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Biomedical subjects

P T So

Publications and source records attributed to P T So.

11 recordsLinked to original sources
PubMed1998

Multiphoton excitation microscopy of in vivo human skin. Functional and morphological optical biopsy based on three-dimensional imaging, lifetime measurements and fluorescence spectroscopy.

Two-photon excitation microscopy has the potential as an effective, noninvasive, diagnostic tool for in vivo examination of human deep tissue structure at the subcellular level. By using infrared photons as the excitation source in two-photon microscopy, a significant improvement in penetration depth can be achieved because of the much lower tissue scattering and absorption coefficients in the infrared wavelengths. Two-photon absorption occurs primarily at the focal point and provides the physical basis for optical sectioning. Multiphoton excitation microscopy at 730 nm was used to image in vivo human skin autofluorescence from the surface to a depth of about 200 microns. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence using 730 nm excitation. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells and provides a functional and morphological optical biopsy.

Biopsy↗
PubMed1997

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin.

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.

Biophysical Phenomena↗
PubMed1997

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Animals↗
PubMed1996

Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation.

Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by monitoring spontaneous equilibrium fluctuations in the local concentration of a fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, submicromolar samples by directly "counting particles". We introduce the application of two-photon excitation to scanning FCS and discuss its important advantages for this technique. We demonstrate the capability of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, and then with B phycoerythrin. The detection of multiple species in a single sample is shown, using mixtures containing both sphere sizes. The method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/ dissociation equilibrium for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good agreement with previously published values. In addition, oligomer dissociation induced by pH titration from pH 8 to pH 5.0 is detectable for the enyme phosphofructokinase. The possibility of measuring dissociation kinetics by scanning two-photon FCS is also demonstrated using phosphofructokinase.

Biophysical Phenomena↗
PubMed1996

Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.

2-Naphthylamine↗
PubMed1996

Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress.

By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.

Animals↗
PubMed1995

Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.

Animals↗
PubMed1995

Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment.

We report on the application of two photon molecular excitation to fluorescence correlation spectroscopy. We demonstrate the first fluorescence correlation spectroscopy measurements of translational mobility in the cytoplasm of living cells. Two-photon excitation inherently excites small sample volumes in three dimensions, providing depth discrimination similar to confocal microscopy, without emission pinholes. We demonstrated accurate measurements of the diffusion constant, D, for particles of several different known sizes, in bulk solutions of different viscosity. We then showed measurements of translational diffusion for 7- and 15-nm radius latex beads in the cytoplasm of mouse fibroblast cells. We measured time-dependent diffusion coefficients. When first injected in the cells, the spheres moved from two to five times slower than in water, with average rates of 18 x 10(-8) cm2/s for the 7 nm and 5 x 10(-8) cm2/s for the 15 nm radius spheres. After a few hours, spheres stick to the cells, and the motion slows down 10 to 100 times.

Animals↗
PubMed1988

X-ray diffraction study of the polymorphic behavior of N-methylated dioleoylphosphatidylethanolamine.

The polymorphic phase behavior of aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and its N-methylated analogues, DOPE-Me, DOPE-Me2, and DOPC, has been investigated by X-ray diffraction. In the fully hydrated lamellar (L alpha) phase at 2 degrees C, the major structural difference is a large increase in the interlamellar water width from DOPE to DOPE-Me, with minor increases with successive methylation. Consistent with earlier reports, inverted hexagonal (HII) phases are observed upon heating at 5-10 degrees C in DOPE and at 65-75 degrees C in DOPE-Me and are not observed to at least 85 degrees C in DOPE-Me2 or DOPC. In DOPE, the L alpha-HII transition is facile and is characterized by a relatively narrow temperature range of coexistence of L alpha and HII domains, each with long-range order. DOPE-Me exhibits complex nonequilibrium behavior below the occurrence of the HII phase: Upon heating, the L alpha lattice spontaneously disorders on a time scale of days; on cooling from the HII phase, the disorder rises on a time scale of minutes. It is shown that, in copious water, the disordered state transforms very slowly into phases with cubic symmetry. This process is assisted by the generation of small amounts of lipid degradation products. The relative magnitudes of the monolayer spontaneous radius of curvature, R0 [Kirk, G. L., Gruner, S. M., & Stein, D. L. (1984) Biochemistry 23, 1093; Gruner, S. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3665], are inferred from the HII lattice spacings vs temperature and are shown to increase with increasing methylation. The relative magnitudes of R0 are categorized as small for DOPE, intermediate for DOPE-Me, and large for DOPC. It is suggested, and examples are used to illustrate, that small R0 lipid systems exhibit facile, low-temperature L alpha-HII transitions, intermediate R0 systems exhibit complex nonequilibrium transition behavior and are likely to form cubic phases, and large R0 systems are stable as L alpha phases. The relationship between the cubic phases and minimal periodic surfaces is discussed. It is suggested that minimal periodic surfaces represent geometries in which near constant, intermediate R0 values can be obtained concomitantly with monolayers of near constant thickness, thereby leading to equilibrium cubic phases. Thus, the relative magnitude of the spontaneous radius of curvature may be used to predict mesomorphic behavior.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗
PubMed1988

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes.

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Models, Biological↗