Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Abstract

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.

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

“…In our images, the NAD(P)H autofluorescence [23] was very low in comparison to that arising from fibers, and not resolvable from the background.…”

“…This microscopy does not require sample treatment such as freezing-thawing, inclusion in matrices, fixation or staining [20 -22], which may produce artifacts, e.g., contraction produced by fixation, which dehydrates the sample. By its deeper penetration in highly scattering tissues vs. light of shorter wavelengths, the near-infrared excitation allows imaging of interior regions of intact tissues, with a penetration of about 200 m [23]. Typical features of two-photon excitation microscopy include the simultaneous absorption of two near-infrared photons with a total energy equivalent to one photon, at half of the wavelength.…”

“…Therefore, absorption in the ultraviolet region can be achieved by near-infrared excitation with the advantage of strongly reducing the sample photodamage and photobleaching, usually associated with UV excitation. In the absence of exogenous fluorophores, a variety of autofluorescence signals can be observed [18,23], arising from molecules absorbing in the UV range (onephoton absorption). We obtained for the first time highresolution images of the intense autofluorescence arising from extracellular matrix proteins of a fresh rat aorta.…”

In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy

“…Nonlinear susceptibility is an optical property characteristic of chiral molecules. The microcrystalline structure of collagen [21][22][23][24] makes it capable of SHG [19,20]. Several researchers have reported endogenous second harmonic generation in biological tissues.…”

“…Many wavelengths can be used; visualization/analysis of deeper subsurface structures such as components of the extracellular matrix, cellular and vascular elements is possible [22][23][24][25][26][27][28].…”