sensitive substrates with superior staining capabilities: a) Metal chelating 2-hydroxy benzylamines are derived from classical aniline substrates (two steps) and utilize metal catalytic effects in an efficient intramolecular way. The enzymatically yielded dark colored polycondensation products are applicable in histochemistry, in colorimetry and especially as precipitating electron opaque labels with enhanced osmiophilic properties for light and electron microscopy. b) Fluorescent 4-hydroxy-styryl derivatives are capable of oxidative selfanchoring reactions at the cellular level close to sites of PO activity. In contrast to deposition of tyramine conjugated fluorochromes an altered fluorochrome with improved fluorescence properties is furnished during oxidative crosslinking of the substrate. This results in a highly specific and photostable fluorescence response and an outstanding low background staining. Histochemical and immunohistochemical applications are presented.

Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death.

Two-photon excitation-based near-infrared (NIR) laser scanning microscopy is currently emerging as a new and versatile alternative to conventional confocal laser scanning microscopy, particularly for vital cell imaging in life sciences. Although this innovative microscopy has several advantages such as highly localized excitation, higher penetration depth, reduced photobleaching and photodamage, and improved signal to noise ratio, it has, however, recently been evidenced that high-power NIR laser irradiation can drastically inhibit cell division and induce cell death. In the present study we have investigated the cellular responses of unlabeled rat kangaroo kidney epithelium (PtK2) cells to NIR femtosecond laser irradiation. We demonstrate that NIR 170-fs laser pulses operating at 80-MHz pulse repetition frequency and at mean power of > or = 7 mW evoke generation of reactive oxygen species (ROS) such as H2O2 that can be visualized in situ by standard in vivo cytochemical analysis using Ni-3,3'-diaminobenzidine (Ni-DAB) as well as with a recently developed fluorescent probe Jenchrom px blue. The formation of the Ni-DAB reaction product as well as that of Jenchrom was relatively more pronounced when irradiated cells were incubated in alkaline solution (pH 8) than in those incubated in acidic solution (pH 6), suggesting peroxisomal localization of these reaction products. Two-photon time-lapse imaging of the internalization of the cell impermeate fluorescent dye propidium iodide revealed that the integrity of the plasma membrane of NIR irradiated cells is drastically compromised. Visualization of the nuclei with DNA-specific fluorescent probes such as 4',6-diamidino-2-phenylindole 24 h postirradiation further provided tangible evidence that the nuclei of these cells undergo several deformations and eventual fragmentation. That these NIR irradiated cells die by apoptosis has been established by in situ detection of DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method. Because the reactive oxygen species such as H2O2 and OH* can cause noxious effects such as cell membrane injury by peroxidation of polyunsaturated lipids and proteins and oxidative phosphorylation, and alterations of ATP-dependent Ca2+ pumps, these ROS are likely to contribute to drastic cytological alterations observed in this study following NIR irradiation. Taken together, we have established that NIR laser irradiations at mean power > or = 7 mW delivered at pulse duration time of 170 fs generally used in two- and multiphoton microscopes cause oxidative stress (1) evoking production of ROS, (2) resulting in membrane barrier dysfunction, (3) inducing structural deformations and fragmentation of the nuclei as well as DNA strand breaks, (4) leading to cell death by apoptosis.

Towards versatile metal associating substrates for the determination of peroxidatic activity/hydrogen peroxide by chemical designing of Schiff base derivatives.

Novel chromogenic N-arylmethyl-aniline-substrates of the general formula R-NH-CH2C6H5-n-Xn (X = OH, NHR) for the localization of peroxidatic activity/hydrogen peroxide were synthesized in two steps from starting amines and aromatic aldehydes. When using 1,2-dinucleophiles (e.g. diaminobenzidine) as starting material there may be limitations resulting from dominant altemative reaction courses (amino-imines vs bis-imines) or tautomerism (amino-imines vs benzimidazolines). This has been investigated in a model study on 1,2-phenylendiamine. All substrates were evaluated for application in histochemistry, electrophoresis, colorimetry and electron microscopy. Thus, 1/ endogenous peroxidatic activity in native cryotome sections of Wistar rats was stained. One selected reagent was used for immuno-histochemical demonstration of vimentine and applied for laser microscopy at 543 nm as well. 2/ Electro-blotted dilution series of horseradish peroxidase were stained and reagents ranked according to their sensitivity. 3/ In test tube experiments precipitation behavior, color and solubility of precipitates was investigated. 4/The chromogens are capable of forming electron opaque final reaction products by way of increased osmiophilicity of the specific reaction product or/and by complexation of electron dense metals as demonstrated by electron microscopical investigations. As a result, two novel reagents derived from 1,2-phenylendiamine and 2-aminophenol are recommended especially for electron microscopy: The discrimination between internum and extemum of specific granules after osmium tetroxide treatment is resolved clearly as compared with results obtained with the standard Kamovsky protocol.

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.

Novel chromogenic substrates with metal chelating properties for the histochemical detection of peroxidasic activity, derived from 3-amino-9-ethylcarbazole (AEC) and 3,6-diamino-9-ethylcarbazole.

For staining of peroxidase activity routinely employed 3-amino-9-ethylcarbazole 1 (AEC) was chemically modified in order to obtain chromogenic enzyme substrates with improved staining properties. In conclusion of systematically structure/staining considerations of a series of novel substrates, it can be generalized that the performance of traditional chromogenic peroxidase amine-substrates is accessible an considerably improvement in terms of sensitivity and adaptibility for various application purposes (solubility and color of the reaction product, electron dense and osmiophilic properties, ...) by attachment of chelating N-benzyl-moieties making available highly efficient the well known metal catalytic effect in a proposed intramolecular way. Thus, the model compounds 3(arylmethyl)amino-9-ethyl-carbazole 4 and 3,6-bis-(arylmethyl)amino-9-ethyl-carbazole 7 were synthesized by condensation of 3-amino-9-ethylcarbazole 1 (AEC) or the corresponding 3,6-diamine 5 with aromatic aldehydes 2. The resulting Schiff bases 3 and 6 were subsequently reduced with sodium borohydride. The obtained benzylamines 4 and 7 were examined as chromogenic substrates: 1) qualitatively in test tube experiments concerning color, precipitation behavior and solubility of the precipitates, 2) quantitatively by means of electroblotted dilution series of horseradish peroxidase, and finally in a biological environment for the localization of endogenous peroxidasic activity 3) in native cryotome tissues of Wistar rats. 4) The usefulness of the new approach for electron microscopy was revealed, too. Thus the discrimination between internum and externum of specific granules after osmium tetroxide contrastate was higher if compared with results obtained by the Karnovsky protocol. The wide spread variation of substitution patterns of the novel reagents gave reason for structure-reactivity considerations and ongoing leading structures. The stereochemical and electronic factors as well as competing reaction pathways governing the reaction course are briefly discussed. In addition, the metal associating reagents are highly effective in oxidative side-coupling reactions with aromatic amine or phenol-additives exemplified here by means of 4-amino-N,N-diphenylamine. The reagents 4 and 7 are obtainable in a simple in situ synthesis, too, offering in principle a 'chemical construction unit'. The demonstrated approach is of general interest for bioanalytical applications offering an access to potentially novel chromogens and electron opaque markers for the detection of peroxidasic activity/hydroperoxides or related redox enzyme systems.

Histochemical method for dipeptidyl aminopeptidase II with a new anthraquinonyl hydrazide substrate.

A new method for the histochemical visualization of lysosomal aminopeptidase dipeptidyl peptidase II activity (DPP II) is developed. The substrate L-Lys-L-Ala-5-chloro-1-anthraquinonylhydrazide-2HBr (Lys-Ala-CAH) is readily hydrolyzed by the enzyme to release 5-Cl-1-anthraquinonylhydrazine (CAH). The last compound is simultaneously coupled to an aromatic aldehyde, e.g. 4-nitrobenzaldehyde (p-NBA) or piperonal (3,4-methylenedioxybenzaldehyde; PPL), to form a highly insoluble deeply colored hydrazone, marking the enzyme locations. Using the new method, DPP II is successfully localyzed in tissue sections from different rat organs.

Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses.

In the present work, the authors investigated ultrastructural changes as well as the reproduction behaviour of preselected single CHO-K1 cells exposed to 170 femtosecond laser pulses at different power output levels in comparison with cells outside the illumination volume. The ultrashort laser pulses were provided by an 80 MHz Ti:sapphire laser at 780 nm. The cells were scanned ten times with a scan rate of 1/16 s(-1). Single CHO-K1 cells exposed to low mean power of 2 mW revealed no significant changes in ultrastructure after laser exposure. In some cases, changes of mitochondria with slight disordering of cristae were found. Cytoplasm was filled with vesicles that seemed to be released from Golgi stacks. Cells irradiated with higher powers demonstrated more dramatic changes in ultrastructure. A considerable number of swollen mitochondria in conjunction with loss of cristae was observed. The main event of mitochondrial changes was the formation of electron dense bodies in the mitochondrial matrix. In addition, lumen of endoplasmatic reticulum was enlarged. Highest applied mean laser power of 12.5 mW lead to complete destruction of mitochondria and their transformation to electron dense structures containing membrane material. Compared with cell targets irradiated with 2 mW mean power, the release of vesicles from Golgi stacks seemed to be rather moderate. Cells localised outside the laser beam revealed no ultrastructural changes. Low mean laser power at 2 mW was unable to impair the reproduction behaviour of CHO-K1 cells. At higher laser power output levels, CHO-K1 cells started to delay cell division. At 12.5 mW, no cell division occurred. The obtained results may be helpful in recommending parameters for safe femtosecond laser microscopy of living specimens.

Intracellular nanosurgery with near infrared femtosecond laser pulses.

We report on laser-assisted knocking out of genomic nanometer-sized regions within the nucleus of living cells. The intranuclear nanosurgery was possible by application of highly intense near infrared femtosecond laser pulses. The non-contact laser treatment was performed within a closed sterile cell chamber. The destructive multiphoton effect was based on 10(12) W/cm2 light intensities and limited to a sub-femtoliter focal volume of a high numerical aperture objective. We used the intracellular nanoscalpel for highly precise non-contact dissection of Hoechst-labelled chromosomes within a nucleus of a living Chinese hamster ovary cell. Following laser treatment, the cell remained alive and did not show any signs of membrane perturbation. The use of near infrared pulses provide the possibility of non-invasive intracellular nanoprocessing also within living tissue in depths of more than 100 microns.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.

New tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase.

A new tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase is proposed. The method is based on a newly synthesized substrate-gamma-L-glutamic acid-1-hydroxy-4-naphthylamide, which upon the enzyme hydrolysis liberates 1,4-aminonaphthol--a powerful reducing agent that reduces tetrazolium salts quickly and quantitatively to deeply colored, water-insoluble formazans, precipitating on the sites of the enzyme activity and marking them accurately. The redox reaction is quick enough and does not need any auxiliary electron-acceptor. The method is very fast and convenient for the histochemical visualization of the enzyme.