OCT

The cellular response to environmental cues is complex, involving both structural and functional changes within the cell. Our understanding of this response is facilitated by microscopy techniques, but has been limited by our ability to image cell structure and function deep in highly-scattering tissues or 3D constructs. A novel multimodal microscopy technique that combines coherent and incoherent imaging for simultaneous visualization of structural and functional properties of cells and engineered tissues is demonstrated. This microscopic technique allows for the simultaneous acquisition of optical coherence microscopy and multiphoton microscopy data with particular emphasis for applications in cell biology and tissue engineering. The capability of this technique is shown using representative 3D cell and tissue engineering cultures consisting of primary fibroblasts from transgenic green fluorescent protein (GFP) mice and GFP-vinculin transfected fibroblasts. Imaging is performed following static and dynamic mechanically-stimulating culture conditions. The microscopy technique presented here reveals unique complementary data on the structure and function of cells and their adhesions and interactions with the surrounding microenvironment.

To achieve high-spectral-resolution multiplex coherent anti-Stokes Raman scattering (CARS), one typically uses a narrowband pump pulse and a broadband Stokes pulse. This is to ensure a correspondence between anti-Stokes and vibrational frequencies. We obtain high-resolution CARS spectra of isopropanol, using a broadband chirped pump pulse and a broadband Stokes pulse, by detecting the anti-Stokes pulse with spectral interferometry. With the temporally resolved anti-Stokes signal, we can remove the chirp of the anti-Stokes pulse and restore high spectral resolution while also rejecting nonresonant scattering. (c) 2006 Optical Society of America.

An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multiphoton microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the contemporary analysis of the spectroscopic features can enhance contrast by differentiating among different tissue components. (c) 2006 American Institute of Physics.

The spectroscopic content within optical coherence tomography (OCT) data can provide a wealth of information. Spectroscopic OCT methods are frequently limited by time-frequency trade-offs that limit high spectral and spatial resolution simultaneously. We present spectroscopic spectral-domain optical coherence microscopy performed with a multimodality microscope. Restricting the spatial extent of the signal by using high-numerical-aperture optics makes high-resolution spectroscopic information accessible, facilitated with spectral-domain detection. Simultaneous acquisition of multiphoton microscopy images is used to validate tissue structure and localization of nuclei within individual cells. (c) 2006 Optical Society of America.

Molecular contrast in optical coherence tomography (OCT) is demonstrated by use of coherent anti-Stokes Raman scattering (CARS) for molecular sensitivity. Femtosecond laser pulses are focused into a sample by use of a low-numerical-aperture lens to generate CARS photons, and the backreflected CARS signal is interferometrically measured. With the chemical selectivity provided by CARS and the advanced imaging capabilities of OCT, this technique may be useful for molecular contrast imaging in biological tissues. CARS can be generated and interferometrically measured over at least 600 mum of the depth of field of a low-numerical-aperture objective. (C) 2005 Optical Society of America.

For decades, Raman scattering has been a useful tool for probing the molecular contents of gases, liquids and biological media.1 Its strength is that it can identify molecules non-invasively based on their resonant vibrational frequencies. The technique uses optical frequency radiation, which is relatively easy to create, manipulate and detect. Unfortunately, the Raman effect is rather weak: Typically only one in a million photons is converted to the detected Raman radiation, so it is too slow to be used as a clinical diagnostic tool or to monitor fast biological processes. Using pulsed laser illumination, Coherent Anti-Stokes Raman Scattering (CARS) can stimulate the production of many more Raman photons by simultaneously exciting the molecular vibrations and detecting the emitted stimulated Raman scattering.

We present a new interferometric technique for measuring Coherent Anti-Stokes Raman Scattering ( CARS) and Second Harmonic Generation (SHG) signals. Heterodyne detection is employed to increase the sensitivity in both CARS and SHG signal detection, which can also be extended to different coherent processes. The exploitation of the mentioned optical nonlinearities for molecular contrast enhancement in Optical Coherence Tomography (OCT) is presented. (C) 2004 Optical Society of America.