Homogeneous fluorescent immunoassay.

A fluorescent immunoassay based on the correlation of fluctuations in particle number measures the amount of tagged species bound to micrometer-sized beads and is insensitive to background fluorescence. Without separation steps, a competitive assay can resolve I nanogram of gentamicin per milliliter from a total sample volume of only 10 microliters.

Atomic-resolution electrochemistry with the atomic force microscope: copper deposition on gold.

The atomic force microscope (AFM) was used to image an electrode surface at atomic resolution while the electrode was under potential control in a fluid electrolyte. A new level of subtlety was observed for each step of a complete electrochemical cycle that started with an Au(111) surface onto which bulk Cu was electrodeposited. The Cu was stripped down to an underpotential-deposited monolayer and finally returned to a bare Au(111) surface. The images revealed that the underpotential-deposited monolayer has different structures in different electrolytes. Specifically, for a perchloric acid electrolyte the Cu atoms are in a close-packed lattice with a spacing of 0.29 +/- 0.02 nanometer (nm). For a sulfate electrolyte they are in a more open lattice with a spacing of 0.49 +/- 0.02 nm. As the deposited Cu layer grew thicker, the Cu atoms converged to a (111)-oriented layer with a lattice spacing of 0.26 +/- 0.02 nm for both electrolytes. A terrace pattern was observed during dissolution of bulk Cu. Images were obtained of an atomically resolved Cu monolayer in one region and an atomically resolved Au substrate in another in which a 30 degrees rotation of the Cu monolayer lattice from the Au lattice is clearly visible.

Scanning tunneling microscopy and atomic force microscopy: application to biology and technology.

The scanning tunneling microscope (STM) and the atomic force microscope (AFM) are scanning probe microscopes capable of resolving surface detail down to the atomic level. The potential of these microscopes for revealing subtle details of structure is illustrated by atomic resolution images including graphite, an organic conductor, an insulating layered compound, and individual adsorbed oxygen atoms on a semiconductor. Application of the STM for imaging biological materials directly has been hampered by the poor electron conductivity of most biological samples. The use of thin conductive metal coatings and replicas has made it possible to image some biological samples, as indicated by recently obtained images of a recA-DNA complex, a phospholipid bilayer, and an enzyme crystal. The potential of the AFM, which does not require a conductive sample, is shown with molecular resolution images of a nonconducting organic monolayer and an amino acid crystal that reveals individual methyl groups on the ends of the amino acids. Applications of these new microscopes to technology are demonstrated with images of an optical disk stamper, a diffraction grating, a thin-film magnetic recording head, and a diamond cutting tool. The STM has even been used to improve the quality of diffraction gratings and magnetic recording heads.

Imaging and manipulating molecules on a zeolite surface with an atomic force microscope.

The adsorption of neutral molecules and ions on the surfaces of zeolites was observed in real time with an atomic force microscope (AFM). Direct imaging of the surface of the zeolite clinoptilolite was possible by using a diluted tert-butyl ammonium chloride solution as a medium. Images of the crystal in different liquids revealed that molecules could be bound to the surface in different ways; neutral molecules of tert-butanol formed an ordered array, whereas tert-butyl ammonium ions formed clusters. These absorbed molecules were not rearranged by the AFM tip when used in an imaging mode. However, when a sufficiently large force was applied, the tip of the AFM could rearrange the tert-butyl ammonium ions on the zeolite surface. This demonstration of molecular manipulation suggests new applications, including biosensors and lithography.

Fluorescence fluctuation immunoassay.

The homogeneous fluorescent immunoassay described above allows one to measure the brightness of fluorescently tagged carrier particles that are suspended in a background of free, unbound fluorescent sources. We have demonstrated the feasibility of our technique using a gentamicin competitive assay as well as idealized model systems. We have seen that the fluctuation-correlation method is able to discriminate against free background sources because each fluorescing particle in solution contributes to the correlation peak [Eq. (4)] with a weighting equal to the square of its respective intensity. Hence, a few very bright sources contribute disproportionately to the "signal" relative to many weak ones. To take advantage of this property, one would therefore design an assay that uses relatively larger carrier particles, each of which is capable of binding on the order of 10(3) to 10(4) tagged antibodies or antigens. Unfortunately, the nonlinear dependence of the correlation peak on the brightness of the fluorescing species causes the technique to be perturbed by carrier particle aggregation; the apparent bound fluorescence intensity increases with the extent of aggregation. The latter may be an unavoidable consequence of performing assays using raw blood serum, for example. The ultimate usefulness of this method will depend on its sensitivity and speed when applied to "real" assays of clinical significance. These characteristics will be influenced by a number of technical details. Given our limited experience with the method thus far, it would appear that its principal drawback is its relatively slow speed. In order to decrease the time needed for a reliable measurement, one must average the random fluctuations in the fluorescent intensity to zero more quickly. In principle, this can be accomplished by decreasing the shot noise by collecting a larger fraction of the fluorescent light, and increasing the sampling rate. The method requires rather complicated instrumentation; it is by no means clear that this level of complexity is justified given the realistic level of sensitivity that will be obtained by this technique.

The measurement of extravascular lung water by thermal-green dye indicator dilution.

The theory and practice of the thermal-dye indicator-dilution method for measurement of EVLW has been discussed, and all available animal data from our laboratory correlating EVTV and gravimetric EVLW have been presented. The method appears to function well over the entire range of edema seen , and to be minimally dependent on cardiac output. Thermal-indicator loss does not seem to be a significant problem and does not impair the accuracy of this method. Out results are consistent with earlier works in the field in identifying significant differences between the isotopic EVLW methods and the thermal-dye method, and it seems likely that these differences are due to the much greater diffusion rate of the thermal indicator.

New uses of fluorescence in the surgical management of necrotizing soft tissue infection.

The planning of incisions in the management of necrotizing soft tissue infections has largely been carried out by subjective methods. Because of disruption of the fasciocutaneous circulation, the perfusion of randomly based flaps is frequently tenuous. A method that provides safe, rapid, and accurate evaluation of tissue perfusion would therefore prove invaluable in the preoperative planning, as well as in the postoperative management of these infections. The digital dermofluorometer is a recently introduced instrument that objectively evaluates skin blood flow based on the cutaneous delivery of sodium fluorescein. We have used the technique successfully and without incident in patients who presented with necrotizing soft tissue infections. The theory, methods, and application of the test have been presented along with two case reports.

Effect of cardiac output on extravascular lung water.

Extravascular lung water (EVLW) and cardiac output (CO) were determined in 21 mongrel dogs using the thermal-green dye double indicator dilution technique. In 12 of the animals the renal vessels were ligated bilaterally to increase peripheral resistance and reduce cardiac output without altering actual EVLW. Measurements before and after renal pedicle ligation revealed an average 47 per cent decrease in cardiac output with an 11 per cent increase in measured lung water. In the remaining nine animals an external arteriovenous fistula was constructed to reduce afterload and increase cardiac output. In the baseline state, opening the fistula caused a 63 per cent increase in cardiac output with a simultaneous five per cent decrease in measured EVLW. This second group of animals was then given intravenous acid sufficient to cause 30-50 per cent increases in EVLW. Measurements of EVLW and CO with the fistula open and closed were continued for three hours. The inverse relation between cardiac output and EVLW continued. The results of these experiments show that cardiac output does exert a small effect on the measurement of EVLW.

Surface quantification of injected fluorescein as a predictor of flap viability.

In a laboratory model, quantitative skin-surface fluorescence has been used to reliably measure skin perfusion in ischemic random flaps and to predict viability. The method is reproducible and allows investigators to sequentially monitor soft-tissue perfusion using a fluorescent indicator. It is superior to the conventional fluorescein test (Wood's lamp method), which allows only a single subjective assessment within a 24-hour period.

A single indicator technique to estimate extravascular lung water.

An estimate of extravascular thermal volume (EVTV) calculated from a single thermal indicator (SI) sampled in the pulmonary artery and the femoral artery was compared with direct gravimetric measurements of extravascular lung water (EVLW) on 47 dogs and with simultaneous double indicator (DI) dilution measurements made on 62 dogs and 38 humans with heat as the diffusible indicator and green dye as the intravascular indicator. For the gravimetric measurements the relation was EVTV(SI) = 1.00 EVLW(grav) + 61 ml r = 0.91. When compared with double indicator dilution measurements, the results were EVTV(SI) = 1.10 EVTV(DI) + 17 ml r = 0.94 for dogs and EVTV(SI) = 0.89 EVTV(DI) + 66 ml r = 0.97 for humans. A total of 1880 measurements were made over a range of EVTV(DI) from 70 to 2150 ml. The single indicator measurements of EVTV agrees with the double indicator measurements of EVTV and with the direct gravimetric measurements of EVLW and can be performed with standard catheters and without the withdrawal of blood.

[Bedside determination of extravascular lung water]. / Bettseitige Bestimmung des extravasalen Lungenwassers.

Extravascular lung water (EVLW) was measured at the bedside in 12 patients with the thermal-green dye double indicator dilution method using a microprocessor. The EVLW ranged from 3.3 to 17.2 ml/kg body weight; in patients without pulmonary problems we have found an average EVLW of 5.7 ml/kg body weight. The method involves easy calculations and is reproducible and accurate.

A theoretical model of regionally ischemic myocardium.

The isometric tension development of a one-dimensional regionally ischemic muscle was analyzed theoretically. The model consist of a one-dimensional normal segment in series with a one-dimensional ischemic segment. Each segment is modeled as a three-element muscle. The inputs to the various elements, except the contractile element in ischemic segment, were obtained from published data for cat papillary muscles. To be consistent with segment length measurements on ischemic canine hearts, it was assumed that the ischemic contractile element contracted normally at the beginning of contraction and then at some tension, TM, fell behind in its rate of tension development compared to the contractile element in the normal segment. Rate of tension development of various lengths of the ischemic segment and strengths of the ischemic contractile element. At the tension, TM, the ischemic segment begins undergoing paradoxical expansion and, simultaneously, as a result of the expansion. the time derivative of the tension produced by the regionally ischemic muscle exhibits a sudden decrease.

Indicator dilution using a fluorescent indicator.

In vitro and in vivo indicator-dilution measurements are made with a fluorescent indicator and a novel detection system using a catheter containing a single optical fiber that carries both the exciting and returning fluorescent light. These fluorescent-dilution measurements are compared with simultaneous green dye-dilution measurements. The double-indicator-dilution measurement of extravascular lung water using heat and fluorescence is compared with gravimetric measurements. Also investigated is the sensitivity of the fluorescent measurement to changes in O2 saturation and hematocrit of the blood. An example of the measurement of a right-to-left heart shunt with this new indicator is given.

Fluorescence immunoassay based on long time correlations of number fluctuations.

We report the development of a fluorescence-based immunoassay technique relying on the physical phenomena of random number fluctuations and diffusion, which we review. By determining the autocorrelation of the fluctuations in the fluorescent intensity, this methid is able to measure the amount of labeled antigen or antibody that is bound to micrometer-sized carrier particles in solution. The principal advantage of this technique is its insensitivity to small, fast-diffusing sources. It also discriminates against weakly fluorescent contaminants of size comparable to the carrier particles. We demonstrate these attributes by using two model systems: a human IgG assay and an idealized system consisting of polystyrene fluorescent spheres and rhodamine dye.

Effect of inhalation injury on lung water accumulation.

Fourteen thermally injured patients with severe inhalation injury were sequentially studied with the thermal-green dye double indicator dilution technique of extravascular lung water (EVLW) measurement. Eight females and six males (average age, 49 years, and average thermal burn, 37% body surface) were studied for 2-31 days postinjury. All were burned in a closed space, had facial burns, soot in their sputum, and a mean carboxyhemoglobin level of 30%. Nine patients died, six of sepsis, one each of acute renal failure, hepatorenal syndrome, and anoxic brain damage. Mean EVLW on admission was 7.0 +/- 2.9 ml/kg and remained normal in the five survivors and in the patients dying of acute renal failure and anoxic brain damage. Six patients had increases in EVLW, caused by altered pulmonary capillary permeability in five and by elevation of hydrostatic pressures in one patient (hepatorenal death). Of the five patients with permeability edema, one appeared to result from a direct early effect of inhalation injury resulting in an EVLW of 13.3 ml/kg on admission. The other four patients had EVLW increases after the onset of sepsis, resulting in a mean EVLW of 23.2 +/0- 7.2 ml/kg at death (p less than 0.01). Seventy-one per cent of all patients developed pneumonia, which appears to have caused an EVLW increase in one patient; the other EVLW increases were caused by systemic sepsis. In our present study of 14 patients with definite severe inhalation injury only one had an early increase in EVLW directly related to the inhalation injury, an early effect on capillary permeability presumably caused by direct chemical toxicity of inhaled gases. The remaining four cases of permeability edema occurred 4-24 days postinjury and resulted from burn wound or pulmonary sepsis. We thus conclude that increases in EVLW after thermal and inhalational injury are primarily caused by systemic or pulmonary sepsis, and have a delayed onset. Early increases in EVLW may be a result of the chemical toxicity of inhaled gases but are very uncommon, moderate in degree, and are seen only with the severest cases of inhalation injury.

Digital cutaneous fluorometry: correlation between blood flow and fluorescence.

Central to the use of fluorescein in vascular diagnosis is the requirement that the intensity of evoked fluorescence be proportional to blood flow. With the introduction of the digital dermofluorometer, a device that quantitates cutaneous fluorescence, establishment of this relationship has become possible. After experimentally producing measured reductions in the distal aortic flow of eight rabbits, the ratio of fluorescence in the flow-restricted and unrestricted areas was obtained by measuring hind- and forelimb fluorescence. At any time between 20 and 60 minutes following a bolus injection of sodium fluorescein (1 mg/kg body weight), there was a significant linear relationship (p less than 0.05, r greater than 0.75) between residual aortic flow and the ratio of hind-/forelimb fluorescence. Simultaneously obtained plasma fluorescein concentrations decayed rapidly by first-order kinetics with a half-life of 12.5 minutes, regardless of the degree of distal aortic occlusion. The time course of the rise and fall of cutaneous fluorescence was slower than that of the plasma fluorescein concentration, proving that interstitial rather than intravascular fluorescein was responsible for the measured fluorescence. We conclude that the intensity of tissue fluorescence is linearly related to blood flow and that conclusions regarding perfusion may be drawn from relative fluorescence at any time between 20 and 60 minutes following a bolus injection of fluorescein. Furthermore, the passage of fluorescein into the interstitium is dependent on a time-limited diffusion process, which along with flow, establishes the time to peak and the absolute amplitude of the tissue fluorescence curve.

Determinants of pulmonary interstitial fluid accumulation after trauma.

We have sequentially measured the daily extravascular lung water (EVLW) changes in 16 severely traumatized patients to better define the principal etiologic factors causing post-traumatic interstitial fluid accumulation and subsequent respiratory failure. We found that severe hemorrhagic shock (mean initial BP = 40 mm Hg), massive transfusion (12.7 liters of blood), and crystalloid resuscitation with resulting hemodilution of plasma colloid osmotic pressure (PCOP) (PCOP less than or equal to 15 mmHg) do not cause EVLW accumulation. Post-traumatic elevations in EVLW were seen after lung contusion (average EVLW = 15.3 +/- 2.5 ml/kg), sepsis (average EVLW = 17.1 +/- 2.9 ml/kg) and cardiac failure (EVLW = 15.3 +/- 0.3 ml/kg). Severe hemorrhagic shock, massive transfusion, and crystalloid resuscitation with resulting hemodilution of plasma colloid oncotic pressure do not cause EVLW accumulation. Post-traumatic elevations in EVLW are seen after lung contusion, sepsis, and cardiac failure. We conclude that after trauma elevations in capillary hydrostatic pressure and capillary permeability alterations resulting from lung contusion or sepsis are the primary determinants of interstitial fluid accumulation.

Lung water changes after thermal injury. The effects of crystalloid resuscitation and sepsis.

Respiratory failure after thermal injury is common, but the etiologic roles of high volume crystalloid resuscitation, hypoproteinemia, inhalation injury, or sepsis have not been specifically defined in human studies. We used the thermal-green dye double indicator dilution measurement of extravascular lung water (EVLW) to follow daily lung water changes in seven severly burned adult patients, resuscitated with only crystalloid solutions. An average weight gain of 21.3 kg, a 30% increase (p < 0.001), was present two to three days after admission. Admission EVLW for all patients was 7.9 +/- 1.2 ml/kg, (means +/- SD), and EVLW at the time of maximal weight gain was 5.9 +/- 1.4 ml/kg, a 25% decrease (p < 0.05). Admission pulmonary artery wedge pressure (PAWP) was 8 +/- 3 mmHG, which was not significantly different from PAWP of 13 +/- 4 mmHg at the time of maximal weight gain. In the three patients who died of sepsis, their terminal weight averaged 17.8 kg (27%) above their admitting weight (p < 0.01) and EVLW was 26.4 +/- 4.4 ml/kg, a 200% increase (p < 0.02) from admission. Their terminal PAWP averaged 22 +/- 2 mmHg, a 170% increase (p < 0.005). None of these patients had an increase in EVLW until clinical signs of sepsis occurred and the rise in EVLW preceded the rise in PAWP. Calculated mean plasma colloid osmotic pressure (PCOP) on admission was 20.7 +/- 4.9 mmHg; at the time of maximal weight gain, it was 8.6 +/- 1.7 mmHg (p < 0.001). The PCOP-PAWP gradient fell to -4 +/- 4 mmHg (p < 0.001) at the time of maximal weight gain and remained less than +4 mmHg throughout the study period in all patients. We conclude that massive crystalloid resuscitation while maintaining PAWP below 15 mmHg does not cause an increase in EVLW during the first four days after thermal injury. EVLW actually decreases slightly in all patients despite marked weight gain, hypoproteinemia and a negative PCOP-PAWP gradient. EVLW does not correlate with the PCOP-PAWP gradient in either septic or nonseptic periods. Three patients had severe inhalational injury and normal EVLW for the first four postburn days. It therefore appears that significant interstitial edema does not result from inhalational injury. There is also no evidence that thermal injury causes an early increase in pulmonary capillary permeability. The occurrence of sepsis, however, results in rapid accumulation of lung water, without any change in hydrostatic or osmotic forces. This study supports the primary role of sepsis in altering pulmonary capillary permeability with resulting pulmonary edema.