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Background: The historical pathological cut-off values for Wiberg's lateral center-edge (LCE) angle and Lequesne's acetabular index (AI) are below 20° and above 12° for the LCE and AI, respectively. The aim of this study was to reassess these two angles more than 50 years after their introduction using a standardized conventional radiological measurement method, considering changing social habits and their associated physiological changes.Methods: A total of 1,226 anteroposterior radiographs of the pelvis (2,452 hips) were obtained according to a strict standardized radiographic technique allowing reliable measurements of the LCE angle and the AI.Results: Distributions of the LCE and AI were pronouncedly Gaussian, with mean values of 33.6° for the LCE and 4.4° for the AI. The 2.5th and 97.5th empirical percentiles were 18.1 and 48.0° for the LCE and -6.9 and 14.9° for the AI. These intervals contained 95 % of the data in our large sample. Small but statistically significant differences between the sexes and right and left hips have been demonstrated. Correlation between age and coxometric indices was low.Conclusion: The above findings do not conflict with the historical benchmarks. Statistical differences between sexes and between right and left hips were not clinically relevant. No conclusion can be drawn about coxometric indices and clinical manifestations of hip dysplasia. [ABSTRACT FROM AUTHOR]

Introduction. The goal of this study was to research the association of femoral bumps and herniation pits with the overlap-ratio of the cross-over sign. Methods. Pelvic X-rays and CT-scans of 2925 patients with good assessment of the anterior and the posterior acetabular wall and absence of neutral pelvic tilt were enrolled in the investigation. Finally pelvic X-rays were assessed for the presence of a positive cross-over sign, and CT-scans for a femoral bump or a herniation pit. Additionally, if a positive cross-over sign was discovered, the overlap-ratio was calculated. Results. A femoral bump was found in 53.3% (n = 1559), and a herniation pit in 27.2% (n = 796) of all hips. The overlap-ratio correlated positively with the presence of a femoral bump, while a negative correlation between the overlap-ratio and the presence of a herniation pit was found. The latter was significantly more often combined with a femoral bump than without. Conclusions. We detected an increased prevalence of femoral bump with increasing overlap-ratios of the cross-over sign indicating a relation to biomechanical stress. The observed decreased prevalence of herniation pits with increasing overlap-ratios could be explained by reduced mechanical stress due to nontightened iliofemoral ligament in the presence of retroversion of the acetabulum.

We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

Purpose: Acetabular fractures typically occur in high energy trauma. Understanding of the various contributing biomechanical factors and trauma mechanisms is still limited. While several investigations figured out what role femoral position during impact plays in distinct fracture patterns, no data exists on the influence of acetabular version on the fracture type. Our study was carried out to clarify this issue., Methods: Radiological data sets of 192 patients (145 male, 47 female, age 14-90 years) sustaining acetabular fractures were assessed retrospectively. The crossover ratio of the crossover sign and presence or absence of the posterior wall sign and ischial spine sign were used to determine acetabular retroversion on conventional radiographs. Acetabular version in the axial plane was measured on a computed tomography (CT) scan. Statistics were then performed to analyse the relationship between the acetabular fracture type according to the Letournel classification and acetabular version., Results: A significant difference (p = 0.029) in acetabular version was found between fractures of the anterior [mean equatorial edge (EE) angle 19.93°] and posterior (mean EE angle 17.53°) acetabulum in the CT scan. No difference was shown on the measurements on conventional radiographs., Conclusions: Acetabular version in the axial plane has an influence on the acetabular fracture pattern. While more anteverted acetabula were frequently associated with anterior fracture types according to the Letournel classification, retroversion of the acetabulum was associated with posterior fracture types.

The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non-membrane-bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non-ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample-related artifacts in the diffusion measurement., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Partial weight bearing is thought to avoid excessive loading that may interfere with the healing process after surgery of the pelvis or the lower extremity. The object of this study was to investigate the relationship between the ability to partially weight bear and the patient's psychomotor skills and an additional evaluation of the possibility to predict this ability with a standardized psychomotor test., Methods: 50 patients with a prescribed partial weight bearing at a target load of 15 kg following surgery were verbally instructed by a physical therapist. After the instruction and sufficient training with the physical therapist vertical ground reaction forces using matrix insoles were measured while walking with forearm crutches. Additionally, psychomotor skills were tested with the Motorische Leistungsserie (MLS). To test for correlations Spearman's Rank correlation was used. For further comparison of the two groups a Mann-Withney test was performed using Bonferroni correction., Results: The patient's age and body weight significantly correlated with the ability to partially weight bear at a 15 kg target load. There were significant correlations between several subtests of the MLS and ground reaction forces measured while walking with crutches. Patients that were able to correctly perform partial weight bearing showed significant better psychomotor skills especially for those subtests where both hands had to be coordinated simultaneously., Conclusions: The ability to partially weight bear is associated with psychomotor skills. The MLS seems to be a tool that helps predicting the ability to keep within the prescribed load limits.

Objective: Knowledge of acetabular anatomy is crucial for cup positioning in total hip replacement. Medial wall thickness of the acetabulum is known to correlate with the degree of developmental dysplasia of the hip (DDH). No data exist about the relationship of routinely used radiographic parameters such as Wiberg's lateral center edge angle (LCE-angle) or Lequesne's acetabular index (AI) with thickness of the medial acetabular wall in the general population. The aim of our study was to clarify the relationship between LCE, AI, and thickness of the medial acetabular wall., Materials and Methods: Measurements on plain radiographs (LCE and AI) and axial CT scans (quadrilateral plate acetabular distance QPAD) of 1,201 individuals (2,402 hips) were obtained using a PACS imaging program and statistical analyses were performed., Results: The mean thickness of the medial acetabulum bone stock (QPAD) was 1.08 mm (95% CI: 1.05-1.10) with a range of 0.1 to 8.8 mm. For pathological values of either the LCE (<20°) or the AI (>12°) the medial acetabular wall showed to be thicker than in radiological normal hips. The overall correlation between coxometric indices and medial acetabular was weak for LCE (r =-0.21. 95% CI [-0.25, -0.17]) and moderate for AI (r = 0.37, [0.33, 0.41])., Conclusions: We did not find a linear relationship between Wiberg's lateral center edge angle, Lequesne's acetabular index and medial acetabular bone stock in radiological normal hips but medial acetabular wall thickness increases with dysplastic indices.

In recent years, new optical systems have been developed with the ability to collect light at very high angles of emission, exceeding the critical angle of total internal reflection. Prominent examples are solid-immersion lenses and paraboloid collectors. These systems achieve high efficiencies in fluorescence detection which is an important issue for sensitive applications in analytical chemistry and biochemical assays. The exclusive collection of supercritical angle fluorescence (SAF) allows for the detection of evanescent modes and thus to confine the detection volume within one wavelength to an interface. For conventional optical systems with high numerical aperture a precise wave-optical theory of imaging was developed by Richards and Wolf in the fifties of the last century. However, their theory is not directly applicable to non-imaging, strongly aberratic light collection systems systems that collect a significant part of light above the critical angle. Here, we extend the theory to describe the optical properties of such systems.

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).

Background: This pilot study was performed to investigate the influence of psychomotor skills on the outcome of a specific nonoperative program (scapular dyskinesis-program) to treat scapular dyskinesis., Materials and Methods: Fifteen patients (group A) with positive and 11 patients (group B) with a negative outcome after physical therapy were included. To test the psychomotor skills of the patients the Motorische Leistungsserie (MLS) was used. The test results of the patients of group A were then compared with those of group B., Results: For 2 parameters, a significant correlation could be detected. However, due to the small sample size, a clear but statistically insignificant difference could be found for several other factors., Conclusion: We, therefore, hypothesize that psychomotor skills testing is a potential method to predict the outcome of nonoperative treatment for scapular dyskinesis. Further investigations with a larger sample size are necessary to confirm this assumption., (Copyright © 2011 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.

Objective: To find a correlation between the cross-over ratio of the cross-over sign on conventional anteroposterior (AP) pelvic radiographs and retroversion measurements ('roof-edge angle' and 'equatorial-edge angle) on computed tomography (CT) scans. This would facilitate the interpretation of the cross-over sign regarding the amount of acetabular retroversion., Materials and Methods: Correctly projected AP pelvic radiographs (2,925 hips) were examined for the presence of the cross-over sign (COS), and the overlap ratio of the COS was measured. On CT scans of the same patients the 'roof-edge angle' (RE angle) and the 'equatorial-edge angle' (EE angle) were also calculated., Results: A statistically significant but only weak relationship could be found between the overlap ratio of the COS and the 'roof-edge angle' (P < 0.0001; correlation coefficient -0.486) and between this ratio and the 'equatorial-edge angle' (P < 0.0001; correlation coefficient -0.395)., Conclusion: A relationship between the overlap ratio and orientation measurements on CT scans could be found, but it was less strong than expected.

Radiological diagnosis of acetabular retroversion is based on the presence of the cross-over sign (COS), the posterior wall sign (PWS), and prominence of the ischial spine (PRISS). The primary purpose of the study was to correlate the quantitative cross-over sign with the presence or absence of the PRISS and PWS signs. The hypothesis was that both, PRISS and PWS are associated with a higher cross-over sign ratio or higher amount of acetabular retroversion. A previous study identified 1417 patients with a positive acetabular cross-over sign. Among these, three radiological parameters were assessed: (1) the amount of acetabular retroversion, quantified as a cross-over sign ratio; (2) the presence of the PRISS sign; (3) the presence of the PWS sign. The relation of these three parameters was analysed using Fisher's exact test, ANOVA, and linear regression analysis. In hips with cross-over sign, the PRISS was present in 61.7%. A direct association between PRISS and the cross-over sign ratio (p < 0.001) was seen. The PWS was positive in 31% of the hips and was also significantly related with the cross-over sign ratio (p < 0.001). In hips with a PRISS, 39.7% had a PWS sign, which was a significant relation (p < 0.001). In patients with positive PWS, 78.8% of the cases also had a PRISS (p < 0.001). Both the PRISS and PWS signs were significantly associated with higher grade cross-over values. Both the PRISS and PWS signs as well as the coexistence of COS, PRISS, and PWS are significantly associated with higher grade of acetabular retroversion. In conjunction with the COS, the PRISS and PWS signs indicate severe acetabular retroversion. Presence and recognition of distinct radiological signs around the hip joint might raise the awareness of possible femoroacetabular impingement (FAI).

The goals of the present investigation were to identify the prevalence of acetabular dome retroversion in a mixed race population, to quantify the average amount of cross-over ratio, and to determine normative values. The presence of the cross-over sign and its overlap ratio was assessed for 2,925 hips meeting strict radiographic criteria of the pelvic radiograph. Fifty-two percent of the hips had no cross-over sign whereas 48% had at least a minimal amount of overlap of the anterior and posterior acetabular wall. Analysis of only those hips with positive cross-over sign revealed a mean cross-over ratio of 26% +/- 11% (range: 3 to 93). Forty-two percent of the patients had no cross-over on either side, 18% on one side, and 40% on both sides. The presence of the cross-over sign is more common than previously expected. Further studies will be necessary to determine the risk of pathological abnormality and to correlate symptoms to crossover ratios. Surgery should not be based solely on the finding of a cross-over sign without clinical correlation.

Surface plasmon-coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer, resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. In addition to high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling diminishes with increasing distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed. We have investigated, both theoretically and experimentally, the SPCE effect for a Cy5-spacer-Ag layer system. Both the angular dependence of emission and the dependence of SPCE emission intensity on Cy5-metal separation were investigated. It is demonstrated that SPCE leads to lower total fluorescence signal than that obtained in the absence of a metal layer. This is the first experimental verification of the reduction in SPCE intensity compared to the metal-free case. Our results are in a good agreement with theoretical models. The validation of the theoretical model provides a basis for optimizing biosensor platform performance, particularly in the context of the advantages offered by SPCE of highly directional emission and surface selectivity.

We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.

This investigation was performed to analyze the influence of innervation and psychomotor skills on the outcome of latissimus dorsi transfer. Patients with the 10 best and 10 worst results after latissimus dorsi transfer for irreparable rotator cuff tears were selected. All patients meeting the inclusion criteria (n = 12) were subject to a psychomotor test battery (Motorische Leistungsserie) and electromyographic innervation assessment. There was no statistical difference between the 2 groups preoperatively in terms of the commonly tested factors known to influence the results of this procedure adversely. There was a significant difference in both the pattern and selectivity of innervation in the group that had better clinical results. The psychomotor findings were negatively correlated with the range of motion and the strength of the operative shoulder. Function of the operative shoulder could also be predicted by psychomotor function of the uninjured contralateral side. Psychomotor skills testing appears to be a new, potential method by which to predict the outcome of latissimus dorsi transfer.

We report a new two-channel fluorescence microscopy technique for surface-generated fluorescence. The realized fluorescence microscope allows high resolution imaging of aqueous samples. The core element of the instrument is a parabolic mirror objective that is used to collect the fluorescence at large surface angles above the critical angle of the waterglass interface. An aspheric lens, incorporated into the solid parabolic element, is used for diffraction-limited laser focusing and for collecting fluorescence at low angles with respect to the optical axis. By separated collection of the fluorescence emitted into supercritical and subcritical angles, two detection volumes strongly differing in their axial resolution are generated at the surface of a glass cover slip. The collection of supercritical angle fluorescence (SAF) results in a strict surface confinement of the detection volume, whereas collecting below the critical angle allows gathering the fluorescence emitted several microns deep inside the sample. Consequently, the signals from surface-bound and unbound diffusing fluorescent molecules can be obtained simultaneously.

To achieve a better understanding of the nonspecific adsorption process of proteins on solid surfaces, the mechanism of this interaction was investigated by a model system comprising the structurally flexible ("soft") protein goat anti-rabbit immunoglobulin G and a set of chemically defined surfaces. The thermodynamic properties of both protein and surfaces were derived from contact angle measurements by applying the Lifshitz-van der Waals acid-base approach, and the Gibbs free enthalpy of interaction was calculated. The protein shows two conformational states, one hydrophobic and the other hydrophilic. The interaction energy indicates that the hydrophobic conformation favorably adsorbs onto the surfaces. With real-time binding kinetics, measured by a supercritical angle fluorescence biosensor, we show that during the nonspecific adsorption the protein performs a reorientation in its three-dimensional amino acid structure from a hydrophilic to a hydrophobic molecular structure. Unlike the rates of adsorption and desorption, the transition rate is independent of the type of surface and only influenced by the structural reorganization of the protein.

New DNA sequencing techniques are currently being developed using single-molecule fluorescence-based detection of enzymatic double-strand synthesis. Such application requires surface architectures on which single-stranded templates can be immobilized. A further important attribute is a very low tendency to attract fluorescently labeled bases nonspecifically. On this account, the adsorption behaviour of Cy5-dNTPs on a variety of surface coatings was studied by performing real-time measurements of the DNA synthesis using a supercritical angle fluorescence biosensor. It is demonstrated that polyacrylic acid coatings are an excellent choice to minimize the nonspecific binding of the bases.

Exact knowledge of the numerical aperture is crucial in many applications using high-aperture objectives such as confocal microscopy, optical trapping, or advanced sub-wavelength imaging methods. We propose and apply a precise and straightforward method for measuring this fundamental parameter of microscope objectives with numerical apertures above unity. Our method exploits the peculiarities of the fluorescence emission of molecules at a glass/air interface.

Surface-plasmon coupled emission (SPCE) has emerged as a new and potentially powerful tool for highly sensitive fluorescence detection. In the case of SPCE, the fluorescence is collected through a semi-transparent thin metal film deposited on glass. We present a theoretical analysis of SPCE, studying the potential enhancement of the fluorescence collection efficiency, brightness, quantum-yield, and photostability. The results are compared with fluorescence detection on a pure glass surface. It is shown that SPCE does not lead to any improvement, but that the metal film actually reduces the sensitivity of fluorescence detection.

A new type of microscope objective is used for the rapid detection of sequence-dependent affinity variations in DNA hybridization. We demonstrate that by performing probe/target hybridization on coverslips at room temperature terminal SNPs (single nucleotide polymorphisms) can be detected within seconds. The study of weak pair interaction, such as the association of very short DNA oligomers, requires the use of high analyte concentrations of both partners to generate a detectable amount of associated pairs. The background of high concentrations of unbound fluorescing analyte can easily hide the low signal of a weakly affine reaction and makes association extremely difficult to detect. Fluorescence detection is a powerful approach to analyze minute amounts of material, even single molecules, but it is usually limited to rather low concentrations. This limitation is now overcome due to the new type of microscope objective, which produces an extremely small detection volume at a water/glass interface.

We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

We explore a new confocal microscope for the detection of surface-generated fluorescence. The instrument is designed for high resolution imaging as well as for the readout of large biochips. Special feature is the separated collection of two different fluorescence emission modes. One optical path covers the emission into the glass at low surface angles, the other captures high angles, exceeding the critical angle of the water/glass interface. Due to the collection of the supercritical angle fluorescence (SAF) the confocal detection volume is strictly confined to the interface, whereas the low angles collect much deeper from the aqueous analyte solution. Hence the system can deliver information about surfacebound and unbound fraction of fluorescent analyte simultaneously.

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-L-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

We present a new optical biosensor for probing molecular binding to a water/glass interface. The system is designed to measure the kinetics of surface reactions down to low analyte concentrations straightforwardly. The selective detection of surface bound fluorescence is achieved by collecting supercritical angle fluorescence (SAF) emission of surface bound molecules into the glass. Thereby the expansion of the detection volume into the aqueous probe is reduced to about one sixth of the fluorescence wavelength, consequently bulk fluorescence from the solution is rejected successfully. The SAF-signal is captured by a parabolic glass lens, which leads to high spatial collection efficiency and detection sensitivity. The sensor has an inverted optical design and is compatible with common glass cover slips, which strongly facilitates operation for the user working in the biological and biochemical fields. The performance of the system is demonstrated by real time measurements of antibody-antigen reactions. Rate constants of the reaction were extracted. Antigen concentrations were detected down to 10(-13) mol/l.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.

We present a theoretical study of a new total-internal-reflection fluorescence microscope for the detection of fluorescence at a water-glass interface. The system is designed for confocal imaging and spectroscopy of nanoparticles and single molecules. Focusing and fluorescence collection through standard glass coverslips is accomplished by a parabolic mirror lens. The large aperture of the element is used to excite fluorescence within the evanescent field of a diffraction-limited focus and to collect focal emission with high efficiency. Tight focusing and supercritical excitation reduce the detection volume for fluorescent analyte molecules well below that of an attoliter (10(-18) L), which can be advantageous for monitoring surface binding of single molecules without interference from fluorescence of the unbound bulk. Calculations of the electric fields in the focus region and simulated confocal imaging demonstrate the performance of the system.

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to more than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to approximately 10(-17) L. This allows for an exceptional discrimination of bulk-generated against surface-generated fluorescence, which may be of great value when surface-binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determining antigen concentrations down to 5 fmol.

We present a theoretical study of a new highly efficient system for optical light collection, designed for ultrasensitive fluorescence detection of surface-bound molecules. The main core of the system is a paraboloid glass segment acting as a mirror for collecting the fluorescence. A special feature of the system is its ability to sample not only fluorescence that is emitted below the angle of total internal reflection (the critical angle) but also particularly the light above the critical angle. As shown, this is especially advantageous for collecting the fluorescence of surface-bound molecules. A comparison is made with conventional high-aperture microscope objectives. Furthermore, it is shown that the system allows not only for highly efficient light collection but also for confocal imaging of the detection region, which is of great importance for rejecting scattered light in potential applications such as the detection of only a few molecules.