Biomedical subjects

Wen Lo

Publications and source records attributed to Wen Lo.

13 recordsLinked to original sources

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

Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin.

In this work, we compared the performance of objectives with similar numerical aperture of 0.75 but different immersion media of air, water, glycerin, and oil in the imaging of human skin epithelium and dermis. In general, we found that the oil immersion objective recorded the strongest intensity at the same mechanical depth. We also characterized the focal shifts and found that with decreasing refractive index, the focal shift becomes increasingly more negative (for both the epithelium and dermis). In imaging the dermis, we estimated the image resolution at the depths of 18.8 and 30.2 microm, and found that the image resolution were comparable at these depths under the four types of immersion conditions. Our results demonstrate that by changing the immersion media, the main microscopic imaging effects are the recorded axial intensities and the focal shifts. The effects on the image resolution are negligible.

Dermis↗

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↗

PubMed2006

Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo.

PURPOSE: To demonstrate the application of multiphoton fluorescence (MF) and second harmonic generation (SHG) microscopy for ex vivo characterization of the structural alterations of human corneas due to keratoconus. METHODS: Buttons of keratoconic corneas derived from penetrating keratoplasty were sent for structural analysis with a custom-built multiphoton microscope. Fluorescence detected within the cytoplasm and the SHG signal obtained from collagen were used to demonstrate the morphologic changes in the corneal specimens. RESULTS: The fluorescent epithelial cells around the apical area were elongated and were aligned parallel to the adjacent collagen fibers. Parallel and centripetal distribution patterns of stromal collagen bundles were demonstrated at different depths within the keratoconic corneas. CONCLUSIONS: MF and SHG microscopy provides three-dimensional structural analysis of keratoconus ex vivo. It may provide important morphologic information for the investigation of the pathogenesis of keratoconus and may have potential in a clinical setting as an in vivo diagnostic and monitoring system for advancing keratoconus.

Cornea↗

PubMed2005

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy.

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.

Adult↗

PubMed2005

Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy.

The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25 degrees-60 degrees C. We find that the SHG signal decreases rapidly starting at 45 degrees C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57 degrees C and worsens with increasing temperature. At 57 degrees C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60 degrees C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.

Animals↗

PubMed2004

Optical biopsy of liver fibrosis by use of multiphoton microscopy.

We demonstrate the application of multiphoton microscopy in diagnosing toxin- (CCl4-) induced liver fibrosis in mice. Although hepatocyte autofluorescence does not vary significantly, different degrees of necrosis and stellate cell proliferation at necrotic sites in livers with fibrosis (ex vivo) can be detected easily from multiphoton-induced autofluorescence images by use of 780-nm excitation. Our result suggests that multiphoton microscopy can be developed into an effective technique for the detection and diagnosis of liver fibrosis in vivo.

Animals↗

PubMed2004

Multiphoton polarization and generalized polarization microscopy reveal oleic-acid-induced structural changes in intercellular lipid layers of the skin.

We have demonstrated that both multiphoton polarization and generalized polarization (GP) microscopy may be combined to characterize the structural changes of intercellular lipids in skin. Both polarization and GP (at 440- and 490-nm emission) images obtained by analysis of Laurdan fluorescence suggest that the treatment of oleic acid results in a skin surface with a more random packing of lipid molecules, which allows easier water penetration. Our results show that combined polarization and GP microscopy can be used to characterize the physical and chemical changes in biological structures.

Culture Techniques↗

PubMed2004

Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy.

Multiphoton microscopy is a powerful technique for achieving three-dimensional submicron imaging in biological specimens. However, specimen optical parameters such as refractive indices and scattering coefficients can result in the loss of image resolution and decreased signal in depth. These factors are coupled to the focusing objective's numerical aperture (NA) in limiting the achievable imaging depths. In this work, we performed multiphoton imaging on aqueous fluorescent solution, human skin, and rat tail tendon to show that, under the same immersion condition, lower NA objectives can examine more deeply into biological specimens and should be used when optimal imaging depths is desired.

Animals↗

PubMedDate not supplied

Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar.

Multiphoton microscopy has evolved into a powerful bioimaging tool in three dimensions. However, the ability to image biological specimens in-depth can be hindered by sample spherical aberration and scattering. These two phenomena can result in the degradation of image resolution and the loss of detected multiphoton signal. In this work, we use the correction collar (for cover glass thickness) associated with a water immersion objective in an attempt to improve multiphoton imaging. In the two samples we examined (human skin and rat tail tendon), we found that while the improvement in image resolution was not visible qualitatively, the measured axial fluorescence or second harmonic generation signal profiles indicate that the use of the correction collar can help to improve the detected multiphoton signals. The maximum increases are 36% and 57% for the skin (sulforhodamine B fluorescence) and tendon (second harmonic generation) specimens, respectively. Our result shows that for in-depth multiphoton imaging, the correction collar may be used to correct for spherical aberration. However, each tissue type needs to be examined to determine the optimal correction collar setting to be used.

Animals↗

PubMedDate not supplied

Characterizing the thermally induced structural changes to intact porcine eye, part 1: second harmonic generation imaging of cornea stroma.

We characterize the structural changes of porcine corneal structures from 25 to 90 degrees C using second harmonic generation (SHG) microscopy. Our results show that porcine stroma undergoes several distinct stages of structural changes between 25 and 90 degrees C. A decrease in SHG intensity from 30 to 45 degrees C and the existence of SHG intensity peaks at 53, 65, and 77 degrees C correlate to distinct structural alterations of the corneal stroma. At higher temperatures, the SHG intensity decreases and a baseline in SHG signal is reached at 90 degrees C. Our results demonstrate that SHG microscopy is a useful technique for obtaining qualitative and quantitative information of thermally treated corneal fibers without histological or labeling procedures. With additional developments, SHG imaging may be developed into an effective imaging technique for in vivo characterization of cornea structural changes.

Animals↗

PubMedDate not supplied

Prediction of heat-induced collagen shrinkage by use of second harmonic generation microscopy.

Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 degrees C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.

Animals↗

PubMedDate not supplied

Evaluation of dermal thermal damage by multiphoton autofluorescence and second-harmonic-generation microscopy.

We attempt to characterize the degree of skin thermal damage by using multiphoton microscopy to characterize dermal thermal damage. Our results show that dermal collagen and elastic fibers display different susceptibility to thermal injury. Morphologically, dermal collagen starts to denature at 60 degrees C while fracture and aggregation of elastic fibers do not occur until 65 degrees C. With increasing temperatures, the structures of both elastic and collagen fibers deteriorate. While second-harmonic-generation (SHG) imaging is helpful in identifying the denaturation temperature of collagen, autofluorescence (AF) imaging can help to identify the structural alternations of tissue at higher temperatures when SHG signals have decayed. We also employ a ratiometric approach based on the AF-to-SHG index of dermis (ASID) to characterize the degree of dermal thermal damage. Use of the ASID index can bypass the difficulty in analyzing inhomogeneous dermal fibers and show that dermal collagen starts to denature at 60 degrees C. Our results suggest that with additional developments, multiphoton microscopy has potential to be developed into an effective in vivo imaging technique to monitor and characterize dermal thermal damage.

Burns↗