Ultrafast nonlinear sub-wavelength solid immersion spectroscopy at T = 8 K: an alternative to nonlinear scanning near-field optical microscopy.

Pump-probe measurements were performed with a subwavelength spatial resolution of 355 nm and a temporal resolution of 130 fs in a multiple quantum well sample at T= 8 K. A solid immersion lens was used to increase the spatial resolution to 0.41 lambda, demonstrating that the limit of conventional microscopy was surpassed.

Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces.

Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.

Direct visualization of surface charge in aqueous solution.

Tapping mode atomic force microscopy operated in the phase shift mode is used to directly visualize the surface charge on biological macromolecules under solution. A simple theory is presented which is qualitatively consistent with experimental observations, although a more complicated theoretical analysis is required for a quantitative comparison.

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air.

Recent developments in profiling optical surfaces.

To understand better the fabrication of optical surfaces and to be able to produce smoother, lower-scatter surfaces, we need to extend characterization techniques to shorter and longer surface spatial wavelengths beyond the conventional 1-µm to 1-mm region. Scanning probe microscopes are now available for profiling optical surfaces with height and lateral resolutions of a few atomic spacings. In this paper we report on measurements made with a Nanoscope II atomic force microscope on a variety of supersmoothoptical surfaces and compare these results with measurements made with a conventional stylus profiling instrument.

Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope.

Reproducible images of uncoated DNA in the atomic force microscope (AFM) have been obtained by imaging plasmid DNA on mica in n-propanol. Specially sharpened AFM tips give images with reproducible features several nanometers in size along the DNA. Plasmids can be dissected in propanol by increasing the force applied by the AFM tip at selected locations.

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.

Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy.

In skeletal muscle the activation of phosphorylase b is catalyzed by phosphorylase kinase. Both enzymes occur in vivo as part of a multienzyme complex. The two enzymes have been imaged by atomic force microscopy and the results compared to those previously found by scanning tunneling microscopy.

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.

Scanning probe microscopy of biological samples and other surfaces.

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive.

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.

Scanning tunneling microscopy of freeze-fracture replicas of biomembranes.

The high resolution of the scanning tunneling microscope (STM) makes it a potentially important tool for the study of biomaterials. Biological materials can be imaged with the STM by a procedure in which fluid, nonconductive biomaterials are replaced by rigid and highly conductive freeze-fracture replicas. The three-dimensional contours of the ripple phase of dimyristoylphosphatidylcholine bilayers were imaged with unprecedented resolution with commercial STMs and standard freeze-fracture techniques.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.

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.