Improved and high throughput quantitative measurements of weak GFP expression in transgenic plant materials

Green fluorescent proteins (GFPs) are widely used in tracing transgene expression and have been known as convenient and efficient markers for plant transformation. However, sometimes researchers are still puzzled by the weak fluorescence since it makes the observation of GFP signals and confirmation of transgenic plants difficult. In this investigation, we explored the possibility of enhancing the weak signals by changing the pH environment of detection and took microplate reader as a more effective instrument compared to traditional fluorescent microscope to detect the weak signals. It was found that the fluorescence intensity of enhanced GFP (EGFP) in transgenic plants can be increased 2–6 folds by altering the environmental pH, and the concentration of EGFP at a large scale (ranged from 20 ng/ml to 20 μg/ml) can be detected and quantified. It can exclude the influence of degradation fragment and hence facilitate later analysis; these advantages were further verified by comparing with western blotting and confocal microscopy. It was reliable and effective for the qualitative and quantitative analysis of transgenic plants and was more suitable for the detection of very weak fluorescent signals.     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

Single actomyosin motor interactions in skeletal muscle

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10⁵. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~ 9 s during muscle contraction.     

On the rotational brownian motion of a bacterial idle motor. II. Theory of fluorescence correlation spectroscopy

The photon flux autocorrelation function of a fluorescent label attached to a bacterial motor shaft is calculated for the case in which the bacterial motor is considered to be actively but idly rotating. It is shown that even when the fluorescent label has a very short lifetime, fluorescence correlation spectroscopy should provide a useful tool for determining the rate of revolution of the bacterial motor under various solution conditions.     

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.     

Intrinsic fluorescence from DNA can be enhanced by metallic particles

High sensitivity detection of DNA is essential for genomics. The intrinsic fluorescence from DNA is very weak and almost all methods for detecting DNA rely on the use of extrinsic fluorescent probes. We show that the intrinsic emission from DNA can be enhanced many-fold by spatial proximity to silver island films. Silver islands are subwavelength size patches of metallic silver on an inert substrate. Time-resolved measurements show a decreased lifetime for the intrinsic DNA emission near the silver islands. These results of increased intensity and decreased lifetime indicate a metal-induced increase in the radiative rate decay of the DNA bases. The possibility of increased radiative decay rates for DNA bases and other fluorophores suggest a wide variety of DNA measurements and other biomedical assays based on metal-induced increases in the fluorescence quantum yield of weakly fluorescent substances.     

Enhancement of imaging depth in turbid media using a wide area detector

The depth of two‐photon fluorescence imaging in turbid media can be significantly enhanced by the use of the here described fluorescence detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector we were able to perform imaging of turbid samples, simulating brain tissue, at depths up to 3 mm, where the two‐photon induced fluorescence signal is too weak to be detected by means used in conventional two‐photon microscopy. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Fluorescence intensity resolution in flow systems.

The factor which can limit fluorescence intensity resolution in a flow cytometer of the type in which cells pass perpendicularly through a focussed laser beam depends on signal intensity. For the brightest sources (e.g. fluorescent DNA stains), the coefficient of variation (CV) is limited in our system to around 3% by stream hydrodynamics, unstable illumination intensity, nonstoichiometric staining, etc. The weakest detectable sources (e.g. fluorescent cell-surface labels) are limited in coefficient of variation by shot noise in the photomultiplier due to constant background light levels. Finally, over a fairly wide brightness range between these extremes, resolution is determined primarily by photoelectron statistical variation on the signal itself (i.e. "photon statistics"). Thus photon collection and detection efficiency (solid angle, barrier filter passband, detector quantum efficiency) become of primary importance.     

Two‐photon excited fluorescence lifetime measurements through a double‐clad photonic crystal fiber for tissue micro‐endoscopy

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two–photon excitation and time‐correlated single photon counting detection through a double‐clad photonic crystal fiber (DC‐PCF) without pre‐chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one‐photon‐based techniques, may be combined with morphology‐yielding techniques such as photoacoustic and ultrasound imaging. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Development of high-sensitivity near-infrared fluorescence imaging device for early cancer detection

We have developed a high-sensitivity near-infrared (NIR) optical imaging system for noninvasive cancer detection based on the molecular-labeled fluorescent contrast agents. Recent developments in molecular beacons offer a way to selectively tag various precancer and cancer signatures and provide high tumor-to-background contrast. Near-infrared imaging can deeply probe tissue up to a couple of centimeters; thus, it possesses the potential for noninvasive detection of breast or lymph node cancer. A phase cancellation (in- and antiphase) device is used to increase the sensitivity in detecting fluorescent photons and the accuracy of tumor localization. The optoelectronic system consists of the laser diode sources, fiber optics, interference filter (to select the fluorescent photons), and the high-sensitivity photon detector (photomultiplier tube). The source-detector pair scans the tissue surface in multiple directions, and the localization image can be obtained by angular back-projection reconstruction. Simulations and experimental data demonstrated the feasibility of detection and localization offluorescent object embedded inside the highly scattering media. Tumor-bearing mouse model with injection of fluorescent contrast agents is used to simulate the human breast tumor labeled with molecular beacons. The system can detect fluorescent contrast agents as small as one nanomole at the depth of three centimeters, with a three-millimeter localization error. This instrument has the potential for tumor diagnosis and imaging, and the accuracy of the localization suggests that this system could help guide the clinical fine-needle biopsy. Also, this portable device would be complementary to x-ray mammography and provide add-on information on early diagnosis and localization of breast tumor.     

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.     

Identification of various lipolytic enzymes in crude porcine pancreatic lipase preparations using covalent fluorescent inhibitors

We developed a specific method for determination and discrimination of lipo-/estero-lytic enzymes in crude lipase preparations. Here we study the composition of commercial porcine pancreatic lipase (PPL), since it is widely used for bioconversions of synthetic and natural substrates. Our method is based on incubation of enzyme samples with fluorescently labeled alkyl- or dialkylglyceryl-phosphonates in an appropriate solvent followed by protein separation by electrophoresis and fluorescence detection with a CCD camera. After incubation with short-chain alkylphosphonate solubilized by taurodeoxycholate, crude PPL preparations showed a very weak band at 50 kDa, which is indicative of low PPL concentrations in these samples. In addition, seven other fluorescent bands were detected. The band at the lowest molecular weight corresponded to alpha-chymotrypsin. Two intensive fluorescent bands were in the molecular weight range of chymotrypsinogen (26 kDa) and four weak bands were in the range 20-24 kDa. Long-chain dialkylglycerophosphonate labeled two protein bands in crude PPL: alpha-chymotrypsin and a very intensive band corresponding to the molecular weight of chymotrypsinogen. Detection of cholesterol esterase (98 kDa) in crude PPL preparations depended on addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to the incubation mix, as demonstrated by spiking with cholesterol esterase. Thus, commercial crude PPL preparations contain a variety of estero-/lipo-lytic enzymes in addition to rather low amounts of active PPL, which should be considered when using crude PPL for bioconversions. Our method can also be used to show whether an isolated esterolytic activity corresponds to a single protein or isoenzymes. Here we confirm by 2D-electrophoretic separation of "pure" PPL that PPL exists as isoenzymes in different glycosylated forms.     

Fluorescence lifetime-based sensing in tissues: a computational study.

We have numerically solved the photon diffusion equation to predict the distribution of light in a tissue model system with a uniform concentration of fluorophore. Our results show that time-dependent measurements of light propagation can be used to monitor the fluorescent lifetimes of a uniformly distributed fluorophore in tissues. With proper referencing, frequency-domain measurements of phase-shift, theta, may allow quantitation of fluorescent lifetimes, tau, independent of changes in the local absorption and scattering properties. These results point to a new approach for noninvasive diagnostic monitoring through quantitation of fluorescent lifetime, tau, when the lifetime of the fluorophore is comparable with photon migration times.     

Molecular biology in living cells by means of digital optical microscopy

The intention of this review is to introduce to microscopists some of the fundamental limits as well as the advances made in digital optical microscopy techniques, also to show their potentialities in the field of molecular cell biology. Several items within the wide field of this subject will be discussed, such as geometric and photometric features, photophysics, statistics of photon flux and detection, spatial and intensity characteristics of digital images, their manipulation and display, fluorescent probes, and measuring techniques.     

DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules

As promising substitutes for organic dyes and quantum dots, few-atom fluorescent silver nanoclusters (Ag NCs) have recently gained much attention in a wide range from cellular imaging to chemical/biological detection applications owing to their ultrasmall size (<2 nm), excellent photostability, good biocompatibility and water solubility. Herein, we design an aptamer, guanine-rich (G-rich) DNA and Ag NCs nanocomplex to investigate its ability for the detection of small molecules. The design contains two DNA strands which are both chimeric conjugates of the DNA aptamer sequence fragment and G-rich sequence fragment. Using cocaine as a model molecule, the two DNA strands are in free state if there is no cocaine present, and the formed Ag NCs through the reduction of Ag(+) by NaBH(4) show weak fluorescence emission. In the presence of cocaine, however, the two aptamer fragments bind cocaine, which in turn puts the two G-rich sequence fragments in proximity and the fluorescent intensity of DNA-Ag NCs enhances greatly. As a result, DNA-Ag NCs are demonstrated as a novel, cost-effective and turn-on fluorescent probe for the analysis of cocaine, with a detection limit of 0.1 μM. Besides, successful detection of adenosine triphosphate (ATP) with detection limit of 0.2 μM demonstrates its potential to be a general method.     

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.     

The photon counting histogram in fluorescence fluctuation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given by Mandel's formula. Based on this equation and considering the fluorescence intensity distribution in the two-photon excitation volume, a theoretical expression for the PCH as a function of the number of molecules in the excitation volume is derived. For a single molecular species two parameters are sufficient to characterize the histogram completely, namely the average number of molecules within the observation volume and the detected photon counts per molecule per sampling time epsilon. The PCH for multiple molecular species, on the other hand, is generated by successively convoluting the photon counting distribution of each species with the others. The influence of the excitation profile upon the photon counting statistics for two relevant point spread functions (PSFs), the three-dimensional Gaussian PSF conventionally employed in confocal detection and the square of the Gaussian-Lorentzian PSF for two photon excitation, is explicitly treated. Measured photon counting distributions obtained with a two-photon excitation source agree, within experimental error with the theoretical PCHs calculated for the square of a Gaussian-Lorentzian beam profile. We demonstrate and discuss the influence of the average number of particles within the observation volume and the detected photon counts per molecule per sampling interval upon the super-Poissonian character of the photon counting distribution.     

Synthesis of pheophorbide-a conjugates with anticancer drugs as potential cancer diagnostic and therapeutic agents

Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells, was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diagnosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also, pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indicated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer therapy as well as for the fluorescence detection of cancer.     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Direct incorporation and extension of a fluorescent nucleotide through rolling circle DNA amplification for the detection of microRNA 24-3P

We designed and synthesized several fluorescent nucleotides from thiophene, anthracene and pyrene, which have different sizes, and screened their incorporation and extension capability during the rolling circle amplification of DNA. The thiophene-based fluorescent nucleotide (dUthioTP) could highly incorporate and extended into the rolling circle DNA product, while other fluorescent nucleotides (dUanthTP, and dUpyrTP) could not. This dUthioTP fluorescent nucleotide could be used for the detection of miRNA 24-3P, which is related PRRSV. This direct labeling system during rolling circle DNA amplification exhibited an increased fluorescence signal showing gel formation for the detection of miRNA 24-3P. This direct labeling system is a very simple and cost-efficient method for the detection miRNA 24-3P and also exhibited highly sensitive and selective detection properties.