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Results for “Microscopy, Fluorescence, Multiphoton”

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PubMed2004

Performances of high numerical aperture water and oil immersion objective in deep-tissue, multi-photon microscopic imaging of excised human skin.

Multi-photon fluorescence microscopy (MPFM) is a powerful technique for imaging scattering, biological specimens in depth. In addition to the sectioning effect generated by the point-like excitation volume, the near-infrared wavelengths used for multi-photon excitation allow deeper penetration into optically turbid specimens. In physiological specimens, the optical properties such as the scattering coefficients and refractive indices are often heterogeneous. In these specimens, it is not clear which type of immersion objective can provide optimized images in-depth. In particular, in-depth dermatological imaging applications using MPFM requires such optimization to obtain qualitative and quantitative information from the skin specimens. In this work, we address this issue by comparing the performances of two common types of high numerical aperture (NA) objectives: water-immersion and oil-immersion. A high-quality water-immersion objective (Zeiss, 40 x C-Apochromat, NA 1.2) and a comparable oil-immersion objective (Zeiss, 40 x Fluar, NA 1.25) were used for in-depth imaging of autofuorescent excised human skin and sulforhodamine B treated human skin specimens. Our results show that in the epidermal layers, the two types of immersion objectives perform comparably. However, in the dermis, multi-photon imaging using the oil immersion objective results in stronger fluorescence detection. These observations are most likely due to the degraded point-spread-function (PSF) caused by refractive index mismatch between the epidermis and the dermis.

Dermis↗

PubMed2004

Saturation modified point spread functions in two-photon microscopy.

Excitation saturation can dramatically alter the effective imaging point spread function (PSF) in two-photon fluorescence microscopy. The saturation-modified PSF can have important implications for resolution in fluorescence imaging as saturation leads to both an increased fluorescence observation volume and an altered spatial profile for the PSF. We introduce here a computational approach to accurately quantify molecular excitation profiles that represent the modified imaging PSF in two-photon microscopy under the influence of excitation saturation. An analytical model that accounts for pulsed laser excitation is developed to calculate the influence of saturation at any location within the excitation laser profile. The overall saturation modified molecular excitation profiles are then evaluated numerically. Our results demonstrate that saturation can play an important role in two-photon fluorescence microscopy even with relatively modest excitation levels.

Fluorescent Dyes↗

PubMed2004

Optical-sectioning improvement in two-color excitation scanning microscopy.

We present a new beam-shaping technique for two-color excitation fluorescence microscopy. We show that by simply inserting a properly designed shaded-ring filter in the illumination beam of smaller wavelength, it is possible to improve the effective optical sectioning capacity of such microscopes by 23%. Such an improvement is obtained at the expense of only a very small increasing of the overall energy in the point-spread-function sidelobes. The performance of this technique is illustrated by a numerical imaging simulation.

Equipment Design↗

PubMed2004

Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

Anisotropy↗

PubMed2004

Single molecule spectroscopic characterization of GFP-MUT2 mutant for two-photon microscopy applications.

Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.

Fluorescence↗

PubMed2005

High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy.

In recent years, nonlinear laser scanning microscopy has gained much attention due to its unique ability of deep optical sectioning. Based on our previous studies, a 1,200-1,300-nm femtosecond laser can provide superior penetration capability with minimized photodamage possibility. However, with the longer wavelength excitation, three-photon-fluorescence (3PF) would be necessary for efficient use of intrinsic and extrinsic visible fluorophores. The three-photon process can provide much better spatial resolution than two-photon-fluorescence due to the cubic power dependency. On the other hand, third-harmonic-generation (THG), another intrinsic three-photon process, is interface-sensitive and can be used as a general structural imaging modality to show the exact location of cellular membranes. The virtual-transition characteristic of THG prevents any excess energy from releasing in bio-tissues and, thus, THG acts as a truly noninvasive imaging tool. Here we demonstrated the first combined 3PF and THG microscopy, which can provide three-dimensional high-resolution images with both functional molecule specificity and sub-micrometer structural mapping capability. The simultaneously acquired 3PF and THG images based on a 1,230-nm Cr:forsterite femtosecond laser are shown with a Hoechst-labeled hepatic cell sample. Strong 3PF around 450 nm from DNA-bounded Hoechst-33258 can be observed inside each nucleus while THG reveals the location of plasma membranes and other membrane-based organelles such as mitochondria. Considering that the maximum-allowable laser power in common nonlinear laser microscopy is less than 10 mW at 800 nm, it is remarkable that even with a 100-mW 1,230-nm incident power, there is no observable photo damage on the cells, demonstrating the noninvasiveness of this novel microscopy technique.

Bisbenzimidazole↗

PubMed2005

Quasi-spherical focal spot in two-photon scanning microscopy by three-ring apodization.

We present a beam-shaping technique for two-photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three-ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi-spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot-fluorescence efficiency.

Algorithms↗

PubMed2006

Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting.

Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.

Cell Line↗

PubMed2006

Probing nucleocytoplasmic transport by two-photon activation of PA-GFP.

Two-photon activation of photoactivatable green fluorescent protein (PA-GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the sub-femtoliter optical volume of the two-photon spot. We demonstrate two-photon activation of PA-GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two-photon excitation. Alternatively, we activate PA-GFP using two-photon excitation and monitor the fluorescence of the photoconverted product with one-photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS-1 cells, by observing the time-dependent fluorescence at various locations within the cell after two-photon activation of PA-GFP in the nucleus and in the cytoplasm. Two-photon activation of a tandem construct of two PA-GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two-photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells.

Active Transport, Cell Nucleus↗

PubMed2003

Evaluation of fluorescent probe surface intensities as an indicator of transdermal permeant distributions using wide-area two-photon fluorescence microscopy.

The application of high-speed two-photon fluorescence microscopy (HTPM) to examine transdermal transport processes has enabled the noninvasive visualization of permeant spatial distributions over a larger, more clinically relevant wide area of the skin. Earlier studies demonstrated that the transdermal fluorescent probe distribution over a 2 x 2 mm skin area was well represented by a significantly reduced sampling of the 400 microscale skin sites (100 x 100 microm) constituting the wide area. In the present study, the 400 microscale skin sites are considered individually, and the site-to-site variability in permeant distributions is used as a model to reflect the range in experimentally measured skin permeabilities resulting from the inherent stratum corneum structural heterogeneity. The correlation established between the permeant surface intensity and the corresponding permeant intensity gradient at each skin site provides an indication of the potential for screening transdermal permeant distributions solely based on the evaluation of microscale permeant surface intensities. The strong linear correlation between the intensity gradient and the surface intensity for the hydrophilic model permeant, sulforhodamine B, demonstrated that surface intensities provide a robust indicator of the corresponding transdermal probe distributions at the microscale. For the hydrophobic model permeant, rhodamine B hexyl ester, however, weak correlations were observed between these two parameters. This result suggests that the stratum corneum microscale surface intensity does not validly capture the corresponding intensity gradients for the entire range of skin permeabilities typically encountered as a result of the inherent stratum corneum heterogeneity.

Administration, Cutaneous↗

PubMedDate not supplied

Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers.

The spectral properties of one-photon, two-photon excited autofluorescence and second harmonic generation (SHG) from ex vivo human skin induced by a femtosecond (fs) laser and three visible lasers in backscattering geometry are systematically investigated. Our experimental results indicate that peak position of autofluorescence spectra from the dermis and epidermis shift toward long wavelengths, and the fluorescent intensity decreases when the excitation wavelength increases due to an effect of the excitation wavelength on autofluorescence signals. However, the intensity of the SHG signal in collagen has the maximal value of 800 nm excitation wavelength. This may be the result that the energy of the SHG signal is in resonance with an electronic absorption band. The two-photon excited autofluorescence and SHG intensity all obey a quadratical dependence on the excitation power. Compared with the two-photon excited fluorescence and SHG, the one-photon excited fluorescence in the dermis and epidermis exhibits different spectral characteristics. The investigation of the spectral characteristics of autofluorescence and SHG from ex vivo human skin can provide new insights into morphologic structures and biochemical components of tissues, which are vital for improving the application of laser-induced autofluorescence and SHG spectroscopy technique for noninvasive in vivo tissue diagnostics.

Epidermis↗

PubMed2005

Fluorescence correlation spectroscopy with autofluorescent proteins.

Fluorescence correlation spectroscopy (FCS) is a versatile technique operating at the single-molecule level, that successfully meets many challenges of modern biological research. Based on the detection of mobile fluorescent molecules diffusing in and out of a diffraction-limited laser focus, the method allows to resolve particle dynamics within cells and their compartments. Previous FCS studies have described various parameters of protein function, namely mobility, transport and localization phenomena, enzymatic turnovers of biochemical substrates and molecular association and dissociation reactions. Recent progress in the application of FCS to intracellular systems has particularly taken advantage of detecting autofluorescent proteins and their genetically encoded fusions to cellular proteins. This review discusses recent applications of FCS analysis with and on fluorescent proteins, particularly highlighting chemical and physical properties. Inherent limitations of the presented approaches are discussed in detail and strategies for optimisation of experimental systems outlined.

Luminescent Proteins↗

PubMed2005

Wavelength- and time-dependence of potentiometric non-linear optical signals from styryl dyes.

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is "resonance-enhanced" SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.

Fiber Optic Technology↗

PubMed2003

Fluorescence imaging with two-photon evanescent wave excitation.

We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.

Animals↗

PubMed2004

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells.

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

Animals↗

PubMed2006

Quantitative detection of the ligand-dependent interaction between the androgen receptor and the co-activator, Tif2, in live cells using two color, two photon fluorescence cross-correlation spectroscopy.

Two-photon, two-color fluorescence cross-correlation spectroscopy (TPTCFCCS) was used to directly detect ligand-dependent interaction between an eCFP-fusion of the androgen receptor (eCFP-AR) and an eYFP fusion of the nuclear receptor co-activator, Tif2 (eYFP-Tif2) in live cells. As expected, these two proteins were co-localized in the nucleus in the presence of ligand. Analysis of the cross-correlation amplitude revealed that AR was on average 81% bound to Tif2 in the presence of agonist, whereas the fractional complex formation decreased to 56% in the presence of antagonist. Residual AR-Tif2 interaction in presence of antagonist is likely mediated by its ligand-independent activation function. These studies demonstrate that using TPTCFCCS it is possible to quantify ligand-dependent interaction of nuclear receptors with co-regulator partners in live cells, making possible a vast array of structure-function studies for these important transcriptional regulators.

Animals↗

PubMed2005

[Two-photon fluorescence of ocular melanomas. Studies on a new diagnostic method].

Choroidal melanoma is the most frequent form of primary neoplasia among malignant ocular tumors. Since it is presumed that metastasis often occurs before the primary tumor is first diagnosed, early detection is exigent. The aim of the studies described in this report was to develop an objective, noninvasive method for the diagnosis of choroidal melanoma. The underlying new principle of fluorescence excitation is presented. This is based on the observation that melanin, due to its unique absorption characteristics, is selectively excited into fluorescence via stepwise absorption of two photons of a femtosecond laser emitting at 800 nm. In the experiment described, the fluorescence of excised tissue from healthy choroidal pigment epithelium was compared to that of excised choroidal melanoma. The fluorescence of choroidal melanomas exhibited a more reddish appearance and less intensity than that of healthy tissue. This implies that the configuration of melanin apparently changes during the process of malignant degeneration. The method described here could thus serve as an evidentiary objective diagnostic technique before initiating treatment for choroidal melanomas.

Animals↗

PubMed2004

Concurrent measurements of the free cytosolic concentrations of H+ and Na+ ions with fluorescent indicators.

We report a method for the concurrent measurement of intracellular [Na+] ([Na+ ]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower p Ka in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.

Animals↗