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Biomedical subjects

Ki Hean Kim

Publications and source records attributed to Ki Hean Kim.

7 recordsLinked to original sources
PubMed2006

Frequency domain lifetime and spectral imaging microscopy.

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.

Journal Article↗
PubMed2006

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging.

The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435-700 nm) and second harmonic generation (SHG, 360-400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea.

Animals↗
PubMed2006

Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.

PURPOSE: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes. METHODS: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye. RESULTS: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes. CONCLUSIONS: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

Animals↗
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↗
PubMed2003

Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy.

In a novel application of dual-channel high-speed two-photon fluorescence microscopy, the skin autofluores-cence and the transdermal fluorescent model drug spatial distributions were imaged simultaneously over precisely the same spatial coordinates. The dual channels enable the detection of the fluorescence emission wavelengths characteristic of the endogenous (intrinsic) skin fluorophores, as well as of the rhodamine-based model drug intensity emission at a different wavelength range of the fluorescence emission spectrum. These fluorescent model drugs delineate the oleic acid induced changes in permeant diffusion with respect to the skin structural features over the 0.3 mm by 0.3 mm skin area imaged per skin sample. The dual-channel high-speed two-photon fluorescence microscopy studies presented here provide evidence for the existence of intracorneocyte diffusion in addition to the commonly cited lipid multilamellar transdermal pathway. The image quantification analysis methodology introduced in this paper reveals that intracorneocyte diffusion exists for the hydrophobic (rhodamine B hexyl ester) and for the hydrophilic (sulforhodamine B) model drugs, in the absence of oleic acid chemical enhancer action. The mechanism of oleic acid chemical enhancer action, however, depends on the model drug physicochemical properties, where the oleic acid induces hydrophobic model drug localization to the lipid multilamellar region, while increasing the hydrophilic model drug lipid to corneocyte partitioning.

Biological Transport↗
PubMed2002

Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: determination of representative skin sample sizes.

A novel application of high-speed two-photon microscopy was utilized to determine the optimum number of skin sites required to accurately determine the changes in transdermal transport properties incurred globally, over a clinically relevant area of skin. In contrast to the four to six skin sites (100 microm by 100 mirom area per site) examined previously, this study accounted for the fluorescent probe distributions at 400 consecutive skin sites, covering a total skin area of 2 mm by 2 mm. The oleic-acid-induced changes in the transdermal transport properties of the model hydrophobic probe, rhodamine B hexyl ester, and of the model hydrophilic probe, sulforhodamine B, for this 400-skin-site study exhibited different dependencies on sample size for each probe. Whereas the examination of six skin sites captures the relative changes in the global transdermal transport properties of the hydrophobic probe, the valid assessment of these changes for the hydrophilic probe requires a significantly larger sample size of at least 24 skin sites.

Biological Transport↗