Weak Fluorescence Signal Research Articles

Active wide-field illumination for high-throughput fluorescence lifetime imaging

Wide-field fluorescence lifetime imaging allows for fast imaging of large sample areas at the cost of low sensitivity to weak fluorescence signals. To overcome this challenge, we developed an active wide-field illumination (AWFI) strategy to optimize the impinging spatial intensity for acquiring optimal fluorescence signals over the whole sample. We demonstrated the ability of AWFI to accurately estimate lifetimes from a multiwell plate sample with concentrations ranging over two orders of magnitude. We further reported its successful application to a quantitative Förster resonance energy transfer lifetime cell-based assay. Overall, this method allows for enhanced accuracy in lifetime-based imaging at high acquisition speed over samples with large fluorescence intensity distributions.

Thousand-fold fluorescent signal amplification for mHealth diagnostics

The low sensitivity of Mobile Health (mHealth) optical detectors, such as those found on mobile phones, is a limiting factor for many mHealth clinical applications. To improve sensitivity, we have combined two approaches for optical signal amplification: (1) a computational approach based on an image stacking algorithm to decrease the image noise and enhance weak signals, and (2) an optical signal amplifier utilizing a capillary tube array. These approaches were used in a detection system which includes multi-wavelength LEDs capable of exciting many fluorophores in multiple wavelengths, a mobile phone or a webcam as a detector, and capillary tube array configured with 36 capillary tubes for signal enhancement.The capillary array enables a ∼100× increase in signal sensitivity for fluorescein, reducing the limit of detection (LOD) for mobile phones and webcams from 1000nM to 10nM. Computational image stacking enables another ∼10× increase in signal sensitivity, further reducing the LOD for webcam from 10nM to 1nM.To demonstrate the feasibility of the device for the detection of disease-related biomarkers, adenovirus DNA labeled with SYBR green or fluorescein was analyzed by both our capillary array and a commercial plate reader. The LOD for the capillary array was 5ug/mL, and that of the plate reader was 1ug/mL. Similar results were obtained using DNA stained with fluorescein.The combination of the two signal amplification approaches enables a ∼1000× increase in LOD for the webcam platform. This brings it into the range of a conventional plate reader while using a smaller sample volume (10ul) than the plate reader requires (100ul). This suggests that such a device could be suitable for biosensing applications where up to 10 fold smaller sample sizes are needed.The simple optical configuration for mHealth described in this paper employing the combined capillary and image processing signal amplification is capable of measuring weak fluorescent signals without the need of dedicated laboratories. It has the potential to be used to increase sensitivity of other optically based mHealth technologies, and may increase mHealth's clinical utility, especially for telemedicine and for resource-poor settings and global health applications.

Capillary array waveguide amplified fluorescence detector for mHealth

Mobile health (mHealth) analytical technologies are potentially useful for carrying out modern medical diagnostics in resource-poor settings. Effective mHealth devices for underserved populations need to be simple, low cost, and portable. Although cell phone cameras have been used for biodetection, their sensitivity is a limiting factor because currently it is too low to be effective for many mHealth applications, which depend on detection of weak fluorescent signals.To improve the sensitivity of portable phones, a capillary tube array was developed to amplify fluorescence signals using their waveguide properties. An array configured with 36 capillary tubes was demonstrated to have a ∼100× increase in sensitivity, lowering the limit of detection (LOD) of mobile phones from 1000nM to 10nM for fluorescein. To confirm that the amplification was due to waveguide behavior, we coated the external surfaces of the capillaries with silver. The silver coating interfered with the waveguide behavior and diminished the fluorescence signal, thereby proving that the waveguide behavior was the main mechanism for enhancing optical sensitivity.The optical configuration described here is novel in several ways. First, the use of capillaries waveguide properties to improve detection of weak florescence signal is new. Second we describe here a three dimensional illumination system, while conventional angular laser waveguide illumination is spot (or line), which is functionally one-dimensional illumination, can illuminate only a single capillary or a single column (when a line generator is used) of capillaries and thus inherently limits the multiplexing capability of detection. The planar illumination demonstrated in this work enables illumination of a two dimensional capillary array (e.g., x columns and y rows of capillaries). In addition, the waveguide light propagation via the capillary wall provides a third dimension for illumination along the axis of the capillaries. Such an array can potentially be used for sensitive analysis of multiple fluorescent detection assays simultaneously.The simple phone based capillary array approach presented in this paper is capable of amplifying weak fluorescent signals thereby improving the sensitivity of optical detectors based on mobile phones. This may allow sensitive biological assays to be measured with low sensitivity detectors and may make mHealth practical for many diagnostics applications, especially in resource-poor and global health settings.

Time Resolved Fluorescence Studies of Genetically Encoded Protein Sensor Frex for Nadh and pH Levels

Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form, NAD+, are the most important coenzymes found in all living cells. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level. We have recently reported a series of genetically encoded fluorescent sensors highly specific for NADH level1. However, those NADH sensors are also found to be very sensitive to pH value of the local environments, which may result in errors in the analysis of NADH level or NADH/NAD+. We here report that the time resolved fluorescence of NADH sensor Frex can simultaneously characterize the pH and NADH level by using a time correlated single photon counting (TCSPC) technique, whose scale is from 100ps to 30ns. Frex sensors were excited at 420 and 485 nm, respectively. Both the total fluorescence photon counting number N and the fluorescence time distribution have been obtained. All fluorescence transients of Frex sensors reveal complex, i.e. multiexponential behavior. Similar to our previous report1, the N ratio under two excitation wavelengths N485/N420 can be used to characterize the NADH level. Meanwhile, the decay associated spectra (DAS) can be fitted by 3 exponential components decay function. We found that the percentage α of the components with the lifetimes of about 0.2, 1 and 3 ns were proportional to the pH values. Therefore, both the NADH and pH levels can be obtained from time resolved fluorescence spectra. No parallel experiment is needed any more to calibrate the pH effects. Candidate mechanisms and further experiments including femtosecond dynamics will be discussed.Reference:1 Zhao, Y. et al. Genetically Encoded Fluorescent Sensors for Intracellular NADH Detection. Cell Metab14, 555-566, (2011).

A nnovel wide field fluorescence sectioning microscope based on polarization filtering image enhancement and dynamic speckle illumination

We propose a novel wide-field fluorescence sectioning microscope based on polarization filtering image enhancement and dynamic speckle illumination in this article. A new type of polarization filtering image enhancement technology, based on the differences in polarization characteristic between excitation light and fluorescence, is used to filter out the excitation light and enhance image quality. The dynamic speckle illumination is employed to achieve the wide field fluorescence sectioning which has many advantages, such as simple configuration, low cost, rapid response and easy operation. The experimental results indicate that our filtering method can be utilized to improve the image quality significantly, and the dynamic using speckle illumination can be employed to achieve optical sectioning with a wide field and a high vertical resolution capability. Our study is not only to enrich the technological method to extract the weak fluorescence signal from the strong excitation light, but also to provide a significant reference for developing a light wavelength tunable multi-spectral fluorescence microscope with rapid response.

In vivo molecular imaging of epidermal growth factor receptor in patients with colorectal neoplasia using confocal laser endomicroscopy

Epidermal growth factor receptor (EGFR) plays an important role in tumorigenesis of colorectal cancer (CRC), and its in vivo molecular imaging in rodent models has become the subject of an increased number of studies using novel imaging techniques for gastrointestinal endoscopy. Current study aimed to evaluate the use of confocal endomicroscopy (CLE) for in vivo molecular imaging of EGFR in patients with colorectal neoplasia. Molecular imaging of colorectal neoplasia in patients was performed by CLE after topical application of a fluorescent-labeled molecular probe against EGFR. Representative images of CLE were chosen to calculate EGFR-specific fluorescence intensity. Targeted biopsy specimens were taken from each examined site during in vivo imaging for histology and immunohistochemistry (IHC). During in vivo molecular imaging in 37 patients, an EGFR-specific fluorescence signal was present in 18/19 CRC, and 12/18 colorectal adenomas. No or only weak fluorescence signal was observed in vivo in 10 cases of normal mucosa. CLE is a novel tool that could be used in molecular imaging with specific targeting of EGFR in patients with colorectal neoplasia. This technique demonstrates a promising imaging approach for targeted therapies of colorectal neoplasia.

In Vivo Virus-Based Macrofluorogenic Probes Target Azide-Labeled Surface Glycans in MCF-7 Breast Cancer Cells

Chemical addressability of viral particles has played a pivotal role in adapting these biogenic macromolecules for various applications ranging from medicine to inorganic catalysis. Cowpea mosaic virus possesses multiple features that are advantageous for the next generation of virus-based nanotechnology: consistent multimeric assemblies dictated by its genetic code, facile large scale production, and lack of observable toxicity in humans. Herein, the chemistry of the viral particles is extended with the use of Cu-free strain-promoted azide-alkyne cycloaddition reaction, or SPAAC reaction. The elimination of Cu, its cocatalyst and reducing agent, simplifies the reaction scheme to a more straightforward approach, which can be directly applied to living systems. As a proof of concept, the viral particles modified with the azadibenzylcyclooctyne functional groups are utilized to trigger and amplify a weak fluorescent signal (azidocoumarin) in live cell cultures to visualize the non-natural sugars. Future adaptations of this platform may be developed to enhance biosensing applications.

A Validated Spectrofluorimetric Method for the Determination of Citalopram in Bulk and Pharmaceutical Preparations Based on the Measurement of the Silver Nanoparticles-Enhanced Fluorescence of Citalopram/Terbium Complexes

A simple, sensitive, and accurate spectrofluorimetric method was developed for the determination of citalopram in bulk and pharmaceutical preparations. The method is based on the enhancement of the weak fluorescence signal (FL) of the Tb (III)-citalopram system in the presence of silver nanoparticles. Fluorescence intensities were measured at 555 nm after excitation at 281 nm. Prepared silver nanoparticles (AgNPs) were characterized by UV-Visible spectra and transmission electron microscopy (TEM). Various factors affecting the formation of citalopram-Tb (III)-AgNPs complexes were studied and optimized. The fluorescence intensity versus concentration plot was linear over the range 0.02-14 μg mL(-1), with an excellent correlation coefficient of 0.9978. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 7.15 × 10(-6) μg mL(-1) and 2.38 × 10(-5) μg mL(-1) respectively. The proposed method was found to have good reproducibility with a relative standard deviation of 3.66% (n = 6). The interference effects of common excipients found in pharmaceutical preparations were studied. The developed method was validated statistically by performing recoveries studies and successfully applied for the assay of citalopram in bulk powder and pharmaceutical preparations. Percent recoveries were found to range from 98.98% to 100.97% for bulk powder and from 96.57% to 101.77% for pharmaceutical preparations.

A fluorescence-based assay for monitoring clinical drug resistance

Background and aimsMultidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor...

Detection of silver(I) ions based on the controlled self-assembly of a perylene fluorescence probe

In the current work, we report a label-free fluorescence turn-on approach for the sensitive and selective sensing of Ag+. A cationic perylene derivative, compound A, was used as the fluorescence probe. Compound A monomer is strongly fluorescent, and the fluorescence can be efficiently quenched through self-aggregation (self-assembly). A cytosine (C)-rich oligonucleotide, oligo-C, was employed. In the absence of Ag+, oligo-C induced strong compound A aggregation due to electrostatic interactions in aqueous media, and very weak fluorescence signal was detected. However, in the presence of Ag+, the specific interactions between oligo-C and Ag+ induced hairpin structure formation of oligo-C through C–Ag+–C bonding interactions. Oligo-C binding to compound A aggregates was weakened; therefore, compound A monomer could be released and detected. The intensity of the fluorescence signal was directly related to the amount of Ag+ added to the assay solution. Our method is highly sensitive—a limit of detection of 5nM was obtained—and also very selective. Ag+ detection in complex sample mixtures was also demonstrated.

High-sensitivity detection of breast tumorsin vivoby use of a pH-sensitive near-infrared fluorescence probe

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Detection of multi-color fluorescent objects with single photon spectrometer

Single photon counting is the most sensitive and accurate method for detection of very weak fluorescent signals obtained in many applications such as DNA sequencing, detection of biological reporters on micro-beads, detection of droplets in micro-fluidic systems, etc. In this paper we describe the use of single photon spectrometer for detection and characterization of very weak multicolor fluorescence produced by mixtures of various fluorescent dyes and quantum dots.

Spectrofluorimetric quantification of bilirubin using yttrium–norfloxacin complex as a fluorescence probe in serum samples

A simple and sensitive spectrofluorimetric method was developed to determine trace amounts of bilirubin (BR) using yttrium (Y3+)–norfloxacin (NFLX) complex as a fluorescence (FL) probe. NFLX can form a stable binary complex with Y3+ and markedly enhances the weak FL signal of the NFLX. The FL intensity of the Y3+–NFLX complex decreased significantly in the presence of BR in a buffer solution at pH=7.2. Under optimal conditions, the FL intensity decreased according to the BR concentration and showed a good linear relationship in the range of 0.03–2.3μgmL−1 of BR with a correlation coefficient of 0.9988. The limit of detection for the determination of BR was 2.8ngmL−1 with a relative standard deviation (RSD) of 1.55% for five replicate determination of 0.05μgmL-1 BR. The presented method offers higher sensitivity with simple instrumentation and was applied successfully in detecting BR at low concentrations.

A Transformer Partial Discharge Measurement System Based on Fluorescent Fiber

Based on the physical phenomena of optical effects produced by the partial discharge (PD) and on the characteristics of fluorescent fiber sensing of weak fluorescent signals, a PD measurement system using a fluorescent fiber sensor was designed. The main parameters of the sensing system were calculated, an experimental testing platform for PD simulation in the lab was established, and PD signals were then detected through ultra-high frequency (UHF) and optical methods under a needle-plate discharge model. PD optical pulses in transformer oil contained signal-peak and multi-peak pulse waveforms. Compared with UHF detection results, the number of PD pulses and the elapsed PD pulse phase time revealed a good corresponding relationship. However, PD signal amplitudes presented the opposite, thus indicating that PD UHF signals reflected pulse amplitude value, whereas PD optical signals reflected pulse energy magnitude. The n-u-φ three-dimensional distributions indicated that most of the PD signals concentrated in the nearby industrial frequency voltage peak value. Overall, the proposed fluorescent fiber sensing system design can be used successfully in transformer PD signal detection.

Background-free fluorescence detection of cold atoms in a two-color magneto-optical trap

A two-color magneto-optical trap (MOT) for trapping cesium (Cs) atoms is experimentally realized. This two-color MOT employs the radiation forces due to photon scattering from the Cs 6P(3/2) F' = 5 - 8S(1/2) F" = 4 excited-state transition, which replaced one pair of the three pairs of cooling/trapping laser beams operating on a single-photon red detuning to the Cs 6S(1/2) F = 4 - 6P(3/2) F' = 5 cycling transition in a standard six-beam Cs MOT, and can cool and trap atoms on both the red-detuning and blue-detuning sides of the two-photon resonance. Employing the two-color MOT, the background-free fluorescence detection of trapped atoms has been demonstrated. This technique will be useful for observation of weak fluorescence signal radiated from single atoms in MOT.

Fabrication and Experimental Characterization of Nanochannels

Nanofluidics is the science and technology involving a fluid flowing in or around structures with a least one dimension in the nanoscale, which is defined as the range from 1 nm to 100 nm. In this paper, we present the fabrication and characterization of nanochannels in silicon and glass. Since the lateral dimension of the channels is limited by the wavelength of UV light used in photolithography, the channel width can only be fabricated in the micrometer scale. However, the depth of the channel can be controlled precisely by the etching rate of reactive ion etching (RIE). Microchannels and access holes were etched with deep reactive ion etching (DRIE). Both nanochannels and microchannels were sealed by a Pyrex glass wafer using anodic bonding. The fabricated nanochannels were characterized by capillary filling and evaporation experiments. Due to the small channel height and weak fluorescent signal, fluorescent techniques are not suitable for the characterization of the nanochannels. A long exposure time is needed because of the limited amount of fluorescent molecules inhibit the measurement of transient and dynamic processes. However, as the channel height is shorter than all visible wavelengths, the contrast in refractive indices of air and liquid allows clear visualization of nanochannels filled with liquids. Automatic image processing with matlab allows the evaluation of capillary filling in nanochannels. Interesting phenomena and discrepancies with conventional theories were observed.

Analysis of crucial factors resulting in microarray hybridization failure

The factors that affect the formation and stability of DNA/DNA duplexes are complicated and still mostly unknown. In this study attempts were made to look for the crucial factor affecting hybridization failure in DNA microarray assays. A comprehensive range of factors were investigated simultaneously using a 25-mer oligonucleotide Potyvirus microarray. These included steric hindrance, direct/indirect labelling types, distance of a probe to the fluorescent labelling end, target (the DNA fragment used to hybridize with microarray probes) strand types either single strand or double strand, probes without mismatch and with different numbers of mismatch nucleotides (up to 36%) and different mismatch locations (5' end, centre and 3' end), probe GC content and T(m), secondary structures of probes and targets, different target lengths (0.277 kb to ~1.3 kb) and concentrations (0.1-30 nM). The results showed that whilst most of these known factors were unlikely to be the main causes of failed hybridization, there was strong evidence suggesting that the viral amplicon target structure is the most crucial factor. However, computing predicted target secondary structures by Mfold showed no correlation with the hybridization results. One explanation is that the predicted target secondary structures are different from the real structures. Here we postulate that the real target structure might be a combination of secondary structures resulting in a three-dimensional structure from exposure to three types of sub-structures: (1) a completely exposed linear structure to allow probes access for the successful hybridization and showing strong fluorescent signals; (2) a partially exposed structure to allow unstable binding and showing weak fluorescent signals; (3) a closed structure resulting in failed hybridization. These results are very important for microarray based studies as they not only provide an explanation for some current controversial results, but also provide potential resolution for the future studies. Due to the lack of available software for predicting the true target structure, development of microarrays should conduct an initial oligonucleotide probe selection procedure and those probes with capacity to hybridize with the target should be considered for the microarray development.

Ultra-Deep Imaging with Cellular Resolution: Enhanced Two-Photon Fluorescence Microscopy with the Use of a Wide Area Photodetector

We have previously shown that the use of a wide photocathode area PMT as a detector in a two-photon fluorescence microscope allowed us to image in turbid samples up to the depth of about 2.5 mm with cellular resolution. This detection scheme enables a very efficient collection of fluorescence photons directly from the wide (1” diameter) area of the sample, which considerably increases the detection system sensitivity in comparison to a traditional two-photon microscope, where fluorescence is collected by the same objective lens used for excitation. Because the imaging depth depends on the ability of the system to sense weak fluorescent signals, this new detection method significantly enhances the imaging depth. We have recently built a new experimental system that works in the upright configuration, which is best suited for experiments on live animals. The system employs a high power Ti:Sa Mai Tai laser with a group velocity dispersion compensator (DeepSee) for two-photon fluorescence excitation that allows us to extend the imaging depth to 3mm in samples simulating brain tissue optical properties. Imaging experiments in vivo and in vitro have also been conducted on live animals (mice) and tissues (skin, colon, small intestine).With the aid of the new high speed response PMT that we are currently incorporating in the system, we will be able to perform fluorescence lifetime imaging microscopy (FLIM) on whole animals and tissue samples at a few mm depth. This double feature will particularly aid in vivo neuron imaging.This work was supported by National Institutes of Health grants: P41-RRO3155, P50-GM076516

Instrument for fluorescence sensing of circulating cells with diffuse light in mice in vivo

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches.

<I>Drosophila</I> spen protein: generation of ployclonal antibodies, functional analysis and tissue-specific expression

The spen family of proteins participates in various biological processes. It is involved in neuronal cell fate, survival and axon guidance, and cell cycle regulation. Recent studies showed the Drosophila spen gene was required for Wnt-dependent signaling in the eye, wing and leg. However, the genetic role and biological function in Drosophila remain largely unclear. A Drosophila C-terminal fragment of spen was cloned and expresed in E. coli. The purified 6×His-spen protein was injected into SD rat to generate polyclonal antibodies. Subcellular localization and tissue-specific expression of spen protein were analysed by immunostaining and histoimmunochemistry. The results indicated that spen protein was localized in the nucleus and expressed at high levels in brain, fat body, hemocyte, gut and salivary gland. To assay the function of mutant hemocytes in vivo, wild-type and spen mutant larvae were infected with fluorescent microspheres. Wild-type hemocytes showed a strong fluorescence signal from the phagocytosed microspheres; however, spen mutant had a weak fluorescence signal, indicating that the mutant hemocytes were defective in the uptake of the microspheres.