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Combining different contrast mechanisms to achieve simultaneous multimodal imaging is always desirable but is challenging due to the various optical and hardware requirements for different imaging systems. We developed a multimodal microscopic optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissues. This imaging system integrated photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and confocal fluorescence microscopy in one platform. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing adjustment. We have conducted phantom, in vivo , and ex vivo tests to demonstrate the capability of the multimodal imaging system for providing comprehensive microscopic information of biological tissues. Integrating all the aforementioned imaging modalities with OCT-guided dynamic focusing for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the future., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

We developed a technology for quantitative retinal autofluorescence (AF, or FAF for fundus AF) imaging for quantifying lipofuscin in the retinal pigment epithelium (RPE). The technology is based on simultaneous visible light optical coherence tomography (VIS-OCT) and AF imaging of the retina and a pair of reference standard targets at the intermediate retinal imaging plane with known reflectivity for the OCT and fluorescence efficiency for the FAF. The technology is able to eliminate the pre-RPE attenuation in FAF imaging by using the simultaneously acquired VIS-OCT image. With the OCT and fluorescence images of the reference targets, the effects of illumination power and detector sensitivity can be eliminated., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

We developed a spectral-domain visible-light optical coherence tomography (VIS-OCT) based multimodal imaging technique which can accomplish simultaneous OCT and fluorescence imaging with a single broadband light source. Phantom experiments showed that by using the simultaneously acquired OCT images as a reference, the effect of light attenuation on the intensity of the fluorescent images by materials in front of the fluorescent target can be compensated. This capability of the multimodal imaging technique is of high importance for achieving quantification of the true intensities of autofluorescence (AF) imaging of the retina. We applied the technique in retinal imaging including AF imaging of the retinal pigment epithelium and fluorescein angiography (FA). We successfully demonstrated the effect of compensation on AF and FA images with the simultaneously acquired VIS-OCT images.

We developed an improved dual band dual focus spectral domain optical coherence tomography (SD-OCT) for in vivo 2D/3D imaging of the whole eye segment, including the whole anterior segment and retina. The system featured two OCT channels with two different bands centered at 840 nm and 1050 nm, which were designed to image the retina and the anterior segments of the eye, respectively. By combing the two probe light beams for co-axial scanning and separating them for focusing at different segments of the eye with a combination of three dichroic mirrors, we not only minimized the loss of the backscattered light from the sample but also improved the imaging depth, scan range and resolution. The full resolved complex (FRC) method was applied to double the imaging depth for the whole anterior segment imaging, with which an imaging depth of 36.71 mm in air was achieved. We demonstrated that this system was capable of measuring the dynamic changes of ocular dimensions, including the asphericity of the cornea and lens, during accommodation.

We developed an optical coherence photoacoustic microscopy (OC-PAM) system, which can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously by using a single pulsed broadband light source. With a center wavelength of 800 nm and a bandwidth of 30 nm, the system is suitable for imaging the retina. Generated from the same group of photons, the OCT and PAM images are intrinsically registered in the lateral directions. To test the capabilities of the system on multimodal ophthalmic imaging, we imaged the retina of pigmented rats. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of absorbers in the retina. Since the absorption of hemoglobin is relatively weak at around 800 nm, the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye, thus providing melanin-specific imaging of the retina.

We accomplished spectral domain optical coherence tomography and auto-fluorescence microscopy for imaging the retina with a single broadband light source centered at 480 nm. This technique is able to provide simultaneous structural imaging and lipofuscin molecular contrast of the retina. Since the two imaging modalities are provided by the same group of photons, their images are intrinsically registered. To test the capabilities of the technique we periodically imaged the retinas of the same rats for four weeks. The images successfully demonstrated lipofuscin accumulation in the retinal pigment epithelium with aging. The experimental results showed that the dual-modal imaging system can be a potentially powerful tool in the study of age-related degenerative retinal diseases.

In this paper, we report our latest progress on proving the concept that ultrasonic phased array can improve the detection sensitivity and field of view (FOV) in laser-scanning photoacoustic microscopy (LS-PAM). A LS-PAM system with a one-dimensional (1D) ultrasonic phased array was built for the experiments. The 1D phased array transducer consists of 64 active elements with an overall active dimension of 3.2 mm × 2 mm. The system was tested on imaging phantom and mouse ear in vivo. Experiments showed a 15 dB increase of the signal-to-noise ratio (SNR) when beamforming was employed compared to the images acquired with each single element. The experimental results demonstrated that ultrasonic phased array can be a better candidate for LS-PAM in high sensitivity applications like ophthalmic imaging.

We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.

We proposed a dual focus dual channel spectral domain optical coherence tomography (SD-OCT) for simultaneous imaging of the whole eye segments from cornea to the retina. By using dual channels the system solved the problem of limited imaging depth of SD-OCT. By using dual focus the system solved the problem of simultaneous light focusing on the anterior segment of the eye and the retina. Dual focusing was achieved by adjusting the collimating lenses so the divergence of the two probing beams was tuned to make them focused at different depth in the eye. We further achieved full range complex (FRC) SD-OCT in one channel to increase the depth range for anterior segment imaging. The system was successfully tested by imaging a human eye in vivo.

The ultimate goal of the study is to provide an imaging tool to detect the earliest signs of glaucoma before clinically visible damage occurs to the retinal nerve fiber layer (RNFL). Studies have shown that the optical reflectance of the damaged RNFL at short wavelength (<560 nm) is reduced much more than that at long wavelength, which provides spectral contrast for imaging the earliest damage to the RNFL. To image the spectral contrast we built a dual-band spectral-domain optical coherence tomography (SD-OCT) centered at 808 nm (NIR) and 415 nm (VIS). The light at the two bands was provided by the fundamental and frequency-doubled outputs of a broadband Ti:Sapphire laser. The depth resolution of the NIR and VIS OCT systems are 4.7 µm and 12.2 µm in the air, respectively. The system was applied to imaging the rat retina in vivo. Significantly different appearances between the OCT cross sectional images at the two bands were observed. The ratio of the light reflected from the RNFL over that reflected from the entire retina at the two bands were quantitatively compared. The experimental results showed that the dual-band OCT system is feasible for imaging the spectral contrasts of the RNFL., (© 2011 Optical Society of America)

We proposed to measure the metabolic rate of oxygen (MRO(2)) in small animals in vivo using a multimodal imaging system that combines laser-scanning optical-resolution photoacoustic microscopy (LSOR-PAM) and spectral-domain optical coherence tomography (SD-OCT). We first tested the capability of the multimodal system to measure flow rate in a phantom made of two capillary tubes of different diameters. We then demonstrated the capability of measuring MRO(2) by imaging two parallel vessels selected from the ear of a Swiss Webster mouse. The hemoglobin oxygen saturation (sO(2)) and the vessel diameter were measured by the LSOR-PAM and the blood flow velocity was measured by the SD-OCT, from which blood flow rate and MRO(2) were further calculated. The measured blood flow rates in the two vessels agreed with each other.

We investigated the feasibility of simultaneously imaging two distinctive molecular contrasts provided by the absorbed photons in biological tissues with a single light source. The molecular contrasts are based on two physical effects induced by the absorbed photons: photoacoustics (PA) and autofluorescence (AF). In an integrated multimodal imaging system, the PA and AF signals were detected by a high-sensitivity ultrasonic transducer and an avalanche photodetector, respectively. The system was tested by imaging ocular tissue samples, including the retinal pigment epithelium and the ciliary body. The acquired images provided information on the spatial distributions of melanin and lipofuscin in these samples.

We have developed an adaptive optics photoacoustic microscope (AO-PAM) for high-resolution imaging of biological tissues, especially the retina. To demonstrate the feasibility of AO-PAM we first designed the AO system to correct the wavefront errors of the illuminating light of PAM. The aberrations of the optical system delivering the illuminating light to the sample in PAM was corrected with a close-loop AO system consisting of a 141-element MEMS-based deformable mirror (DM) and a Shack-Hartmann (SH) wavefront sensor operating at 15 Hz. The photoacoustic signal induced by the illuminating laser beam was detected by a custom-built needle ultrasonic transducer. When the wavefront errors were corrected by the AO system, the lateral resolution of PAM was measured to be better than 2.5 µm using a low NA objective lens. We tested the system on imaging ex vivo ocular samples, e.g., the ciliary body and retinal pigment epithelium (RPE) of a pig eye. The AO-PAM images showed significant quality improvement. For the first time we were able to resolve single RPE cells with PAM.

We have developed a non-invasive photoacoustic ophthalmoscopy (PAOM) for in vivo retinal imaging. PAOM detects the photoacoustic signal induced by pulsed laser light shined onto the retina. By using a stationary ultrasonic transducer in contact with the eyelids and scanning only the laser light across the retina, PAOM provides volumetric imaging of the retinal micro-vasculature and retinal pigment epithelium at a high speed. For B-scan frames containing 256 A-lines, the current PAOM has a frame rate of 93 Hz, which is comparable with state-of-the-art commercial spectral-domain optical coherence tomography (SD-OCT). By integrating PAOM with SD-OCT, we further achieved OCT-guided PAOM, which can provide multi-modal retinal imaging simultaneously. The capabilities of this novel technology were demonstrated by imaging both the microanatomy and microvasculature of the rat retina in vivo.

Since the photoacoustic effect relies only on the absorbed optical energy, the back-reflected photons from samples in optical-resolution photoacoustic microscopy are usually discarded. By employing a 2 x 2 single-mode fiber optical coupler in a laser-scanning optical-resolution photoacoustic microscope for delivering the illuminating laser light and collecting the back reflected photons, a fiber-optic confocal microscope is integrated with the photoacoustic microscope. Thus, simultaneous multimodal imaging can be achieved with a single light source and images from the two modalities are intrinsically registered. Such capabilities are demonstrated in imaging both phantoms and small animals in vivo.

We have developed a multimodal imaging technique by integrating photoacoustic microscopy and spectral-domain optical coherence tomography to provide simultaneous volumetric microscopic imaging of both optical absorption and scattering contrasts in biological tissues. In the integrated system, the two imaging modalities share the same optical scanning and delivery mechanisms after their probing and illumination light beams are combined. By further synchronizing the image acquisitions, the images from the two modalities are intrinsically registered. The capabilities of this novel technique were demonstrated by imaging both the microanatomy and microvasculature in mouse ears in vivo.

We have developed a laser-scanning optical-resolution photoacoustic microscopy method that can potentially fuse with existing optical microscopic imaging modalities. To acquire an image, the ultrasonic transducer is kept stationary during data acquisition, and only the laser light is raster scanned by an x-y galvanometer scanner. A lateral resolution of 7.8 microm and a circular field of view with a diameter of 6 mm were achieved in an optically clear medium. Using a laser system working at a pulse repetition rate of 1,024 Hz, the data acquisition time for an image consisting of 256 x 256 pixels was less than 2 min.

A dual channel dual focus spectral-domain optical coherence tomography was developed for imaging the accommodation of the eye in real time. The system can provide simultaneous cross-sectional imaging of all the surfaces of the anterior segment of the eye including the cornea, anterior chamber, anterior and posterior surfaces of the crystalline lens. Thus, the modification of the curvatures of the anterior and posterior surfaces of the crystalline lens and the dimensions of the anterior segment of the eye with accommodation can be calculated. The system was successfully tested in imaging accommodation. The preliminary results demonstrated the feasibility of this novel approach.

We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.

Optical Doppler tomography (ODT) is a branch of optical coherence tomography (OCT) that can measure the speed of a blood flow by measuring the Doppler shift impinged on the probing sample light by the moving blood cells. However, the measured speed of blood flow is a function of the Doppler angle, which needs to be determined in order to calculate the absolute velocity of the blood flow inside a vessel. We developed a technique that can extract the Doppler angle from the 3D data measured with spectral-domain OCT, which needs to extract the lateral and depth coordinates of a vessel in each measured ODT and OCT image. The lateral coordinates and the diameter of a blood vessel were first extracted in each OCT structural image by using the technique of blood vessel shadowgram, a technique first developed by us for enhancing the retinal blood vessel contrast in the en face view of the 3D OCT. The depth coordinate of a vessel was then determined by using a circular averaging filter moving in the depth direction along the axis passing through the vessel center in the ODT image. The Doppler angle was then calculated from the extracted coordinates of the blood vessel. The technique was applied in blood flow measurements in retinal blood vessels, which has potential impact on the study and diagnosis of blinding diseases like glaucoma.

We previously developed a technique to acquire a SLO (scanning laser ophthalmoscope) like fundus intensity image from the raw spectra measured with spectral-domain optical coherence tomography (OCT), the same spectra used to generate a 3D OCT data set. This technique offers simultaneous fundus and OCT images and, therefore, solves the problem of registering a cross sectional OCT image to fundus features. However, the registration of high density OCT images is still an unsolved problem because no useful fundus image can be generated from the high density scans. High density OCT images can significantly improve the image quality and enhance the visualization of retinal structure, especially the structure of small lesions. We have developed a feature-based algorithm, which can register a high density OCT image on the fundus image generated from normal density scans. The algorithm was successfully tested for both normal and diseased eyes.

We report on a new configuration of fiber-based polarization-sensitive Mueller matrix optical coherence tomography that permits the acquisition of the round-trip Jones matrix of a biological sample using only one light source and a single depth scan. In this new configuration, a polarization modulator is used in the source arm to continuously modulate the incident polarization state for both the reference and the sample arms. The Jones matrix of the sample can be calculated from the two frequency terms in the two detection channels. The first term is modulated by the carrier frequency, which is determined by the longitudinal scanning mechanism, whereas the other term is modulated by the beat frequency between the carrier frequency and the second harmonic of the modulation frequency of the polarization modulator. One important feature of this system is that, for the first time to our knowledge, the Jones matrix of the sample can be calculated with a single detection channel and a single measurement when diattenuation is negligible. The system was successfully tested by imaging both standard polarization elements and biological samples.

A high-speed spectral-domain optical coherence tomography (OCT) system was built to image the human retina in vivo. A fundus image similar to the intensity image produced by a scanning laser ophthalmoscope (SLO) was generated from the same spectra that were used for generating the OCT sectional images immediately after the spectra were collected. This function offers perfect spatial registration between the sectional OCT images and the fundus image, which is desired in ophthalmology for monitoring data quality, locating pathology, and increasing reproducibility. This function also offers a practical way to detect eye movements that occur during the acquisition of the OCT image. The system was successfully applied to imaging human retina in vivo.

A unique feature of polarization-sensitive Mueller optical coherence tomography is that, by measuring Jones or Mueller matrices, it can reveal the complete polarization properties of biological samples, even in the presence of diattenuation. We map local polarization properties for the first time to our knowledge by using polar decomposition in combination with least-squares fitting to differentiate measured integrated Jones matrices with respect to depth. We also introduce the new concept of dual attenuation coefficients to characterize diattenuation per unit infinitesimal length in tissues. We experimentally verify the algorithm using measurements of a section of porcine tendon and the septum of a rat heart.

An optical-fiber-based multichannel polarization-sensitive Mueller optical coherence tomography (OCT) system was built to acquire the Jones or Mueller matrix of a scattering medium, such as biological tissue. For the first time to our knowledge, fiber-based polarization-sensitive OCT was dynamically calibrated to eliminate the polarization distortion caused by the single-mode optical fiber in the sample arm, thereby overcoming a key technical impediment to the application of optical fibers in this technology. The round-trip Jones matrix of the sampling fiber was acquired from the reflecting surface of the sample for each depth scan (A scan) with our OCT system. A new rigorous algorithm was then used to retrieve the calibrated polarization properties of the sample. This algorithm was validated with experimental data. The skin of a rat was imaged with this fiber-based system.

A double-beam polarization-sensitive system based on optical coherence tomography was built to measure the Mueller matrix of scattering biological tissue with high spatial resolution. The Jones matrix of a sample can be determined with a single scan and subsequently converted into an equivalent nondepolarizing Mueller matrix. As a result, the system can be used to measure the Mueller matrix of an unstable sample, such as soft tissue. The polarization parameters of a porcine tendon, including magnitude and orientation of birefringence and diattenuation, were extracted by decomposition of the measured Mueller matrix.