Omid Masihzadeh

Multiphoton Microscopy for Ophthalmic Imaging

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

Label-free second harmonic generation holographic microscopy of biological specimens.

Second-order nonlinear holographic microscopy for high-speed, three-dimensional imaging is demonstrated. The use of harmonic generation allows image formation of endogenous features in biological samples such as muscle tissue. We have acquired holograms with acquisition times as short as 10 ms, limited by the switching speed of our shutter; frame rates of 100s of Hz are expected to be possible. The samples are imaged with a Yb:KGW femtosecond laser oscillator, whose 1027 nm wavelength is well suited to minimize absorption and scattering. The low average power of the oscillator prevents damage to the sample.

Simultaneous spatial and temporal focusing for tissue ablation

Simultaneous spatial and temporal focusing (SSTF) of femtosecond pulses was originally conceived as a novel method for increasing the field-of-view in multiphoton imaging applications. Multiphoton imaging with SSTF deviated from traditional nonlinear systems in that it enabled the use of low numerical aperture beams to be used to increase the field-of-view, but retain the axial sectioning of a high numerical aperture beam. In this manner efficiency gains in the imaging process were achieved without compromising axial resolution [1,2].

Complete polarization state control of ultrafast laser pulses with a single linear spatial light modulator.

Shaping of the phase, amplitude, and polarization state of an ultrashort pulse is demonstrated using a novel arrangement of a single, linear, high-resolution liquid crystal array. Orthogonal polarization components, separated by a Wollaston prism, are manipulated independently and re-combined in a near-common path, common-optic geometry.

Enhanced spatial resolution in third-harmonic microscopy through polarization switching

Enhanced spatial resolution in third-harmonic generation (THG) microscopy is demonstrated through manipulation of the polarization state across the focal field of a microscope. Enhancements in resolution of up to a factor of 2 are observed for a focal field linearly polarized at the center and switched to circularly polarized at the beam edges. As THG scattering is suppressed for circular polarization, the THG signal diameter is reduced, improving spatial resolution.

Tomographic retrieval of the polarization state of an ultrafast laser pulse

We introduce a self-referenced method for determining the complete polarization state of an ultrafast pulse field. The algorithm is based on any well-established technique that measures both the intensity and phase of a single polarization, such as frequency-resolved optical gating (FROG). We demonstrate the retrieval of nontrivial fields generated using a polarization-amplitude-phase ultrafast pulse shaper using four standard FROG measurements.

Direct trabecular meshwork imaging in porcine eyes through multiphoton gonioscopy

Abstract. The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy: A Novel Technique for Imaging the Retina

PURPOSE To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. METHODS Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. RESULTS The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CONCLUSIONS CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

Control and measurement of spatially inhomogeneous polarization distributions in third-harmonic generation microscopy

We demonstrate in situ characterization of a spatially varying polarization state of an optical field at the focus of a scanning third-harmonic generation (THG) optical microscope. Polarization projections are measured by forming THG images of a polystyrene microsphere scanned through the focused beam and combined in a noniterative phase-retrieval algorithm. Controlled spatially varying polarization states are introduced by imaging spatially inhomogeneous polarization distributions constructed with reflective spatial light modulator to the focal plane of a microscope objective.

Imaging the intact mouse cornea using coherent anti-stokes Raman scattering (CARS).

PURPOSE The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon-hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents. METHODS Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed. RESULTS Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers. CONCLUSIONS The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques.