[Show abstract][Hide abstract] ABSTRACT: Multi-scale multimodal microscopy is a very useful technique by providing multiple imaging contrasts with adjustable field of views and spatial resolutions. Here, we present a tri-modal microscope combining multiphoton microscopy (MPM), optical coherence microscopy (OCM) and optical coherence tomography (OCT) for subsurface visualization of biological tissues. The advantages of the tri-modal system are demonstrated on various biological samples. It enables the visualization of multiple intrinsic contrasts including scattering, two-photon excitation fluorescence (TPEF), and second harmonic generation (SHG). It also enables a rapid scanning over a large tissue area and a high resolution zoom-in for cellular-level structures on regions of interest. The tri-modal microscope can be important for label-free imaging to obtain a sufficient set of parameters for reliable sample analysis.
[Show abstract][Hide abstract] ABSTRACT: Multimodal MPM/OCT is developed for tissue/cellular imaging with femtosecond laser. Cross-sectional OCT and en face MPM imaging are achieved with one platform. Imaging is demonstrated on cornea and its refractive index and thickness are characterized.
[Show abstract][Hide abstract] ABSTRACT: Refractive index (RI) is the optical property of a medium that describes
its ability to bend incident light. The corneal refractive index is an
especially important measurement in corneal and intraocular refractive
surgery where its precise estimation is necessary to obtain accurate
surgical outcomes. In this study, we calculated the corneal RI using a
combined multiphoton microscopy (MPM) and optical coherence tomography
(OCT) system. MPM excites and detects nonlinear signals including two
photon excitation fluorescence (TPEF) and second harmonic generation
(SHG). TPEF signals are observed from NADH in the cytoplasm, allowing
MPM to image the cellular structures in the corneal epithelium and
endothelium. SHG signals are observed from collagen, an abundant
connective tissue found in the stroma. Optical coherence tomography
(OCT) produces cross-sectional, structural images based on the
interference fringes created by the reflected light from the sample and
reference arms. Our system uses a single sub-10 fs Ti: sapphire laser
source which is good for both MPM excitation and OCT resolution. The MPM
and OCT images are coregistered when they are taken successively because
their axial resolutions are similar and the system shares the laser
source and the scanning unit. We can calculate the RI by measuring the
optical thickness and the optical path length of the cornea from the MPM
and OCT images respectively. We have imaged and calculated the RI of
murine and piscine corneas. We were able to see the epithelial, stromal,
and endothelial layers and compare their relative thicknesses and the
organization of the stromal collagen lamellae. Our results showed that
our system can provide both functional and structural information about
the cornea and measure the RI of multi-layered tissues.
Proceedings of SPIE - The International Society for Optical Engineering 02/2013; DOI:10.1117/12.2005483 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a noninvasive method for characterizing the refractive index (RI) and thickness distribution in biological tissues using a combined multiphoton microscopy (MPM) and optical coherence tomography (OCT) system. Tissue layers are distinguished by the MPM and OCT images, and the RI and thickness of each layer are determined by analyzing the co-registered MPM and OCT images. The precision of this method is evaluated on four standard samples which are water, air, immersion oil and cover glass. Precision of within ~1% error compared to reference values is obtained. Biological tissue measurement is demonstrated on fish cornea. Three layers are detected, which are identified as the epithelium and stroma I and II of the cornea. The corresponding RI of each layer is measured to be ~1.446-1.448, 1.345-1.372, and 1.392-1.436, respectively. The difference of RI in the three layers correlates with the tissue compositions including cells in epithelium, large collagen fiber bundles in stroma I, and small collagen fibers in stroma II. The combined MPM/OCT technique is shown to be able to distinguish tissue layers through biochemically specific contrasts and measure RI and thickness of tissue layers at different depths.
[Show abstract][Hide abstract] ABSTRACT: Two types of combined multiphoton microscopy and optical coherence tomography (MPM/OCT) are compared for multimodal optical imaging. Single-scale multiphoton microscopy and optical coherence microscopy (MPM/OCM) is shown to acquire multiple contrasts from MPM and OCT simultaneously. Multi-scale MPM/OCT is shown to provide multiple field-of-views (FOVs), where OCT provides tissue level imaging and MPM provides cellular level imaging. In both types, the MPM includes two channels which are two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Representative images using each system are demonstrated on biological specimens. A detailed comparison of the two types of MPM/OCT shows that each system has its own pros and cons. MPM/OCM is high-resolution but with limited FOV, and OCM may or may not provide additional information than MPM depending on the samples. Multi-scale MPM/OCT can change FOV but need both low and high NA objectives. For future development, the two types of MPM/OCT can be further integrated to achieve both functions on a single system.
Journal of Biophotonics 05/2012; 5(5-6):396-403. DOI:10.1002/jbio.201100138 · 3.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper presents tissue imaging with a multimodal multiphoton microscopy (MPM) and optical coherence tomography (OCT) system. The multimodal system can acquire multiple contrasts including two-photon excited fluorescence, second harmonic generation, and scattering. The system can also acquire tissue level imaging with OCT and cellular level imaging with MPM. The MPM/OCT imaging is demonstrated on onion skin and cornea samples. The multimodal MPM/OCT provides a multi-contrast and multi-field-of-view imaging system which has potential applications in cancer detection and diagnosis.
Photonics and Optoelectronics (SOPO), 2012 Symposium on; 01/2012
[Show abstract][Hide abstract] ABSTRACT: A multiscale multiphoton microscopy (MPM) and optical coherence tomography (OCT) system has been developed using a sub-10 fs Ti:sapphire laser. The system performs cross-sectional OCT imaging over millimeter field-of-view and en-face high-resolution MPM imaging with submicrometer resolution from the same sample location. With fish cornea, we have demonstrated cross-sectional imaging of cornea tissue layers using OCT, and the zoom-in imaging of cells and collagen fibers in each layer using MPM. The multiscale MPM/OCT system shows the potential of a rapid coarse scan to search for abnormal regions and the subsequent fine zoom-in imaging for diagnosis.