Digital microspectrofluorometry by multichannel scaling and single photon detection

Synopsis A careful analysis of the requirements for microspectrofluorometric measurements has been made. General problems encountered in the measurement of light intensity and spectral distributions are considered, together with some special factors arising when measuring the fluorescence emitted by single cells. Criteria for evaluating microspectrofluorometric apparatus are given, and the relative merits of analog and digital methods are compared.A fully digital instrument using single-photon detection and a multichannel analyser is described. Automatic procedures for obtaining photodecomposition-time curves and emission spectra are illustrated in detail. Other possible applications of the instrument are also outlined. The experimental results reported show that microspectrofluorometric measurements of high sensitivity are possible, as light intensities down to a few photons per second can be measured.     

Real-time detection of changes in the electrophoretic mobility of a single cell induced by hyperosmotic stress

Living cells survive environmentally stressful conditions by initiating a stress response. We monitored changes in the electrophoretic mobility (EPM) of single, optically trapped yeast cells under hyperosmotic stress conditions using optical tweezers combined with a position detector. We studied the dynamics of the EPM stress response for cells at different phases of the cell cycle.     

Measurement of myo-inositol in single cells and defined areas of the nervous system by selected ion monitoring

In this work we want to bring attention to the potential value of the direct analysis of tissue constituents using two well established techniques: the selected ion monitoring method of gas chromatography-mass spectrometry and the techniques of quantitative histochemistry. The substrate analyzed is myo-inositol which is measured in single cells of rabbit Deiters' nucleus, spinal cord anterior horn, and dorsal root ganglion. Comparison of inositol levels in neurons vs neuropil showed a significantly greater level in anterior horn neurons; however, no difference was found in Deiters' nucleus. In dorsal root ganglion cells, 5-μm sections through a portion of the cell not containing the nucleus were compared with sections through a portion of the nucleus; no difference in inositol levels was found. Two hypothalamic nuclei were examined and found to have very different concentrations of inositol. In the samples examined, there was a threefold inositol concentration range.     

Measurement of propericiazine in plasma by capillary gas chromatography and selected ion monitoring detection

A new method, based upon a selective extraction and gas chromatographic/mass spectrometric analysis, was developed to monitor the effect of combined haemoperfusion-haemodialysis treatment in a case of propericiazine poisoning. The method relies on the selected ion monitoring of the acetate derivatives of propericiazine and its internal standard fluphenazine, after their extraction from 1 ml of alkalinized plasma with n-hexane:isopropanol (8:2, v/v), back-extraction into an acidified water phase, realkalinization and extraction with n-hexane: isopropanol, derivatization with acetic anhydride and gas chromatography on a short (12 m) OV-101 fused silica capillary column. The described procedure is specific and provides between-assay variability of 4.8% CV at 5 micrograms 1(-1) plasma concentration. The method enables quantification down to 1 microgram 1(-1) and hence demonstrates sufficient sensitivity to permit pharmacokinetic or drug monitoring studies.     

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.     

Biological monitoring of occupational exposure to 1-bromopropane by means of urinalysis for 1-bromopropane and bromide ion

The purposes of the present study are (1) to develop a sensitive analytical method to measure 1-bromopropane (1-BP) in urine, (2) to examine if 1-BP or bromide ion (Br) in urine is a useful biomarker of exposure to 1-BP, and (3) to identify the lowest 1-BP exposure concentration the method thus established can biomonitor. A factory survey was carried out on Friday, and 33 workers (all men) in cleaning and painting workshops participated; each worker was equipped with a diffusive sampler (carbon cloth KF-1500 as an adsorbent) to monitor 1-BP vapour for an 8-h shift, and offered a urine sample at the end of the shift for measurement of 1-BP and Br in urine. In addition, 10 non-exposed men offered urine samples as controls. The performance of the carbon cloth diffusive sampler was examined to confirm that the sampler is suitable for monitoring time-weighted average 1-BP vapour exposure. A head-space GC technique was employed for analysis of 1-BP in urine, whereas Br in urine was analysed by ECD-GC after derivatization to methyl bromide. The workers were exposed to vapours of seven other solvents (i.e. toluene, xylenes, ethylbenzene, acetone, etc.) in addition to 1-BP vapour; the 1-BP vapour concentration was 1.4 ppm as GM and 28 ppm as the maximum. Multiple regression analysis however showed that 1-BP was the only variable that influenced urinary 1-BP significantly. There was a close correlation between 1-BP in urine and 1-BP in air; the correlation coefficient (r) was >0.9 with a narrow variation range, and the regression line passed very close to the origin so that 2 ppm 1-BP exposure can be readily biomonitored. The correlation of Br in urine with 1-BP in air was also significant, but the r (about 0.7) was smaller than that for 1-BP, and the background Br level was also substantial (about 8 mg l^-1). Thus, it was concluded that 1-BP in end-of-shift urine is a reliable biomarker of occupational exposure to 1-BP vapour, and that Br in urine is less reliable.     

High-sensitivity blood-based detection of breast cancer by multi photon detection diagnostic proteomics

We have developed several new methods for blood-based cancer detection by diagnostic proteomics. Ultrasensitive methods of immunoassay using multiphoton-detection (IA/MPD) increase sensitivity by 200- to 1,000-fold (1 femtogram/mL). This has allowed the measurement of cancer biomarkers with very low concentrations in blood that could not be measured for full patient cohorts with conventional immunoassays. Sensitivity and specificity in cancer detection have been found to be potentiated by use of immunoassay panels which include tissue-specific cancer biomarkers as well as cytokines and angiogenic factors. The ultrasensitive immunoassays revealed that patient to patient variations in the concentrations of individual biomarkers in blood can extend over many orders of magnitude (up to six) and that the distributions of biomarker concentrations over patient cohorts are non-Gaussian. New methods of data analysis which correlate abundances of multiple, different biomarkers have been developed to deal with such data sets. Sensitivity and specificity of about 95% have been achieved for blood-based detection of breast cancer in pilot studies on 250 patients and 95 controls. Pilot studies indicate that this methodology may also allow differentiation of malignant breast cancer from benign lesions and can provide similar sensitivity and specificity for other epithelial cancers such as prostate cancer, ovarian cancer and melanoma. The methods developed for selection, application, and evaluation of very high sensitivity biomarker panels are expected to have general relevance for diagnostic proteomics.     

Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection

Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.     

Bone mineral content of the hyperprolactinemic rat femur by single photon absorptiometry

Chronic hyperprolactinemia was obtained in the rat by an implantation of two adenohypophyses under the kidney capsule. This experimental model was set to evaluate the influence that high prolactin levels may have on bone mineralization when an intervention of estrogens can reasonably be excluded. Bone density was measured by single photon absorptiometry at the femur level. Our results indicate that the increase in PRL throughout the period of observation was associated with a significant decrease in bone mineral content, while calcium and parathyroid hormone serum levels remained unchanged. This would suggest that PRL plays a role in bone calcium metabolism, that might be included among the effects of this hormone.     

Real-time monitoring of polyacrylamide gel electrophoresis by schlieren optics

A modernized schlieren optics was applied to follow protein bands visually during polyacrylamide gel electrophoresis. A band containing as little as 0.3 microgram of a protein could be detected. Besides the protein bands, usually overlooked phenomena such as boundary migration of the buffer components could be visualized. As an example, electrophoretic patterns thus obtained for SDS-polyacrylamide gel electrophoresis with a discontinuous buffer system are presented. The use of a split-type colored filter was found to be useful for coloring a particular location on the gel red or blue depending on the sign of the refractive index gradient. This means of detection also seems useful in that, firstly, it makes electrophoretic mobility measurement more quantitative and, secondly, it allows localization of a protein band and sampling in the intact state.     

Real-time monitoring of recombinant bacterial proteins by mass spectrometry

Genetically altered bacteria manipulated to express green fluorescent protein (GFP) were used in an investigation of real-time monitoring for recombinant protein expression in cell by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A significant advantage to whole cell MALDI MS is its ability to analyze bacterial cultures without pretreatment other than concentration. This paper describes the simultaneous analysis of overexpressed GFP recombinant Escherichia coli JM101 by MALDI-TOF MS and standard fluorescence measurements. Cells were harvested from liquid culture media during a 12 h GFP induced expression cycle to demonstrate the feasibility of near real-time monitoring of induced protein expression. The results show that although MALDI MS is not as sensitive as fluorescence measurements, expression levels of the targeted protein can easily be determined. Data available only through MALDI MS measurements reveal the presence of both native GFP and GFP-(histidine)(6) proteins. Additionally, biochemical processes not yet fully understood are observed in the presence and absence of ribosomal protein constituents. Thus, the work presented here demonstrates the ability of MALDI MS to monitor and characterize in real time the expression of targeted and unexpected genetically recombinant proteins in active cell cultures.     

Real-time optical detection of single human and bacterial viruses based on dark-field interferometry

The rapid and sensitive detection and characterization of human viruses and bacteriophage is extremely important in a variety of fields, such as medical diagnostics, immunology and vaccine research, and environmental contamination and quality control. We introduce an optical detection scheme for real-time and label-free detection of human viruses and bacteriophage as small as ~24 nm in radius. Combining the advantages of heterodyne interferometry and dark-field microscopy, this label-free method enables us to detect and characterize various biological nanoparticles with unsurpassed sensitivity and selectivity. We demonstrate the high sensitivity and precision of the method by analyzing a mixture containing HIV virus and bacteriophage. The method also resolves the distribution of small nano-impurities (~20-30 nm) in clinically relevant virus samples.     

Real-time detection of viable microorganisms by intracellular phototautomerism

Background  

To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner.     

Determinants of single photon response variability

The responses to single photon absorptions (quantum bumps) vary randomly in size in Limulus photoreceptors. This variability is a natural consequence of simple chemical reactions involving a small number of molecules. The measured size distributions differ significantly from the exponential distribution predicted by the simplest transduction cascade models, one feature of which is that light-activated rhodopsin (R*) is turned off in a single step process. As shown in the companion paper, the nonexponential size distributions can be accounted for if R* is turned off in a multi-step process. This would lead to a nonexponential (peaked) distribution in the number of G- protein molecules activated during a quantum bump and to a nonexponential distribution in the size of bumps. To test this possibility we measured the distribution of quantum bump size under two conditions in which the variability in the number of activated G- proteins was eliminated. eliminated. In one method, bumps were produced by direct activation of single G-proteins using GTP-gamma-S; in the second GDP-beta-S reduced the R* gain to the point where most quantal events were due to activation of a single G-protein. In both cases the size distribution of bumps became much closer to an exponential distribution than that of normal light-induced bumps. These results support the idea that the size distribution of light-induced bumps is dependent on events at the R* level and reflects to the multi-step deactivation of R*.     

A simple extraction procedure for prostaglandins in human seminal plasma with specific detection by selected ion monitoring

The extraction of prostaglandins (PGs) from biological samples and in particular from human seminal plasma becomes in practice a rather complex process, usually requiring a significant degree of sample manipulation and clean up procedures. Our work in this field has led to a very simple method based on a direct ultrafiltration of samples of human seminal plasma and extraction of the PGs in the ultrafiltrate into ethylacetate. The type of ultrafiltration membranes used for this purpose retain all substances of molecular weight over 1000, effectively removing proteins and other heavy interfering material. Recoveries of PGs in the first step are of the order of 81,5 to 86,8% as verified with tritiated PGs while the second step is virtually quantitative (>99%). These extractions are both quantitatively and qualitatively reproducible judging from the recovery values obtained from replicate determinations of the same samples as well as from the pattern reproducibility of the corresponding gas chromatographic and selected ion and profiles. The latter are identical to the profiles obtained from samples processed by a different extraction method of proven efficacy which in principle would validate the procedure and results herein described.     

Selective transmission of single photon responses by saturation at the rod-to-rod bipolar synapse

A threshold-like nonlinearity in signal transfer from mouse rod photoreceptors to rod bipolar cells dramatically improves the absolute sensitivity of the rod signals. The work described here reaches three conclusions about the mechanisms generating this nonlinearity. (1) The nonlinearity is caused primarily by saturation of the feedforward rod-to-rod bipolar synapse and not by feedback from horizontal or amacrine cells. This saturation renders the rod bipolar current insensitive to small changes in transmitter release from the rod. (2) Saturation occurs within the G protein cascade that couples receptors to channels in the rod bipolar dendrites, with little or no contribution from presynaptic mechanisms or saturation of the postsynaptic receptors. (3) Between 0.5 and 2 bipolar transduction channels are open in darkness at each synapse, compared to the approximately 30 channels open at the peak of the single photon response.     

Real-time dynamic monitoring of nonprotein-bound copper in the blood

To obtain real-time dynamic changes of non-protein-bound copper in the blood, we have developed an online microdialysis sampling system coupled with a flow-injection graphite furnace-atomic absorption spectrometer (FI-GFAAS). The analytical performances of the online system such as linearity, limit of detection, precision, and spiked recoveries were validated. Before the in vivo experiments, the in vivo recovery was conducted. The levels of non-protein-bound Cu in the blood of living rabbits were evaluated before and after administering them with 5 mg/kg body weight of CuSO4 by the online microdialysis-FI-GFAAS system. The results showed that the average basal concentration of non-protein-bound Cu in the blood of living rabbits was 16.2 microg/L (n = 3). Furthermore, the levels of non-protein-bound Cu in the blood of living rabbits were observed after a long delay following intravenous injection of CuSO4. The non-protein-bound Cu reached the maximum value at 125 min after injection. Our present study might provide the in vivo, direct observation that different metals have their own binding characteristics with proteins when transported into the blood of living organisms.