Fluorescence Light Collection Research Articles

Additive GaN Solid Immersion Lenses for Enhanced Photon Extraction Efficiency from Diamond Color Centers.

Effective light extraction from optically active solid-state spin centers inside high-index semiconductor host crystals is an important factor in integrating these pseudo-atomic centers in wider quantum systems. Here, we report increased fluorescent light collection efficiency from laser-written nitrogen-vacancy (NV) centers in bulk diamond facilitated by micro-transfer printed GaN solid immersion lenses. Both laser-writing of NV centers and transfer printing of micro-lens structures are compatible with high spatial resolution, enabling deterministic fabrication routes toward future scalable systems development. The micro-lenses are integrated in a noninvasive manner, as they are added on top of the unstructured diamond surface and bonded by van der Waals forces. For emitters at 5 μm depth, we find approximately 2× improvement of fluorescent light collection using an air objective with a numerical aperture of NA = 0.95 in good agreement with simulations. Similarly, the solid immersion lenses strongly enhance light collection when using an objective with NA = 0.5, significantly improving the signal-to-noise ratio of the NV center emission while maintaining the NV's quantum properties after integration.

A Counter Propagating Lens-Mirror System for Ultrahigh Throughput Single Droplet Detection.

Fluorescence-based detection schemes provide for multiparameter analysis in a broad range of applications in the chemical and biological sciences. Toward the realization of fully portable analysis systems, microfluidic devices integrating diverse functional components have been implemented in a range of out-of-lab environments. That said, there still exits an unmet and recognized need for miniaturized, low-cost, and sensitive optical detection systems, which provide not only for efficient molecular excitation, but also enhanced photon collection capabilities. To this end, an optofluidic platform that is adept at enhancing fluorescence light collection from microfluidic channels is presented. The central component of the detection module is a monolithic parabolic mirror located directly above the microfluidic channel, which acts to enhance the number of emitted photons reflected toward the detector. In addition, two-photon polymerization is used to print a microscale-lens below the microfluidic flow channel and directly opposite the mirror, to enhance the delivery of excitation radiation into the channel. Using such an approach, it is demonstrated that fluorescence signals can be enhanced by over two orders of magnitude, with component parallelization enabling the detection of pL-volume droplets at rates up to 40 000 droplets per second.

Design and field application of a UV-LED based optical fiber biofilm sensor

Detecting changes in the formation dynamics of biofilms stemming from bacteria and unicellular microorganisms in their natural environment is of prime interest for biological, ecological as well as anti-fouling technology research. We developed a robust optical fiber-based biofilm sensor ready to be applied in natural aquatic environments for on-line, in situ and non-destructive monitoring of large-area biofilms. The device is based on the detection of the natural fluorescence of microorganisms constituting the biofilm. Basically, the intrinsic fluorescence of the amino acid tryptophan is excited at a wavelength of λ=280nm and detected at λ=350nm utilising a numerically optimized sensor head equipped with a UV-LED light source and optical fiber bundles for efficient fluorescence light collection. Calibration was carried out with tryptophan solutions and two characteristic marine bacteria strains revealing linear signal response, satisfactory background suppression, wide dynamic range, and an experimental detection limit of 4×103cells/cm2. Successful field experiments in the Baltic Sea accomplished over a period of twenty-one days provided for the first time continuous observation of biofilm formation dynamics in a natural habitat. Starting from the first adhering bacteria, the measurement yielded the characteristic three phases of biofilm formation up to a fully developed biofilm. The sensor system holds potential for applications in aquatic sciences including deep sea research and, after further miniaturisation, in the industrial and biomedical field.

Long range surface plasmon-coupled fluorescence emission for biosensor applications

A biosensor scheme that employs long range surface plasmons (LRSPs) for the efficient excitation and collection of fluorescence light from fluorophore-labeled biomolecules captured in a three-dimensional hydrogel matrix is discussed. This new approach to plasmon-enhanced fluorescence (PEF) is experimentally and theoretically investigated by using the Kretschmann configuration of attenuated total reflection (ATR) method. A layer structure supporting LRSPs that consists of a low refractive index fluoropolymer layer, a thin gold film and a large binding capacity N-isopropylacrylamide (NIPAAm)-based hydrogel matrix swollen in an aqueous sample is employed. By using this layer architecture, the extended field of LRSPs probes the binding of biomolecules in the binding matrix at up to micrometer distances from the gold surface. With respect to regular surface plasmon-enhanced fluorescence spectroscopy (SPFS) and surface plasmon-coupled emission (SPCE), a narrower angular distribution of the fluorescence light intensity, a larger peak intensity and the excitation and emission at lower angles were observed.

Aberration-free lithography setup for fabrication of holographic diffractive optical elements

A holographic optical lithography setup with extremely small longitudinal spherical aberration is described. The setup is used for the fabrication of holographic diffractive optical elements intended to collect light emitted from a fluorescence spot located on a biochip surface. A key feature of the optical setup is its ability to simulate a point-source-like fluorescence spot. A detailed description of the setup together with its optical properties are provided. The fluorescence light collection efficiency of the holographic diffractive optical elements produced using this setup is demonstrated.

Microlens array fabrication by enhanced thermal reflow process: Towards efficient collection of fluorescence light from microarrays

This paper describes two enhanced resist reflow methods for the fabrication of microlens arrays and demonstrates their use for integrated biomolecular fluorescence detection on printed microarrays. A PDMS (polydimethylsiloxane) microlens array was fabricated by a double soft lithography approach using a photoresist microlens array as master mold. Additionally, by using both a careful control of the surface wettability and thermal treatments, we demonstrate the possibility to extend the resist reflow process in order to tune the diameters of microlens array over a large range by using a unique photomask pattern. We introduce an enhanced reflow on hydrophobic surfaces obtained by fluorosilane treatment and identify a threshold shrinkage temperature (T shrinkage) of 140 °C, above which the diameter of microlenses can be then reduced down to 40% compared with the initial pattern on the photomask. Furthermore, on hydrophilic substrates, achieved by an accurate incomplete development of the photoresist, we demonstrate a nearly perfect linear dependency (1.4 μm/°C) of microlens diameter spreading up to 70% the initial diameter inside a temperature reflow window of 110–140 °C. For both approaches, above a freezing temperature (T freezing) of 170 °C, the microlens profile characteristics are temperature independent. By using high numerical aperture microlens array, we provide a proof of concept for the integration and enhanced light collection of the fluorescent signals collected form a microarray of fluorescent spots thus showing the potential of the concept for biophotonic integration.

Addressing Factors Affecting Fluorescent Signal Collection of a Multimode Photonic Crystal Fiber Fluorometer

A multimode photonic crystal fiber (PCF) fluorometer interrogated by a spectrometer is investigated. By adding a glass tip at the end of the PCF, we found that this fluorometer significantly enhances the fluorescent light collection efficiency (CE). Factors associated with the CE and stray light rejection are addressed. The experimental study verifies the proposed PCF probe based on the consideration of solely meridian rays. However, our further study in this paper shows the significance of skew rays under total internal reflection (TIR), which in fact carry 53% of the total guidable fluorescent signal power and are blocked in our experiment. We highlight that this missing power is strongly associated with the numerical aperture (NA) of each optical component in the system and suggest further improvement on the components in the system. The proposed PCF fluorometer is particularly efficient and cost effective for an immersion medium with a high attenuation.

Modeling of photonic crystal fiber with air holes sealed at the fiber end and its application to fluorescent light collection efficiency enhancement

A model of large core and multimode photonic crystal fiber (PCF) with sealed air holes at its end face is proposed for the first time. The model indicates that this PCF can be replaced by another equivalent fiber with complete holes at a new end-face position. Instead of a segment of glass rod between the old and new end faces, light rays travel in a virtual medium with the loss rating of pure glass but the refractive index of the immersion medium. For a two-fiber structure, this segment of pure glass has the capability of enhancing the light collection efficiency, which is investigated using a specifically designed fiber probe and fluorescent samples with different concentrations.

Influence of interface-dipole interactions on the efficiency of fluorescence light collection near surfaces.

Recently Török and colleagues published in a series of papers a general wave-optical approach to calculating the light-collection efficiency function (CEF) for confocal imaging of dipole emitters [J. Mod. Opt. 45, 1681 (1998); J. Microec. 194, 127 (1999); Opt. Lett. 25, 1463 (2000)]. In their theory they did not address the possibility that the lifetimes of fluorescing molecules can change significantly near interfaces, which has a direct effect on the CEF. The research of Török and colleagues is extended here to include this effect, which may become important for imaging near surfaces.

Rare Gas Quenching of Metastable O2 at 295 K

The rare gas quenching rate constants of the metastable species, O2 was measured using an induced fluorescence technique that utilizes an incoherent broadband light source and an integrating sphere to maximize excitation efficiency and fluorescence light collection. The measured rate constants at 295 K were the following (in units of 10-20 cm3 molecule-1 s-1): He, 430 ± 13; Ne, 26 ± 5; Ar, 9 ± 5; Kr, 32 ± 3; Xe, 67 ± 5. These values are of much lower magnitude than previous results indicate. However, except for that of helium, these rate constants should be regarded as upper limits, given that water vapor and hydrogen (both highly efficient quenchers) are ubiquitous contaminants of gas samples. The quenching rate constant with ground-state oxygen derived from these experiments, (4.25 ± 0.52) × 10-17 cm3 molecule s-1, is in excellent agreement with previous results.

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds

Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.

Laser-Based Fluorescence Eem Instrument forIn-SituGroundwater Monitoring

I. ABSTRACT A fluorescence excitation-emission matrix (EEM) instrument is described which employs laser excitation and optical fiber light delivery for remote sensing applications. Ultraviolet laser light is generated at a variety of discrete wavelengths by pumping a Raman shifter with the third or fourth harmonic of a Nd:YAG laser, and this Raman-shifted light is used for fluorescence excitation to generate EEM spectra. Seven-meter optical fibers are used for laser beam delivery and fluorescence light collection, and a diode array detector is used to detect fluorescence emission spectra. As a test of the capability of this instrument, the composition of a two-component mixture of the groundwater pollutants carbazole and p-cresol is determined quantitatively by least-squares analysis of the mixture's fluorescence EEM. Detection limits were in the ppb range and the linear dynamic range was better than 3 orders of magnitude.

Flow cytometry of mammalian sperm: progress in DNA and morphology measurement.

Variability in DNA content and head shape of mammalian sperm are potentially useful markers for flow cytometric monitoring of genetic damage in spermatogenic cells. The high refractive index and extreme flatness of the sperm heads produce an optical effect which interferes with DNA measurements in flow cytometers which have dye excitation and fluorescence light collection normal to the axis of flow. Orientation of sperm in flow controls this effect and results in coefficients of variation of 2.5% and 4.2%, respectively, for DNA measurements of mouse and human sperm. Alternatively, the optical effect can be used to generate shape-related information. Measurements on randomly oriented sperm from three mammalian species using a pair of fluorescence detectors indicate that large shape differences are detectable. Acriflavine-Feulgen stained sperm nuclei are significantly bleached during flow cytometric measurements at power levels routinely used in many flow cytometers. Dual beam studies of this phenomenon indicate it may be useful in detecting abnormally shaped sperm.