Ultraviolet Fluorescence Microscopy Research Articles

Review of some Hystricosporites representatives from Bolivia

A taxonomic study of megaspores with bifurcate-tipped processes from the Devonian–Carboniferous transition of Bolivia was performed. Megaspore specimens were isolated from palynological samples of the Pando X1 and Manuripi X1 boreholes. Their morphological characteristics and wall structures were described and illustrated with transmitted optical light, ultraviolet fluorescence, and scanning electron microscopy. This megaspore assemblage comprised six fossil species of Hystricosporites (H. costatus, H. delectabilis, H. elongatus, H. expandus, H. furcatus and H. spiralis). The morphologic features of their sporoderms confirm that these megaspores with bifurcate-tipped processes could have had a botanical affinity related to lycopsids. Additional future studies of wall ultrastructure using transmission electron microscopy will provide more information about their phylogenetic relationships with fossil and extant lycopsids. Keywords: megaspores, lycopsids, Carboniferous, Devonian.

Deep ultraviolet fluorescence microscopy of three-dimensional structures in the mouse brain

Three-dimensional (3D) imaging at cellular resolution improves our understanding of the brain architecture and is crucial for structural and functional integration as well as for the understanding of normal and pathological conditions in the brain. We developed a wide-field fluorescent microscope for 3D imaging of the brain structures using deep ultraviolet (DUV) light. This microscope allowed fluorescence imaging with optical sectioning due to the large absorption at the surface of the tissue and hence low tissue penetration of DUV light. Multiple channels of fluorophore signals were detected using single or a combination of dyes emitting fluorescence in the visible range of spectrum upon DUV excitation. Combination of this DUV microscope with microcontroller-based motorized stage enabled wide-field imaging of a coronal section of the cerebral hemisphere in mouse for deciphering cytoarchitecture of each substructure in detail. We extended this by integrating vibrating microtome which allowed serial block-face imaging of the brain structure such as the habenula in mouse. Acquired images were with resolution high enough for quantification of the cell numbers and density in the mouse habenula. Upon block-face imaging of the tissues covering entire extent of the cerebral hemisphere of the mouse brain, acquired data were registered and segmented for quantification of cell number in each brain regions. Results in the current analysis indicated that this novel microscope could be a convenient tool for large-scale 3D analysis of the brain in mice.

APPLICATION STUDY OF TEMPORARY PLUGGING ASSISTED FRACTURING IN TRANSITION ZONES OF SABEI OILFIELD

The northern oil and water transition zone of the Sabei Oilfield is the largest development area of the whole Daqing Changyuan transition zone. At present, it is producing with ultra-high water cut, and both water flooding and polymer flooding are under operation. In order to clarify the distribution characteristics and existing state of the remaining oil in the transitional zone, liquid nitrogen freezing and ultraviolet fluorescence microscopy are used to quantitatively characterize the microscopic occurrence of residual oil in cores after different volumes of water flooding, the relationship between water flooding intensity and microscopic residual oil distribution in free state and immobile state is established. Analysis results of residual oil in SII layer of the Sabei Oilfield show more immobile residual oil. Conventional fracturing measures have been done to improve oil recovery, but its efficiency deteriorated year by year, which is only 51.1% of the measures done for the oil-rich zones. Therefore, temporary plugging assisted fracturing technology using fiber was carried out to improve the stimulation efficiency in the transition zone. Also, an improved permeability calculation model was established to analyze the influence of the fracture length, fracture height, and oil viscosity on productivity ratio and recovery factor. Based on these results, the optimal value ranges of operational parameters were obtained. Then, eight wells were stimulated using the temporary plugging assisted fracturing technology in the field. The results show that after fracturing the average daily oil production rate increased by 6.3 t and the cumulative oil production from eight wells increased by 17386 t in 720 days, demonstrating the efficacy of the temporary plugging assisted fracturing technology compared with conventional fracturing.

Ultraviolet Autofluorescence Microscopy of Nanotyrannus lancensis Sections Reveals Blood Clots in Vessel Canals

Abstract: Theropod dinosaurs have captured the imagination of the public and paleontologists alike. Histology of the bones of theropods has revealed much about dinosaur physiology, behavior, and growth. Histology and ultraviolet fluorescence (UVFL) microscopy of one controversial dinosaur, Nanotyrannus lancensis, reveals the presence of blood clots in post-fixed vessel canals of claw, vertebra, and other isolated post-cranial elements collected at Hell Creek, MT. These clots are thicker, more closely adherent to canal walls, and more reactive to 347 nm UVFL incident light than unfixed specimens. Theropod histology images in the literature display similar clots, and those should be subjected to UVFL for confirmation. In addition, nematodes are evidently preserved in vessel canals of dinosaurs.

Response strategies of N-fixation by epiphytic bryophytes to water change in a subtropical montane cloud forest

Water changes are predicted to regulate physiological activities of bryophytes characterized by poikilohydric gametophytes. In montane forest ecosystems, nitrogen(N)–fixing bryophyte–cyanobacteria associations are main N resources. The aim of this study was to assess how bryophyte–associated microbiomes and their nitrogenase activity response to instant or long-term water content changes. We investigated the cyanobacterial colonization and nitrogenase activity of four epiphytic bryophyte species in a subtropical montane cloud forest during the dry and rainy seasons in Ailao Mountains, Yunnan, southwestern China. We also evaluated the nitrogenase activity of bryophyte–cyanobacteria associations in response to different water contents in laboratory experiment. The degree of cyanobacterial colonization was evaluated using ultraviolet fluorescence microscopy, and nitrogenase activity of bryophyte–cyanobacteria associations were measured using the acetylene reduction assay (ARA). Cyanobacteria showed an association with all four bryophyte species, with 1.04–3.37% area colonized of the shoot and 10.16–20.21 nmol C2H4·g−1·h−1 average nitrogenase activity. Nitrogenase activity was positively related to cyanobacterial colonization (R = 0.742; P = 0.0349). The relationship between water content and nitrogenase activity was unimodal, and both water surplus and shortage inhibited N fixation. Furthermore, long–term drought conditions reduced the degree of cyanobacteria colonization on bryophyte shoots, resulting in decreased nitrogenase activity in the dry season. These results indicate that different response strategies of N fixation operate in bryophyte–cyanobacteria associations to cope with instant and long–term changes in water availability. Our data suggest that both extreme precipitation and drought have a negative impact on N fixation of cyanobacteria–bryophyte associations.

Imaging Diagnostics Coupled with Non-Invasive and Micro-Invasive Analyses for the Restoration of Ethnographic Artifacts from French Polynesia

In this paper, two different objects from the ethnographic collection of the museum of the Congregation of the Sacred Hearts of Jesus and Mary (Rome), a Polynesian barkcloth (tapa) and a Polynesian headdress in feathers (pa’e ku’a), were investigated to characterize the materials, to evaluate their state of conservation and address the restoration activities. Imaging methods such as multispectral imaging, 3D ultraviolet induced fluorescence and scanning electron microscopy have been integrated with analytical techniques such as X-ray fluorescence spectroscopy, Fourier transform infrared and surface enhanced Raman spectroscopy. Imaging investigations allowed us to differentiate constitutive materials and study their distribution, such as the yellow dye in the tapa used to trace the geometrical pattern and the psittacofulvins responsible for the feathers’ colors in the headdress. The combination of molecular spectroscopy, supported by observation under a scanning electron microscope, allowed us to propose a characterization of the organic painting materials (Morinda citrifolia, Curcuma longa) used for the tapa, and of the type of feathers (from Vini kuhlii bird) and vegetal fibers (Cocos nucifera L.) used to realize the headdress, as well as enabling the identification of degradation products and microorganisms affecting the artifacts before restoration. Fourier transform infrared spectroscopy detected the organic materials used as adhesives for the tapa and headdress: a polysaccharide, probably starch, for the tapa and a natural rubber from Cerbera manghas L. for the headdress. The results of the multi-analytic diagnostic campaign enabled the choice of proper restoration materials, compatible with the original ones, and helped us develop effective protocols for the artifacts’ conservation, such as laser cleaning of the feathers.

Detection and localization of calcium oxalate in kidney using synchrotron deep ultraviolet fluorescence microscopy.

Renal oxalosis is a rare cause of renal failure whose diagnosis can be challenging. Synchrotron deep ultraviolet (UV) fluorescence was assayed to improve oxalosis detection on kidney biopsies spatial resolution and sensitivity compared with the Fourier transform infrared microspectroscopy gold standard. The fluorescence spectrum of synthetic mono-, di- and tri-hydrated calcium oxalate was investigated using a microspectrometer coupled to the synchrotron UV beamline DISCO, Synchrotron SOLEIL, France. The obtained spectra were used to detect oxalocalcic crystals in a case control study of 42 human kidney biopsies including 19 renal oxalosis due to primary (PHO, n = 11) and secondary hyperoxaluria (SHO, n = 8), seven samples from PHO patients who received combined kidney and liver transplants, and 16 controls. For all oxalocalcic hydrates samples, a fluorescence signal is detected at 420 nm. These spectra were used to identify standard oxalocalcic crystals in patients with PHO or SHO. They also revealed micrometric crystallites as well as non-aggregated oxalate accumulation in tubular cells. A nine-points histological score was established for the diagnosis of renal oxalosis with 100% specificity (76-100) and a 73% sensitivity (43-90). Oxalate tubular accumulation and higher histological score were correlated to lower estimated glomerular filtration rate and higher urinary oxalate over creatinine ratio.

Cell-by-cell estimation of PAH sorption and subsequent toxicity in marine phytoplankton

Polycyclic Aromatic Hydrocarbons (PAHs) have elicited increasing concern due to their ubiquitous occurrence in coastal marine environments and resultant toxicity in organisms. Due to their lipophilic nature, PAHs tend to accumulate in phytoplankton cells and thus subsequently transfer to other compartments of the marine ecosystem. The intrinsic fluorescence properties of PAHs in the ultraviolet (UV)/blue spectral range have recently been exploited to investigate their uptake modes, localization, and aggregation in various biological tissues. Here, we quantitatively evaluate the sorption of two model PAHs (phenanthrene and pyrene) in three marine phytoplankton species (Chaetoceros tenuissimus, Thalassiosira sp. and Proteomonas sp.) using a combined approach of UV excitation flow cytometry and fluorescence microscopy. Over a 48-h exposure to a gradient of PAHs, Thalassiosira sp. showed the highest proportion of PAH-sorbed cells (29% and 97% of total abundance for phenanthrene and pyrene, respectively), which may be attributed to its relatively high total lipid content (33.87 percent dry weight). Moreover, cell-specific pulse amplitude modulation (PAM) microscope fluorometry revealed that PAH sorption significantly reduced the photosynthetic quantum efficiency (Fv/Fm) of individual phytoplankton cells. We describe a rapid and precise hybrid method for the detection of sorption of PAHs on phytoplankton cells. Our results emphasize the ecologically relevant sub-lethal effects of PAHs in phytoplankton at the cellular level, even at concentrations where no growth inhibition was apparent. This work is the first study to address the cell-specific impacts of fluorescent toxicants in a more relevant toxicant-sorbed subpopulation; these cell-specific impacts have to date been unidentified in traditional population-based phytoplankton toxicity assays.

Multiplex protein-specific microscopy with ultraviolet surface excitation.

Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as microscopy with ultraviolet surface excitation (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.

Characterization of Phase Transformations for Amorphous Solid Dispersions of a Weakly Basic Drug upon Dissolution in Biorelevant Media.

The overall goal of this study was to investigate the dissolution performance and crystallization kinetics of amorphous solid dispersions (ASDs) of a weakly basic compound, posaconazole, dispersed in a pH-sensitive polymeric matrix consisting of hydroxypropyl methylcellulose acetate succinate (HPMC-AS), using fasted-state simulated media. ASDs with three different drug loadings, 10, 25 and 50wt.%, and the commercially available tablets were exposed to acidic media (pH1.6), followed by transfer to, and dissolution in, intestinal media (pH6.5). Parallel single stage dissolution experiments in only simulated intestinal media were also performed to better understand the impact of the gastric stage. Different analytical methods, including nanoparticle tracking analysis, powder x-ray diffraction, second harmonic generation and two-photon excitation ultraviolet fluorescence microscopy, were used to characterize the phase behavior of these systems at different stages of dissolution. Results revealed that all ASDs exhibited some degree of drug release upon suspension in acidic media, and were also vulnerable to matrix crystallization. Upon transfer to intestinal media conditions, supersaturation was observed. This was short-lived for some dispersions due to the release of the crystals formed in the acid immersion stage which acted as seeds for crystal growth. Lower drug loading ASDs also exhibited transient formation of amorphous nanodroplets prior to crystallization. This work emphasizes the significance of assessing the impact of pH change on dissolution and provides a fundamental basis of understanding the phase behavior kinetics of ASDs of weakly basic drugs when formulated with pH sensitive polymers.

A physicochemical characterisation of a medieval herbal ink, Mahī, of Assam, India

Mahī, a unique herbal ink used in medieval Assam in India for writing and illustrating manuscripts, is known for its intense colour and durability for centuries, and its non‐corrosive nature. Mahī is distinctly different from the other contemporary inks used elsewhere in the world, including other parts of India. The interesting features of Mahī are due to its special recipe involving several herbal ingredients in addition to iron sourced from fish blood or the rust of iron tools, with cow urine as an extractant and solvent. The objective of the current study is an in‐depth understanding of the traditional method of the preparation of Mahī, and its characterisation through analysis of the physicochemical properties using spectroscopic and imaging techniques as well as biochemical analysis. A series of model Mahī samples are prepared following the traditional method with varying ingredients, and their compositions and physicochemical properties are evaluated with respect to the ingredients using standard methods and analytical tools including atomic absorption spectroscopy, tensiometry, ultraviolet ( UV )‐visible, Fourier Transform‐infrared ( FT‐IR), Raman, steady‐state fluorescence, UV fluorescence and emission microscopy. The colour of the ink has been attributed to various polyphenols, gallic acid, epigallocatechin gallate, epicatechin, quercetin, kaempferol and tannic acid, and their iron complexes. UV fluorescence and emission microscopy confirmed an autofluorescence indicative of the presence of phenolic and chlorophyll pigments. Solubilisation by glycosidic biosurfactant contributes to stabilisation of Mahī. The non‐corrosive nature of Mahī has been attributed to its neutral pH and absence of free iron and copper ions.

Microfacies, diagenesis and oil emplacement of the Upper Jurassic Arab-D carbonate reservoir in an oil field in central Saudi Arabia (Khurais Complex)

The Upper Jurassic (Kimmeridgian) Arab-D reservoir of the Arab Formation in the Arabian Peninsula is considered to be the largest hydrocarbon carbonate reservoir in the world. Despite a relative large number of studies dealing with lithofacies and depositional sequences, there are still few studies dealing with diagenesis of this supergiant reservoir. In this study, petrographic, cathodoluminescence and ultra-violet light fluorescence microscopy analyses of the Arab-D reservoir in an oil field of Saudi Arabia (Khurais Complex) have been combined with isotope geochemistry (δ13C, δ18O and 87Sr/86Sr) to decipher the sequence of diagenetic events affecting the reservoir before and during oil emplacements, and the impact of depositional mineralogies and their modifications during the diagenesis on the reservoir quality and heterogeneity. All the analyzed samples correspond to lithofacies that can be ascribed to nine microfacies types (MFT-1 to MFT-9). Three stages of carbonate dissolution (d1 to d3), four stages of calcite cements (C1 to C4) and 5 zones of replacive dolomite and dolomite cement (D1 to D5) have been documented from diagenetic environments ranging from marine to late burial. Abundant oil fluid inclusions have been recognized in calcite C3 and C4 and dolomite D5 from wells located in the flanks of the structure (Group 2 of cements). Cements from wells located in the center of the structure are largely oil-inclusions free (Group 1 of cements). This distribution suggests that Group 2 of cements formed within the oil-water transition zone of the reservoir during the oil infilling of the structure, allowing to constrain the timing of oil emplacement and suggesting at least two phases of oil charging. Porosity enhancement by corrosive dissolution (d3) occurred during late burial and took place just before precipitation of dolomite D5 and calcite C4, and just before the second stage of hydrocarbon arrival, suggesting the presence of aggressive fluids preceding the late phase of hydrocarbon migration. Cross-plot of carbon vs. oxygen stable isotopes shows a general trend toward decreasing δ18O values with increasing burial depth, whereas all δ13C values remain positive except for Group 2 of C3 and C4 calcites, which exhibit negative (up to −4.6‰) δ13C values. The later suggest precipitation from pore waters with increased volumes of 12C as a by-product of hydrocarbon maturation. Except for C4, all the 87Sr/86Sr values are within the range of the Late Jurassic seawater, suggesting precipitation from Jurassic marine or evolved marine pore waters. In contrast, the more radiogenic calcite C4 suggests a source from fluids that interacted with siliciclastics or clay-rich sequences. Depositional mineralogy of the grainy lithofacies is interpreted to have had an impact on the effectiveness of cementation and dissolution, controlling the reservoir heterogeneity. Due to excess of cementation, reservoir quality is worse in grainstone microfacies rich in aragonitic components and better in grainstone and packstone microfacies poorer in aragonitic grains but affected by late dissolution of micrite. The integration of all these data resulted in a more precise understanding of the reservoir quality and heterogeneity.

Diagenetic fluids evolution of Oligocene Huagang Formation sandstone reservoir in the south of Xihu Sag, the East China Sea Shelf Basin: constraints from petrology, mineralogy, and isotope geochemistry

The Oligocene Huagang Formation is the main sandstone reservoir in the Xihu Sag, situated in the east of the East China Sea Shelf Basin. With an integrated approach of thin-section petrography, ultra-violet fluorescence microscopy, scanning electron microscopy, and isotope geochemistry, the different diagenetic features were identified, the typical diagenetic parasequences were established, and the diagenetic fluids evolution history were reconstructed for the Oligocene Huagang Formation sandstone reservoir in the south of Xihu Sag. The Huagang Formation sandstone reservoir is now in Period B of the mesodiagenesis, which has undergone significant diagenetic alterations such as mechanical compaction, Pore-lining chlorite cement, feldspar dissolution, quartz cementation and dissolution, and carbonate cementation. Three types of carbonate cements (early siderite, medium ferrocalcite and late ankerite) were identified in the Huagang Formation sandstone reservoir. The carbon and oxygen isotopic compositions of carbonate cements show that the early calcite precipitate from alkaline lacustrine environment whereas the late carbonate cements were closely related to the organic acids. To the Huagang Formation sandstone reservoir, it has experienced two main episodes of dissolution during diagenesis. The early dissolution is that unstable components such as feldspar, lithic fragments, and carbonate cement were dissolved by acidic water. The second dissolution is that quartz and other silicate minerals were dissolved under the alkaline condition. Two main phases of hydrocarbon charging occurred in this study area. The first hydrocarbon emplacement was prior to the medium carbonate cementation but posterior to feldspar dissolution and the onset of quartz cementation at the end of the Miocene. The second hydrocarbon charging occurred in the Quaternary period after the late carbonate precipitation.

Direct Optofluidic Measurement of the Lipid Permeability of Fluoroquinolones

Quantifying drug permeability across lipid membranes is crucial for drug development. In addition, reduced membrane permeability is a leading cause of antibiotic resistance in bacteria, and hence there is a need for new technologies that can quantify antibiotic transport across biological membranes. We recently developed an optofluidic assay that directly determines the permeability coefficient of autofluorescent drug molecules across lipid membranes. Using ultraviolet fluorescence microscopy, we directly track drug accumulation in giant lipid vesicles as they traverse a microfluidic device while exposed to the drug. Importantly, our measurement does not require the knowledge of the octanol partition coefficient of the drug – we directly determine the permeability coefficient for the specific drug-lipid system. In this work, we report measurements on a range of fluoroquinolone antibiotics and find that their pH dependent lipid permeability can span over two orders of magnitude. We describe various technical improvements for our assay, and provide a new graphical user interface for data analysis to make the technology easier to use for the wider community.

Ultraviolet femtosecond Kerr-gated wide-field fluorescence microscopy.

A Kerr-gated microscope capable of imaging ultraviolet luminescence with femtosecond time resolution has been developed. The system allows the spatial, spectral, and temporal measurement of UV-emitting samples. The instrumentation was optimized for emission collection in the UV, resulting in sub 90 fs time resolution of gated signals. ZnO nanowires were used to demonstrate the performance of the instrument. The evolution of the emission from a single nanowire was tracked via ultrafast transient spectroscopy and through sequential imaging. Transient dynamics were extracted from a region of intense emission on a single ZnO nanowire. This technique is a powerful tool capable of contactless ultrafast measurements of charge carrier dynamics in single nanoparticles.

Cyanobacteria associations in temperate forest bryophytes revealed by δ15N analysis1

Abstract Bryophytes have an important biogeochemical role in many forest ecosystems, where they regulate soil temperature and moisture and influence carbon and nitrogen (N) cycling. Associations between bryophytes and N2-fixing cyanobacteria have been reported in northern latitude forests and are shown to provide a substantial N input to these ecosystems, but whether the association extends into temperate forests remains largely unknown. We investigated the extent of bryophyte-cyanobacteria associations in common understory bryophytes in a temperate forest in New York using δ15N analyses and ultraviolet fluorescence microscopy. Of the seven taxa examined, three (Fissidens taxifolius, Marchantia polymorpha, and Thuidium delicatulum) were associated with cyanobacteria, with colonization observed in 15–85% of the leaves examined. The taxa with cyanobacterial associations exhibited δ15N values that were 3.9‰ higher compared with taxa that did not, and the δ15N signatures were close to the atmospheric N2 signa...

Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering

Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein-dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano-sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein-dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X-ray powder diffraction, second harmonic generation, ultraviolet two-photon excited fluorescence and scanning electron microscopy.

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼10(3)-10(4)-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

Two-photon excited UV fluorescence for protein crystal detection

Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging ofchiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers.