Fluorescence Intensity Calibration Research Articles

Fabrication of calibration plate for total internal reflection fluorescence microscopy using roll-type liquid transfer imprint lithography

A strong demand exists for the visualization of the fluid flow in microchannels, such as those in labs-on-a-chip. Multilayer nanoparticle image velocimetry (MnPIV) is a promising technique for this purpose, wherein the x- and y-positions of a nanoparticle are measured using a camera and its z-position is estimated via its fluorescence intensity. To accurately estimate the z-position, however, MnPIV requires a fluorescence intensity calibration process. Water is typically used in the microfluidic devices and a nanoscale level with a refractive index matching method is conventionally used for the calibration of MnPIV. Therefore, a calibration plate with nanoscale levels whose refractive index is matched to water is necessary to calibrate the z-position of fluorescent particles in water. In this study, a fabrication technique of the calibration plate was performed using ultraviolet (UV) nanoimprint with a UV-curable resin, whose refractive index is matched to water and which possesses a high viscosity of 2900cps. Although the residual layer thickness (RLT) of the level must be less than 100nm to perform MnPIV, it is challenging to reduce the RLT with such a high viscosity of the resin using conventional UV nanoimprint lithography. To reduce the RLT, therefore, in this study roll-type liquid-transfer imprint lithography (LTIL) was used with the high-viscosity UV-curable resin, where a two-fold repetition of the roll-type LTIL process demonstrated a calibration pattern RLT of 40nm or less. As a result, calibration of the MnPIV system was achieved with the calibration plate developed herein and fabricated by UV nanoimprint lithography.

Quantitative Flow Cytometry Measurements in Antibodies Bound per Cell Based on a CD4 Reference.

Multicolor flow cytometer assays with fluorescently labeled antibodies are routinely used in clinical laboratories to measure the cell number of specific immunophenotypes and to estimate expression levels of specific receptors/antigens either on the cell surface or intracellularly. The cell number and specific receptors/antigens serve as biomarkers for pathological conditions at various stages of a disease. Existing methods and cell reference materials for quantitative expression measurements have not yet produced results that are of wide clinical interest or are instrument-independent across all fluorescence channels. This unit details a procedure for quantifying surface and intracellular biomarkers by calibrating the output of a multicolor flow cytometer in units of antibody bound per cell (ABC). The procedure includes (1) quality control of the flow cytometer, (2) fluorescence intensity calibration using hard dyed microspheres assigned with fluorescence intensity values, (3) compensation for fluorescence spillover between adjacent fluorescence channels, and (4) application of a biological reference calibrator to establish an ABC scale. The unit also points out current efforts for quantifying biomarkers in a manner that is independent of instrument platforms and reagent differences.

Noninvasive Quantification of Green Fluorescent Protein Labeled Escherichia coli in a Dynamic Capillary Fringe by Fluorescence Intensity

Quantification of bacteria attached to sand grains is usually not possible without destroying biofilms or the sand structure. In situ quantification of autofluorescing or of fluorescence‐carrying bacteria might be an alternative if fluorescence intensity (FI) was strong enough and could be related to cell numbers. For calibration, a Hele–Shaw cell was filled with sterile quartz sand and sectioned for inoculation with increasing cell densities of Escherichia coli strain HB101 K12 pGLO, overexpressing green fluorescent protein (GFP). Digital photographs of the single sections were taken under ultraviolet light at wavelength λ = 365 nm for fluorescence quantification by TotalLab Quant software. The FI was related to respective cell densities per quartz sand volume. Growth of GFP‐labeled E. coli, suspended in Lysogeny broth (LB) medium (dissolved organic C = 6.8 g L−1), could thus be quantified noninvasively in capillary fringe (CF) laboratory systems simulating static or hydrodynamic conditions. Best growth conditions existed in the transition zone of the CF, where after 3 d the highest cell densities of 5.0 to 6.5 × 108 cells cm−3 were determined. The E. coli cells that grew attached as a biofilm in the different sections of the CF could also be quantified under flow‐through conditions and after fluctuation of the water table. For reliable results, FI calibration must be performed in the same setup as used for experiments, and fluorescence characteristics of the bacterial strain should be well known. This noninvasive method using bright fluorescing bacteria can be advantageous for the determination of bacterial growth and biofilm formation in laboratory experiments with unsaturated soil under changing hydraulic conditions.

Rapid analysis of barley straw before and after dilute sulfuric acid pretreatment by photoluminescence

The fluorescence intensities (FIs) of raw and pretreated barley straws were measured by fluorescence microscopy, and the difference in the fluorescence intensity of barley straw before and after dilute acid pretreatment was analyzed by investigation of the major compounds of barley straw. The difference in fluorescence intensity was due to the difference in xylan content. Barley straw was pretreated using dilute sulfuric acid at various conditions and the correlation between the fluorescence intensity and glucose yield of barley straw was investigated. The coefficient of determination (R(2)) of the correlation was found to be 72.28%. Also the calibration of fluorescence intensity with the xylan content was performed. In addition, the absorption and emission spectra of the raw and the pretreated barley straw were examined to verify the proposed method. The absorption and emission wave lengths were 550 nm and 665 nm, respectively.

Hydrodynamic optical alignment for microflow cytometry.

A microfabricated flow cytometer has been developed that is capable of detecting nearly all of the microparticles in an aqueous suspension. Current design allows for integrated coupling between an optical fiber-based detection system and the particle stream via hydrodynamic focusing. By adjusting the relative flow-rates at the auxiliary inputs of the focusing manifold, the particle stream can be steered out-of-plane relative to the illuminating laser, and similarly the particle stream can be squeezed or expanded. The microfabricated device was constructed in polydimethylsiloxane with cross-sectional microfluidic dimensions of 125 µm×125 µm. Using the present device and method, fluorescent microparticles in aqueous solution were counted at an absolute counting efficiency of 91±4%. The coefficient of variation of the fluorescence pulse-heights for far-red fluorescent microparticles was 15%. The device exhibited a linear response to fluorescence intensity calibration microparticles as shown by comparison with a commercial cytometer instrument.

Fluorescence Intensity Calibration Using the Raman Scatter Peak of Water

Fluorescence data of replicate samples obtained from different fluorescence spectrometers or by the same spectrometer but with different instrument settings can have great intensity differences. In order to compare such data an intensity calibration must be applied. Here we explain a simple calibration method for fluorescence intensity using only the integrated area of a water Raman peak. By applying this method to data from three different instruments, we show that it is possible to remove instrument-dependent intensity factors, and we present results on a unified scale of Raman units. The method presented is a rapid and simple approach suitable for routine measurements with no need for hazardous chemicals.

Dark proteins: Effect of inclusion body formation on quantification of protein expression

Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of "dark" proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells.

Toward instrument-independent quantitative measurement of fluorescence intensity in fiber-optic spectrometer systems

Fluorescence has been widely used in biological research and clinical diagnosis. One challenge facing the rapid growth of fluorescence application is the inability to make comparable fluorescence intensity measurements during a long period of clinical study and across laboratories. We propose a method to implement system-independent fluorescence intensity calibration in fiber-optic fluorescence spectrometer systems. This method is based on a National Institute for Standards and Technology traceable standard light source for system spectral response calibration, and a fluorescence reference standard for fluorescence intensity calibration. Human skin in vivo and a fluorescence phantom made of fluorescent microspheres were measured with two different system configurations and at different probe-sample distances. Experimental results showed very good agreement with theory after system-independent fluorescence intensity calibration.

Ex Vivo Expanded Dendritic Cells Home to T-Cell Zones of Lymphoid Organs and Survive in Vivo after Allogeneic Bone Marrow Transplantation

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

Fluorescence Intensity Calibration for Immunophenotyping by Flow Cytometry

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.

Visualization of oxygen concentration in water bodies using a fluorescence technique

Spatial variations of oxygen concentration were measured instantaneously in water body using the fluorescence intensity image technology. The movement of a fluorescent solution was visualized according to the oxygen concentration difference. Pyrenebutyric acid (PBA) was used as a fluorescence and a charge coupled-device (CCD) camera as an image detector. The calibration of fluorescence intensity as a function of oxygen concentration and temperature was utilized for a direct measurement of oxygen concentration at every pixel in an image of light intensity. Light intensity decreased about 40% while the amount of oxygen in water increased from zero to the air-saturated concentration (8.5 mg/l at 21°C), suggesting that the technique is sufficiently sensitive to determine the variation of oxygen concentration. Consecutive images of the oxygen content of a submerged jet solution were provided to quantify the spatial variation of oxygen in a water body. Analysis of these images also described the movement of solution, suggesting that the visualization technique could be applied more widely for research in experimental fluid flow and chemical transport in water bodies.

Model system evaluating fluorescein-labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers.

Fluorescence intensity calibration was evaluated in a model system for flow cytometers using commercially available fluorescein-labeled microbeads as internal standards and stabilized fluoresceinated thymus cell nuclei (Fluorotrol) as surrogates for stained mononuclear cells. Spectrophotometrically determined calibration values for the microbeads were used to generate a standard curve that converted green fluorescence histogram channels into molecular equivalents of soluble fluorescein (MESF). In 19 analyses repeated during a single run, the coefficients of variation (CVs) for the derived MESF values on both dimly and brightly stained Fluorotrol populations were less than 2%. In 26 separate determinations over 14 weeks, the CVs of the derived MESF values were less than 3%. The MESF values of the dim and bright Fluorotrol populations derived from the microbead standard curves were both about 50% lower than those determined by direct spectrophotometric analysis of Fluorotrol. The analytical imprecision of fluorescence intensity measurements in this idealized model system has a CV less than 3%, and the analytical inaccuracy shows that calibration in MESF units remains uncertain over about a two-fold range.

Kinetic studies of ground state atoms, Sb(54S), by time-resolved resonance fluorescence

Ground state antimony atoms, Sb[5p3(4S)], have been studied by time-resolved resonance fluorescence at λ= 231.1 nm (64P½→ 54S) in the “single-shot” mode. The antimony atoms were generated by pulsed irradiation of SbMe3 using a “magnetically-pinched”, Garton light source with a high spectral output in the vacuum ultraviolet. Optical excitation of the resulting Sb(54S) was effected by means of a sealed microwave-powered atomic emission source, and the resonance fluorescence was monitored photoelectrically with a photomultiplier tube gated with a “pre-trigger” system. Kinetic studies of Sb(54S) in the presence of NO and of O2 are described and the resulting rate constants are compared with analogous data derived previously using time-resolved resonance absorption at the same wavelength, and discussed in terms of departures from the standard Beer–Lambert law for light absorption. Radiation trapping calculations are also described which involve summation of the Voigt profiles over all the individual nuclear hyperfine components that contribute to the λ= 231.1 nm transition. Solutions of the diffusion equation for radiation with different boundary conditions are employed in the calculations of the relationship between fluorescence intensity and particle density. The results are compared with the experimental fluorescence intensity calibration for this system.