Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source

Abstract

Abstract.A 12-fs broadband ͑100-nm͒ source is used to combine multiphoton microscopy ͑MPM͒ and optical coherence tomography ͑OCT͒ in a single platform. An ultrafast Ti:sapphire laser simultaneously provides short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. Using 0.3-m microspheres and a 63ϫ, 0.95 numerical aperture objective, we demonstrate that MPM and OCT channels are coregistered with lateral resolution of approximately 0.5 m and axial resolution of approximately 1.5 m. Preliminary studies of a 3-D organotypic epithelial tissue model show that multiphoton images of fluorescence and second harmonic signals are derived from cellular and extracellular matrix structures, respectively, while OCT images are generated from scattering interfaces due to tissue variations in refractive index. The combined MPM/OCT microscope is capable of providing simultaneous functional and structural information from cells and extracellular matrix and is potentially a powerful tool for studying biological processes in thick tissues. High-resolution imaging techniques derived from complementary signals can be combined to provide 3-D structural and functional information about biological tissues. A critical technical challenge for these "multimodality" imaging methods is to ensure simultaneous, coregistered information content from each image voxel. For example, optical coherence tomography ͑OCT͒ and confocal fluorescence microscopy ͑CM͒ have been integrated into a single platform 1 using both coherence and pinhole gating to achieve image coregistration. Recently, conventional OCT and second-harmonic OCT have been combined together to study reflectance and nonlinear properties of samples using coherence gating strategies. 2Multiphoton microscopy ͑MPM͒ 3 and OCT 4 are capable of noninvasive, high-resolution imaging in thick, scattering biological tissues. Two major signals detected in MPM are twophoton excited fluorescence ͑TPEF͒ and second-harmonic generation ͑SHG͒. TPEF can be detected from intrinsic sources ͑e.g., cofactors, proteins͒ and exogenous fluorophores, while strong SHG signals can be obtained from noncentrosymmetric molecules such as collagen, a common structural protein. OCT detects backscattered light from refractive index discontinuities that occur between tissues of different structure or composition. Using two separate MPM and OCT systems, our previous research on wound healing and oral cancer progression has demonstrated that MPM and OCT can provide complementary structural and functional information. 5,6 However, this study was performed on two separate systems with different sampling scales so that coregistration between the MPM and OCT images was difficult.A combined MPM/OCT system has been previously reported by Beaurepaire et al.7 using an 80-fs Ti:sapphire laser. Because the coherence length of their source was 15 m, the intrinsic OCT axial resolution was not matched with the MPM channel. In order to improve the resolution of the OCT channel, a pinhole ...

Optical coherence tomography today: speed, contrast, and multimodality

“…Ex vivo ocular tissues and a rat retina in vivo were successfully imaged. A single 12 fs broadband (100 nm) source (Femtolasers) was used to combine threechannel MPM and OCT in a single platform by Tang et al 195 The ultrafast Ti:sapphire laser was dispersion compensated to simultaneously provide short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. MPM and OCT channels were coregistered with a lateral resolution of ∼0.5 μm and an axial resolution of ∼1.5 μm, showing the potential of providing simultaneous functional and structural information from cells and an extracellular matrix with a 3-D organotypic epithelial tissue model. SHG revealed information about the cellular and extracellular collagen matrix, the autofluorescence signal added information from the cell body of fibroblasts and OCT visualized the internal structure.…”

Imaging subcellular scattering contrast by using combined optical coherence and multiphoton microscopy

“…Combined MPM-OCT can acquire coregistered two-photonexcited fluorescence (TPEF) and backscattered light simultaneously from the same sampling volume. [6][7][8] This allows the use of fluorescence targeted probes to identify molecular components of subcellular scattering structures. Details of the combined MPM-OCT system used in our experiment are described in Ref.…”

“…Details of the combined MPM-OCT system used in our experiment are described in Ref. 6. Briefly, a 12 fs Ti:sapphire laser (Femtolasers) excites MPM and OCT signals simultaneously.…”

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

“…Another implementation for multi-scale imaging with high transverse resolution is to perform imaging with high and low numerical aperture objectives sequentially [28]. Femtosecond laser sources that can be simultaneously used for both MPT and OCT measurements have already been reported [23]. However, further improvements in optical design, image acquisition speed and patient interface have to be achieved before performing successful in vivo clinical measurements using a combined MPT/OCT system.…”

“…Multimodal MPT/ OCM systems capable of obtaining MPT and OCM images simultaneously have been previously reported in cell cultures and tissue models, and have demonstrated that complimentary information can be obtained from biological tissues [22][23][24][25]. Significant improvements in terms of system design, light source, image resolution, acquisition speed and postprocessing algorithm have since been achieved [26,27].…”