Silk fibroin is emerging as an important biomaterial for tissue engineering applications. The ability to monitor non-invasively the structural conformation of silk matrices prior to and following cell seeding could provide important insights with regards to matrix remodeling and cell-matrix interactions that are critical for the functional development of silk-based engineered tissues. Thus, we examined the potential of intrinsic fluorescence as a tool for assessing the structural conformation of silk proteins. Specifically, we characterized the intrinsic fluorescence spectra of silk in solution, gel and scaffold configurations for excitation in the 250 to 335 nm range and emission from 265 to 600 nm. We have identified spectral components that are attributed to tyrosine, tryptophan and crosslinks based on their excitation-emission profiles. We have discovered significant spectral shifts in the emission profiles and relative contributions of these components among the silk solution, gel and scaffold samples that represent enhancements in the levels of crosslinking, hydrophobic and intermolecular interactions that are consistent with an increase in the levels of ss-sheet formation and stacking. This information can be easily utilized for the development of simple, non-invasive, ratiometric methods to assess and monitor the structural conformation of silk in engineered tissues.

The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a) excite and detect fluorescence at two distinct colors simultaneously, and b) perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein (GFP)-labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies. Keywordsin vivo cytometry; confocal optics; circulating cancer cells; circulating stem cells; fluorescence; cancer; flow cytometry; confocal detection Standard flow cytometry is currently the method of choice for rapid quantification of cells, but it can only be performed in vitro. 1 Analysis of circulating cells in the bloodstream by this method has generally been performed after withdrawal of blood samples, but this method is not effective for studies on rapidly changing cell populations, since blood samples cannot be drawn often enough. Confocal microscopy and two-photon imaging techniques have been able to detect fluorescently labeled cells in vivo, but the equipment required for these techniques is prohibitively expensive when used for video rate image capture, and the velocity of blood flow makes the quantification of cells extremely difficult. 2 In 2004, Novak et al. described the first in vivo flow cytometer (IVFC), which was capable of real-time confocal detection of fluorescently labeled cells in both the arterial and venous circulation of small animals. 3 This instrument has been used to study the circulation kinetics of prostate cancer and leukemia cells labeled with a single chromophore either before or after intravenous injection into mice or rats.4 -6 A lab-bench design for two-color in vivo flow cytometry has also been described. 7 Here, we introduce a portable version of the two-color in vivo flow cytometer with additional hardware and software improvements that enable real-time cell counting and enhance the range of fluorophores that can be detected, as well as the specificity with which cell populations can be monitored.Specifically, in the portable, two-color IVFC system, excitation is provided by the 633-nm line of a HeNe laser and the 473-nm line of a diode-pumped solid state (DPSS) laser. Incorporation of the blue DPSS laser allows fluorescence detection of additional common chromophores such as fluorescein isothiocyanate (FITC) and phyco-erythrin (PE), as well as green fluorescent protein (GFP). Simultaneous detection of two chromophores is possible and relevant for enhancing the specificity of detection. Portability allows flexibility in the location ...

Cellular transformation is associated with a number of phenotypic, cell biological, biochemical and metabolic alterations. The detection and classification of morphological cellular abnormalities represents the foundation of classical histopathology and remains an important mainstay in the clinic. More recently, significant effort is being expended towards the development of noninvasive modalities for the detection of cancer at an early stage, when therapeutic interventions are highly successful. Methods that rely on the detection of optical signatures represent one class of such approaches that have yielded promising results. In our study, we have applied two spectroscopic imaging approaches to systematically identify in a quantitative manner the fluorescence and light scattering signatures of subcellular abnormalities that are associated with cellular transformation. Notably, we find that tryptophan images reveal not only intensity but also localization differences between normal and human papillomavirus immortalized cells, possibly originating from changes in the expression, 3D packing and organization of proteins and protein-rich subcellular organelles. Additionally, we detect alterations in cellular metabolism through quantitative evaluation of the NADH, FAD fluorescence and the corresponding redox ratio. Finally, we use light scattering spectroscopy to identify differences in nuclear morphology and subcellular organization that occur from the nanometer to the micrometer scale. Thus, these optical approaches provide complementary biomarkers based on endogenous fluorescence and scattering cellular changes that occur at the molecular, biochemical and morphological level. Since they obviate the need for staining and tissue removal and can be easily combined, they provide desirable options for further clinical development and assessment. ' 2007 Wiley-Liss, Inc.Key words: cancer biomarkers; noninvasive imaging; cancer diagnosis; spectroscopy; fluorescence; light scattering; HPV; cervical cancer Clinical cancer detection has relied heavily on the use of simple stains to visualize and classify morphological cellular abnormalities. The development of more sophisticated probes to evaluate specific oncogenic events continues to revolutionize early detection of various cancers. Nevertheless, a simple morphological evaluation of exfoliated cervical epithelial cells, the Papanicolaou (''Pap'') smear has been exceedingly useful for detection of precancerous cervical lesions and may have saved more lives than any other commonly used cancer screening procedure. Many of the more sophisticated diagnostic tests involve physical removal of suspect tissue or other invasive or physically unpleasant procedures. Hence, the development of rapid, noninvasive methods is of paramount importance for successful early detection. Relatively simple spectroscopic procedures are available to measure alterations in a number of cellular parameters and may be well suited for this purpose.Indeed, optical spectroscopy and imaging techniques exhib...

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We have designed and implemented a dual-mode adaptive optics (AO) imaging system that combines spectral domain optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) for in vivo imaging of the human retina. The system simultaneously acquires SLO frames and OCT B-scans at 60 Hz with an OCT volume acquisition time of 4.2 s. Transverse eye motion measured from the SLO is used to register the OCT B-scans to generate three-dimensional (3-D) volumes. Key optical design considerations include: minimizing system aberrations through the use of off-axis relay telescopes, conjugate pupil plane requirements, and the use of dichroic beam splitters to separate and recombine the OCT and SLO beams around the nonshared horizontal scanning mirrors. To demonstrate system performance, AO-OCT-SLO images and measurements are taken from three normal human subjects ranging in retinal eccentricity from the fovea out to 15-deg temporal and 20-deg superior. Also presented are en face OCT projections generated from the registered 3-D volumes. The ability to acquire high-resolution 3-D images of the human retina in the midperiphery and beyond has clinical importance in diseases, such as retinitis pigmentosa and cone-rod dystrophy.