Lily H. Laiho

Mechanotransduction through growth-factor shedding into the extracellular space.

Physical forces elicit biochemical signalling in a diverse array of cells, tissues and organisms, helping to govern fundamental biological processes. Several hypotheses have been advanced that link physical forces to intracellular signalling pathways, but in many cases the molecular mechanisms of mechanotransduction remain elusive. Here we find that compressive stress shrinks the lateral intercellular space surrounding epithelial cells, and triggers cellular signalling via autocrine binding of epidermal growth factor family ligands to the epidermal growth factor receptor. Mathematical analysis predicts that constant rate shedding of autocrine ligands into a collapsing lateral intercellular space leads to increased local ligand concentrations that are sufficient to account for the observed receptor signalling; direct experimental comparison of signalling stimulated by compressive stress versus exogenous soluble ligand supports this prediction. These findings establish a mechanism by which mechanotransduction arises from an autocrine ligand–receptor circuit operating in a dynamically regulated extracellular volume, not requiring induction of force-dependent biochemical processes within the cell or cell membrane.

Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.

Interferometric second harmonic generation microscopy

We describe a novel second harmonic generation (SHG) microscope that employs heterodyne detection by interfering the epi directed SHG from a sample with SHG from a reference crystal. In addition, the microscope provides complementary reflectance information based on optical coherence microscopy (OCM). The instrument features dual balanced detection to minimize the effect of source fluctuations, and polarization-sensitive detection to measure the nonlinear susceptibility of the sample. Interferometric detection can potentially improve the sensitivity and thus extend the imaging depth as compared with direct detection of SHG.

Deconvolution of Skin Images with Multivariate Curve Resolution

Two-photon fluorescence microscopy has been demonstrated as a high-resolution 3-D tissue imaging technique. If the dream of a noninvasive optical biopsy is to be realized upon this technology, biochemically relevant information must be extracted from the data thus obtained. The current study investigated the ability of different Gram-Schmidt modifications to orthogonal projection multivariate curve resolution (MCR) to find the emission spectra and distributions of fluorophores in volume images. A two-photon microscope, incorporating a high-power femtosecond Ti:Sapphire laser and a high sensitivity multianode photomultiplier spectrally resolved detector, was used to obtain spectroscopic data for each point in a volume of the sample. The MCR algorithm was developed and tested by first examining fluorescent beads. MCR analysis on images obtained from ex vivo human skin provided recognizable structural resolution with no a priori information, suggesting that the resolved spectra correspond to different endogenous fluorophores.

Tissue imaging based on two-photon autofluorescence and second harmonic generation

While autofluorescence is observed in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. Image guided spectral analysis is being developed to aid in extracting spectroscopic components from two-photon images. This methodology is being applied to the study of human skin. In ex vivo specimens, the overall bulk emission spectrum of the skin and the layer-resolved emission spectra of the stratum corneum, stratum spinosum, basal layer, and dermis have been measured. From the image guided spectral analysis, it was determined that there are approximately five factors that contribute to most of the luminescence signals from human skin. The autofluorescent species identified include tryptophan, NAD(P)H, melanin (or localizing species), and elastin. The collagen matrix produced a significant second harmonic signal. Because of the coherent nature of second harmonic emission, we are exploring the use of phase sensitive detection techniques to extend the imaging depth for collagen distribution.

Spectrally resolved and interferometric detection in second harmonic generation microscopy

We demonstrate two imaging systems for spectrally resolved and heterodyne detection in SHG microscopy of biological media. Coherent detection is achieved by mixing the SHG from a biological sample with SHG from a reference crystal.