We report the synthesis and optical characterization of two new photoactivators and demonstrate their use for multiphoton excited three-dimensional free-form fabrication with proteins. These reagents were developed with the goal of cross-linking Type 1 collagen. This cross-linking process produces structures on the micron and submicron size scales. A rose bengal diisopropyl amine derivative combines the classic photoactivator and co-initiator system into one molecule, reducing the reaction kinetics and increasing cross-linking efficiency. This derivative was successful at producing stable structures from collagen, whereas rose bengal alone was not effective. A benzophenone dimer connected by a flexible diamine tether was also synthesized. This activator has two photochemically reactive groups and is highly efficient in cross-linking bovine serum albumin and Type 1 collagen to form stable, robust structures. This approach is more flexible in terms of cross-linking a variety of proteins than by traditional benzophenone photochemistry. The photophysical properties vary greatly from that of benzophenone, with the appearance of a new, lower energy absorption band (lambda max approximately 370 nm in water) and broad, visible emission band (approximately 500 nm maximum). This absorption band is highly solvatochromic, suggesting it arises, at least in part, from a charge transfer interaction. Collagens are typically difficult to cross-link photochemically, and the results here suggest that these two new activators will be suitable for cross-linking other forms of collagen and additional proteins for biomedical applications such as the de novo assembly of biomimetic tissue scaffolds.

Acetowhitening is a phenomenon that can be observed when applying acetic acid to cervices. The way tissues whiten is observed and rated in colposcopy to help diagnose any precancerous changes in the cervix. This paper reports a study designed to measure multiple parameters of the acetowhitening process from a sequence of images captured with an improved colposcope after the application of acetic acid. Clinical examination was recorded on video tape in order to allow for the registration of the image data with the biopsy locations for which a histopatology conrmed diagnosis was available. Image processing techniques were used to align, lter , and segment the images of size 640 480 captured every 5 seconds during 5 minutes. A whiteness signal was then computed for each biopsy location. After tting a polynomial-based model to the data and computing different features, a linear discriminant analysis was performed to separate high-grade lesions from other types of lesions. In this study we used 26 high grade (CIN II-III) pathology-conrme d

Fluorescence emission and diffuse reflectance spectra of freshly excised cervical tissue were studied with two specially designed contact probes. The objective of the study was to reach a better understanding of the relationship between spectroscopic measurements and cervical tissue morphology. Tissue samples from loop electro-surgical excision and hysterectomy specimens were measured within 20 to 90 minutes of excision. Emission spectra with 337 nm excitation, and reflectance spectra were collected at wavelengths between 370 and 720 nm from different tissue sites. Hematoxylin-eosin stained slides of the measured zones were obtained and compared to the spectra.In one experiment, a contact probe with a central illumination fiber and two concentric rings of detection fibers (radii 0.1 and 1 mm), was placed in contact with the epithelium and used to measure spectra from ectocervix and endocervix. The influence of 5% acetic acid on fluorescence and reflectance spectra was also investigated. In another experiment, a single 100-micron fiber probe was placed perpendicular to a cut edge of tissue and scanned to measure spectra in depth. Depth scans were made over various areas of the cervix. Data were analyzed to estimate the distribution of common tissue fluorophores as a function of depth from the surface and location on the cervix.Significant differences in shape and in absolute intensity were observed in fluorescence and reflectance spectra taken from endocervical tissue and ectocervical tissue. Hemoglobin absorption was more pronounced for endocervical tissue. Changes with depth in NADH fluorescence were less pronounced with endocervical tissue. The influence of NADH decreases while collagen increases is one way to describe the differences observed in the shape of the spectra observed as a function of depth. The probe geometry, in particular the source/detector separation distance, plays an important role in defining the shape of fluorescence and reflectance spectra. Data collected in this study from nonneoplastic cervical tissue reveal changes after the application of acetic acid that are not negligible, the amplitude of fluorescence spectra decreases while the amplitude of reflectance spectra increases.

We report the design, development, and characterization of a sensitive, time-resolved fluorescence spectrometer capable of measuring fluorescence spectra and transient decays simultaneously, with data acquisition times less than 1 s. The spectrometer, a portable fluorescence lifetime spectrometer (FLS), was designed to be compatible with both laboratory and clinical research studies on biological systems, and was applied to the study of several biological fluorophores in vitro and human tissue in vivo. The instrument consisted of a nitrogen laser pumping a dye laser for excitation from 337.1 nm through the near infrared, a quartz fiber-optic probe for remote light delivery and collection, and amplified detectors for rapid spectral and temporal detection from 350 to 800 nm. The spectral resolution of the FLS was determined to be 3 nm, which is sufficient for accurately detecting the broad spectral bands associated with biological fluorophores. The FLS was able to detect 5×10−7 M fluorescein dye concentrations with spectral signal-to-noise ratios (SNRs) of 29. Time-resolved detection with the FLS had a dynamic range of approximately three decades with a SNR of 200. Using fluorescence lifetime standards, the FLS was determined to be capable of accurately resolving fluorophore lifetimes from hundreds of picoseconds to tens of nanoseconds in duration, with an ultimate temporal resolution of 360 ps.

Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogen-transformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore; No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coup[ed instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogen-transformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed. (C) 2001 Society of Photo-Optical Instrumentation Engineers.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.

The tryptophan derivative 3-ethylindole was studied in the first excited electronic state and the cation ground state using resonance enhanced multiphoton ionization (REMPI) and zero electron kinetic energy (ZEKE) spectroscopy. Weakly bound clusters of 3-ethylindole with argon (n=1–3) have also been studied. The monomer spectroscopy revealed that two conformations of the 3-ethylindole exist in the jet cooled sample. Density-functional theory (DFT) calculations have been used to calculate the ground- and ionic-state geometries and energies. The calculations reveal the two conformations to be a planar and a nonplanar orientation of the ethyl substituent relative to the indole plane. The ZEKE spectrum of the nonplanar form has an extensive progression in the vibration associated with ethyl torsion and indicates a significant geometry change in the ethyl chain torsion upon ionization. The ethyl chain torsional potential is mapped out using DFT calculations, and the ion surface is adjusted such that calculated frequencies and Franck–Condon factors reproduce the experimental ZEKE spectra. The conformer interconversion barrier height is calculated in the ground and ion states. This is compared to an experimentally determined barrier height in the ion. The spectroscopy of the argon complexes reveals interesting results with regard to the cooling of the 3-ethylindole conformations.

ADVERTISEMENT RETURN TO ISSUEPREVCommunication to the...Communication to the EditorNEXT3-Dimensional Submicron Polymerization of Acrylamide by Multiphoton Excitation of Xanthene DyesPaul J. Campagnola, David M. Delguidice, Gary A. Epling, Kurt D. Hoffacker, Amy R. Howell, Jonathan D. Pitts, and Steven L. GoodmanView Author Information Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030, Department of Chemistry, Stamford High, Stamford, Connecticut, Department of Chemistry, University of Connecticut, Storrs, Connecticut, and Center for Biomaterials, University of Connecticut Health Center, Farmington, Connecticut 06030 Cite this: Macromolecules 2000, 33, 5, 1511–1513Publication Date (Web):February 4, 2000Publication History Received29 June 1999Revised9 November 1999Published online4 February 2000Published inissue 1 March 2000https://doi.org/10.1021/ma991042eCopyright © 2000 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views976Altmetric-Citations83LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (89 KB) Get e-AlertsSUBJECTS:Fluorescence,Lasers,Manufacturing,Polymerization,Power Get e-Alerts

Nonlinear multiphoton photo-cross-linking and photopolymerization of proteins and polymers in solution have been used to direct the three-dimensional assembly of micron scale objects. Two aspects of fabricated proteinatious matrixes are examined in this paper: the efficiency of protein photopolymerization and the application of fabricated matrixes as sustained release devices. The efficiency of photoactivated cross-linking of the proteins bovine serum albumin and fibrinogen, using rose bengal, have been determined and found to vary with photosensitizer concentration. This concentration dependence suggests that the mechanism for protein cross-linking is a direct hydrogen transfer between an amino acid residue of the protein and the dye molecule itself. A comparison of the surface structure of single and multiple protein oligomers is undertaken and shown to Vary significantly depending on fabrication materials. Alkaline phosphatase bioactivity, upon entrapment in a protein structure, is maintained. The properties of fabricated protein matrixes as sustained release devices is also examined. The rates of diffusion of fluorescently labeled dextrans (10 and 40 kDa) from an optically fabricated BSA matrix vary with molecular weight and are linear with cross-link density. The half-life of release of 10 kDa dextran-TMR from a BSA micron scale structure is less than or equal to 6 min while 40 kDa dextran-TMR half-life of release is 25 min. Finally, rhodamine 610, a typical drug size molecule, was entrapped in an acrylamide structure, and its release is found to be diffusion-limited with half-lives of 10-31 min, depending on cross-link density.

The neutral S1 excited state and the ion ground state of 9-ethylfluorene-Arn van der Waals complexes have been studied for n=1–3. Resonance enhanced multiphoton ionization spectroscopy of the S1 state of the argon clusters reveals multiple isomeric structures for each of the cluster sizes studies coupled with the two monomer conformations. The n=1 cluster shows three isomers, one of the symmetric 9-ethylfluorene and two of the unsymmetric. The n=2 clusters has four possible isomers all of which are assigned to a (1|1) conformation, although each represents a unique structure with different argon binding sites. The n=3 cluster collapses down to two dominate isomers, one for each conformation of the parent. Mass analyzed threshold ionization (MATI) spectroscopy was used to investigate the ion, as well as assisting in isomer assignment of the S1 spectrum. IVR and dissociation of the argon complexes have also been studied with MATI spectroscopy. Ab initio calculations are used to determine the binding energy for all conformers and isomers of the n=1 complex. These values are in excellent agreement with the experimentally bracketed values, and prove useful in isomer assignments. Redistribution of the 208 cm−1 band of the n=2 symmetric conformation shows dynamics suggesting interconversion of all isomers to a new unassigned structure. Preferential dissociation of the argon located on the ethyl chain side of the sym-9-ethylfluorene-Ar2 complex is observed in both isomers.

The S1 excited state and cation ground state of jet cooled 9-ethylfluorene have been studied experimentally using resonant enhanced multiphoton ionization and zero electron kinetic energy (ZEKE) photoelectron spectroscopy. The spectroscopy has identified two conformations of the ethyl chain which are labeled symmetric and unsymmetric both of which exist in the supersonic expansion. Density functional quantum chemical calculations are used to calculate the ground state and cation energies of each conformer as well as the barrier to conformer interconversion via a bond rotation. Dynamics on the S1 surface are measured using picosecond and nanosecond ZEKE photoelectron spectroscopy. Fast irreversible vibrational redistribution is measured at energies ⩾990 cm−1 and the ZEKE spectra are shown to have a unique signature for each of the two isomers. Picosecond and nanosecond ZEKE spectroscopy are used to search for conformer interconversion but even at the highest energy probed (2648 cm−1) no evidence is seen for a dynamic barrier crossing. Statistical density of states calculations are used to predict the relative populations of each conformer expected as a function of excess energy as well as related Rice–Ramsperger–Kassel–Marcus calculations to predict the expected isomerization rates.

The spectrum of the S1 electronic state of jet-cooled 9-phenylfluorene–Arn, n=0–4, has been measured by two color resonant enhanced multiphoton ionization spectroscopy. The cation ground states of these complexes have also been studied by mass analyzed threshold ionization (MATI) spectroscopy in a 1+1 excitation process with various intermediate states in S1. Ab initio calculations in conjunction with the spectroscopy have determined that the phenyl ring at the 9 position is perpendicular to the plane of the fluorene moiety yielding an overall symmetry of Cs. The Ar complexes for n=1–3 exhibit multiple isomers which are identified in the S1 spectrum and confirmed by MATI spectroscopy. The structure of these isomers is determined by spectral analysis and additivity rules as well as atom–atom calculations using a Lennard-Jones potential. Vibrational dynamics from selected S1 vibronic levels are observed by the appearance of the picosecond or nanosecond time delayed MATI spectra. Vibrational redistribution and dissociation of the clusters are measured with nanosecond and picosecond time resolution. It is found that different isomers of the n=1 cluster show dramatically different rates of redistribution for several vibronic bands.

The spectrum of the S-1 electronic state of jet-cooled 9-phenylfluorene-Ar-n, n=0-4, has been measured by two color resonant enhanced multiphoton ionization spectroscopy. The cation ground states of these complexes have also been studied by mass analyzed threshold ionization (MATI) spectroscopy in a 1+1 excitation process with various intermediate states in S-1. Ab initio calculations in conjunction with the spectroscopy have determined that the phenyl ring at the 9 position is perpendicular to the plane of the fluorene moiety yielding an overall symmetry of C-s. The Ar complexes for n = 1-3 exhibit multiple isomers which are identified in the S1 spectrum and confirmed by MATI spectroscopy. The structure of these isomers is determined by spectral analysis and additivity rules as well as atom-atom calculations using a Lennard-Jones potential. Vibrational dynamics from selected SI vibronic levels are observed by the appearance of the picosecond or nanosecond time delayed MATI spectra. Vibrational redistribution and dissociation of the clusters are measured with nanosecond and picosecond time resolution. It is found that different isomers of the n=1 cluster show dramatically different rates of redistribution for several vibronic bands. (C) 1998 American Institute of Physics. [S0021-9606(98)00641-2].

The fluorene–Ar4 cluster has been shown to exhibit two distinct isomers when formed in a molecular beam. Resonance enhanced multiphoton ionization and mass analyzed threshold ionization experiments have been performed to investigate the structural properties, energetics and dynamics of these clusters when excited to vibronic bands in the S1 electronic state, with a specific interest in measuring isomer interconversion. At 208 cm−1 excess energy in the S1 isomer interconversion is not observed in the Ar4 cluster. Dissociation of the Ar5 cluster from the 722 cm−1 band is shown to produce both Ar4 isomers.

Fluorene–Arn complexes formed in a pulsed supersonic jet have been studied in their S1 state using two color REMPI spectroscopy with mass resolved detection. The appearance and shifts of the S1 origins relative to the fluorene monomer are measured for cluster sizes up to n = 30. The shifts and appearance of these bands are used to identify multiple conformations at low n and have indicated a shift from two sided clustering by Ar at low n to primarily one sided clustering at large n. The ionic ground state of the smaller clusters (n⩽6) are studied using mass analyzed threshold ionization (MATI) spectroscopy. The change of the ionization potentials as a function of cluster size has been determined. In the case of the fluorene–Ar4 cluster, the MATI spectrum of two separate cluster conformations was measured, revealing significantly different ionization potentials. Vibrational dynamics has been studied in several smaller clusters (n⩽3) by measuring MATI and ZEKE spectra when pumping vibronic transitions in the fluorene chromophore. Significantly enhanced coupling of the chromophore to van der Waals modes is observed in going from n = 1 to n = 3.

High resolution photoelectron spectroscopy is applied to the study of molecular clusters. The primary species studied are fluorene-Arn complexes. Spectroscopy of the neutral S1 state has been performed on clusters as large as n equals 30. In order to study the photoelectron spectra of the clusters size selectively mass analyzed threshold ionization (MATI) is used which is a mass resolved version of the ZEKE technique. MATI spectroscopy has been applied to clusters up to n equals 5. The spectral shifts in the S1 origin and ion threshold are used as a measure of the relative stability of the different clusters. Using previous experimental and theoretical work on related clusters the structures of the clusters are inferred from the observed spectral shifts. In some cases multiple conformations of a particular cluster size are identified.