Depth-Encoded Spectral Domain Phase Microscopy for Simultaneous Multi-Site Nanoscale Optical Measurements.

Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion.

Correlation of pathologic features in spectral domain optical coherence tomography with conventional retinal studies.

PURPOSE: To delineate pathologic changes in retinal cross sections obtained with spectral (Fourier) domain optical coherence tomography (SDOCT), so that the findings are maintained when collapsed into a two-dimensional fundus image for comparison with conventional retinal studies. METHODS: SDOCT of the posterior pole of 12 eyes (5 with neovascular age-related macular degeneration [AMD]; 7 with nonneovascular AMD) produced three-dimensional stacks of scans. Location of pathologic features was delineated with color markings in each scan before the stack was collapsed along the depth axis. This en face image contained retinal vessel shadowing and preserved color markings of delineated pathologic features relative to the vessel pattern and was superimposed onto conventional studies. RESULTS: For patients with neovascular AMD, location and extent of choroidal neovascularization, macular edema, and subretinal fluid were visible on the two-dimensional summed images and, in some cases, involved sites not suspected with conventional imaging. For patients with nonneovascular AMD, the location of drusen and geographic atrophy were correlated with autofluorescence images. For one eye with drusen and three eyes with neovascular AMD, presence or extent of subretinal fluid identified by SDOCT was not visible using other imaging methods. CONCLUSIONS: In this pilot AMD study, pathologic features within SDOCT scans were transferred into two-dimensional en face projections, enabling researchers to correlate lateral extent of pathologic features from SDOCT with conventional studies. This integration of SDOCT with other retinal studies is promising and will be useful to study the relationship between local OCT morphology and other parameters of retinal disease or function.

Retinal blood flow measurement by circumpapillary Fourier domain Doppler optical coherence tomography.

We present in vivo human total retinal blood flow measurements using Doppler Fourier domain optical coherence tomography (OCT). The scan pattern consisted of two concentric circles around the optic nerve head, transecting all retinal branch arteries and veins. The relative positions of each blood vessel in the two OCT conic cross sections were measured and used to determine the angle between the OCT beam and the vessel. The measured angle and the Doppler shift profile were used to compute blood flow in the blood vessel. The flows in the branch veins was summed to give the total retinal blood flow at one time point. Each measurement of total retinal blood flow was completed within 2 s and averaged. The total retinal venous flow was measured in one eye each of two volunteers. The results were 52.90+/-2.75 and 45.23+/-3.18 microlmin, respectively. Volumetric flow rate positively correlated with vessel diameter. This new technique may be useful in the diagnosis and treatment of optic nerve and retinal diseases that are associated with poor blood flow, such as glaucoma and diabetic retinopathy.

In vivo total retinal blood flow measurement by Fourier domain Doppler optical coherence tomography.

There is considerable interest in new methods for the assessment of retinal blood flow for the diagnosis of eye diseases. We present in vivo normal human volumetric retinal flow measurement using Fourier domain Doppler optical coherence tomography. We used a dual-plane scanning pattern to determine the angle between the blood flow and the scanning beam in order to measure total flow velocity. Volumetric flow in each blood vessel around the optic nerve head was integrated in one cardiac cycle in each measurement. Measurements were performed in the right eye of one human subject. The measured venous flow velocity ranged from 16.26 mm/s to 29.7 mm/s. The arterial flow velocity ranged from 38.35 mm/s to 51.13 mm/s. The total retinal venous and arterial flow both added up to approximately 54 microl/min. We believe this is the first demonstration of total retinal blood flow measurement using the OCT technique.

Real-time spectral domain Doppler optical coherence tomography and investigation of human retinal vessel autoregulation.

Investigation of the autoregulatory mechanism of human retinal perfusion is conducted with a real-time spectral domain Doppler optical coherence tomography (SDOCT) system. Volumetric, time-sequential, and Doppler flow imaging are performed in the inferior arcade region on normal healthy subjects breathing normal room air and 100% oxygen. The real-time Doppler SDOCT system displays fully processed, high-resolution [512 (axial) x 1000 (lateral) pixels] B scans at 17 frames/sec in volumetric and time-sequential imaging modes, and also displays fully processed overlaid color Doppler flow images comprising 512 (axial) x 500 (lateral) pixels at 6 frames/sec. Data acquired following 5 min of 100% oxygen inhalation is compared with that acquired 5 min postinhalation for four healthy subjects. The average vessel constriction across the population is -16+/-26% after oxygen inhalation with a dilation of 36+/-54% after a return to room air. The flow decreases by -6+/-20% in response to oxygen and in turn increases by 21+/-28% as flow returns to normal in response to room air. These trends are in agreement with those previously reported using laser Doppler velocimetry to study retinal vessel autoregulation. Doppler flow repeatability data are presented to address the high standard deviations in the measurements.

Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy.

We present a low-cost, high-speed, retrofitted laser scanning module for microscopy. The cage-mounted system, with various available fiber-coupled sources, offers a real-time imaging alternative to costly commercial systems with capabilities for conventional or confocal reflectance and fluorescence applications as well as advanced laser scanning microscopy implementations. Reflectance images of a resolution target and confocal images of fluorescent polystyrene beads are presented for system characterization. Confocal fluorescence image stacks of T84 epithelial cancer cells are presented to demonstrate application to biological studies. This laser scanning module is a flexible, scalable, high-speed alternative to commercial laser scanning systems suitable for applications requiring a simple imaging tool and for teaching laboratories.

Adaptive-optics optical coherence tomography for high-resolution and high-speed 3D retinal in vivo imaging.

We have combined Fourier-domain optical coherence tomography (FD-OCT) with a closed-loop adaptive optics (AO) system using a Hartmann-Shack wavefront sensor and a bimorph deformable mirror. The adaptive optics system measures and corrects the wavefront aberration of the human eye for improved lateral resolution (~4 µm) of retinal images, while maintaining the high axial resolution (~6 µm) of stand alone OCT. The AO-OCT instrument enables the three-dimensional (3D) visualization of different retinal structures in vivo with high 3D resolution (4×4×6 µm). Using this system, we have demonstrated the ability to image microscopic blood vessels and the cone photoreceptor mosaic.