Optical coherence tomography (OCT), based on low coherence interferometry, offers the unique advantage of high spatial and temporal resolution [5]. This technique has been recently applied to perform depth-resolved measurements of blood vessel diameter, blood flow and total hemoglobin in the rat brain [6]. In this and other works, Boas’ group move beyond surface scatter changes reported by others and demonstrate methods for three-dimensional imaging of the rat cortex and provide measurements of two dimensional blood flow that correspond with autoradiographic techniques [6–8]. Several other important methods have recently been put forward, each aiming to maximize both temporal and spatial resolution at the tissue depth required for the intended application. Optical microangiography (OMAG) uses intrinsic optical scattering signals backscattered from tissue to gauge microvascular caliber, microvascular density and the changes that occur to vessels in response to physiological challenges such as hypoxia or hyperoxia [9]. Optical frequency domain imaging (OFDI) is another technique that has emerged to investigate aspects of tissue biology requiring high spatial and temporal resolution without altering the natural state of the tissue. The combination of optical contrast with ultrasonic detection has also been successfully adopted in the form of both photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) [10–12]. These techniques use an exciting laser on tissue samples to generate thermal expansion that can be subsequently detected as an ultrasound wave. PAM was most recently used to obtain high-resolution images of living zebrafish larva [13], an application that could be easily expanded to other animal models. Our understanding of tumor angiogenesis and morphological characteristics of growing microvascular networks has benefited [10]. Diffuse correlation spectroscopy (DCS) has recently progressed from bench to bedside where quantitative measurements of blood flow in the human brain have been compared with multiple existing techniques including arterial spin labeled MRI, xenon-CT and Doppler ultrasound [14]. Moving beyond the characteristics of changes in flow and morphology, the combination of hyperspectral imaging with spectral domain OCT provide information on the sheer rate on vessel walls during tumor growth [12]. A deeper understanding of the biophysical forces involved with changing vessels could certainly prove valuable beyond a richer understanding of antiangiogenesis therapies, and would be particularly useful in understanding the pathobiology of vascular malformation diseases such as arteriovenous malformations or hereditary aneurysm syndromes. Another important aspect of imaging biological tissues is the consideration of motion. An elegant report by Chen’s group [15] describes the use of Doppler-variance OCT whereby they perform retinal vascular imaging that is largely insensitive to bulk motion, a clear advantage for future applications to ophthalmology centered diagnosis and patient management.

Speckle-variance OCT (SV-OCT) has the capability of depth resolved three-dimensional imaging over a period of time, usually measured in seconds. The speckle pattern relies on the spatial coherence properties of the optical signal back reflected from the tissue being sampled. This endogenous contrast mechanism relies on the mathematical analysis of the dynamic speckle pattern generated by the motion of erythrocytes in the artery, vein or capillary bed. Recently, Mariampillai et al. introduced microvascular imaging based on inter-frame speckle variance in a swept-source OCT system [16,17]. This method calculates the inter-frame intensity variance of a sequence of structural images. The main advantage is its depth resolution and insensitivity to Doppler angle-dependent contrast allowing for three-dimensional imaging of microvascular networks at greater tissue depths. The obvious downside of the method is its sensitivity to bulk motion.

Rat spinal cord preparation: Male Wistar rats weighing approximately 150g were induced with 2.5% isoflurane inhalational anesthesia. A rectal temperature probe was inserted and body temperature was maintained at 37°C ± 0.5°C using a heated gel pack. A dorsal tail vein catheter was inserted for intravenous access. Maintenance fluids were given at a rate of 2mL/kg/hr. A dorsal laminectomy was performed at the L1 to L2 level according to previous published methods [16]. Following the laminectomy, a tracheostomy was performed and mechanical ventilation (2% isoflurane at 2 mL tidal volume and 55 breaths per minute) was initiated using a Harvard Rodent Ventilator. The animal was then paralyzed using an infusion of pancuronium bromide (2 mg/kg/hr). This was performed so as to conduct breath-hold periods during the imaging protocol so as to stabilize the spine. The spine was then mechanically fixed by performing an extra-cavitary dissection around the transverse process of the T12 vertebral body and fixing halo pins (DePuy) into the anterior vertebral body and fixed to a standard imaging breadboard using a homemade jig (Figs. 2A and ​and2B2B below). The imaging protocol, as described below, was initiated

Mouse spinal cord preparation: C57BL/6 mice weighing approximately 35g were anesthetized with a mixture of xylaxine (10 mg/kg) and ketamine (70 mg/kg) intraperitoneal (i.p.). The mouse spinal cord was prepared in a similar fashion to that of the rat with the following exceptions: Cardio-respiratory motion was found to be less in the lumbar spine of a mouse than a rat, therefore we did not utilize a breath-hold paradigm; the animal was maintained on i.p. anesthesia and a tracheostomy was not performed; the mouse spinal column was held in position using a homemade jig as outlined in Fig. 3A below. Briefly, we used two pairs of forceps to stabilize the spinal column above and below the exposed spinal cord. These forceps were gently held in position using a fastening device so as to put gentle pressure on the vertebral bodies without damaging the bony spine. These forceps were then fixed to the imaging breadboard.

Histology: Spinal cord specimens were removed from the examined animals after completion of the imaging protocol and placed in formalin. Standard H&E stains were performed to compare histological anatomy with OCT derived anatomical information.

Imaging System: A schematic of the swept source OCT system is illustrated in Fig. 1 . The laser, data acquisition and software were specifically designed to enable continuous real-time processing and display of both structural and speckle variance data while simultaneously saving recalibrated fringe data at an A-scan rate of 108 kHz. Briefly, speckle variance OCT is an inter-frame vascular detection technique that unlike phase sensitive processing is not affected by Doppler angle or the flow velocity. The technique is similar to power Doppler and OMAG techniques in that it measures residual signal power after filtering out static tissue components. The algorithm performs a straightforward variance calculation on the non-log compressed intensity data at each pixel location within a set of N frames (referred to as the gate length). The resulting set of variance images can either be thresholded manually or by using the structural data, which is typically more effective in the presence of residual motion [18]. The swept source laser design is a buffered polygon ring cavity laser where a 600 lines/mm diffraction grating (GR25-0613, Thorlabs) was chosen to create the necessary dead time between laser sweeps to enable buffering from 54 kHz to 108 kHz [19]. Standard techniques for triggering and recalibration were used in this system [16]. The analog signals were fed in to a data acquisition system and digitized at 250 MS/s (ATS9350, Alazartech), which was transferred to the memory on a video graphics card (GeForce GTX 460 1GB, NVIDIA). Custom-written kernels together with built-in CUDA libraries (i.e. cudpp, cufft) were used to manipulate and reconstruct structural and speckle variance OCT images on the multiple cores (336) of the GPU, which enables massive parallel data processing and not only dramatically enhance the data throughput when compared to processing using only the CPU but also concurrently free up CPU resources [20]. Another advantage for processing data directly on the graphics card is that the reconstructed OCT images can be displayed without any additional memory transactions. The above setup allows real-time acquisition, processing, and display only limited by the A-scan rate of the laser. Finally, the recalibrated fringe data was saved to a PCIe solid state drive (RevoDrive3 X2 series, OCZ) for post processing and 3D visualization.

Imaging protocol: The anesthetized animal was placed under the scan head of the imaging system and properly positioned using the real-time 2D-display for feedback. For both 2D and 3D imaging we chose to use 512 A-scans per image over a scan distance of 3mm, this resulted in an image acquisition rate of greater than 200 fps. We found that this imaging speed was sufficient to eliminate most of the effects of residual bulk tissue motion. Average structural and speckle variance images were calculated using a gate length of N = 4 and displayed in real-time at 50 fps. For 3D imaging we acquired 3000 B-mode images over a 3mm(x) × 3mm (y) × 2mm (z) volume in approximately 15 seconds for post processing. The large amount of oversampling done in the y-direction minimized the amount of static structure decorrelation when calculating the speckle variance image.

The mouse spinal cord exhibited both less cardio-respiratory motion and a less extensive network of dorsal venous tributaries. We were thus able to image approximately 500μm into the mouse spinal cord, visualizing the entire dorsal gray matter and a portion of the ventral gray matter. The gray matter of the spinal cord contains neuronal cell bodies, synapses and interneurons. The surrounding white matter is composed of myelinated axons responsible for transmitting neuronal signals to and from the brain. Given the bulk motion correction and depth resolved imaging in the mouse spinal cord, it will be possible to investigate microvascular changes in the dorsal horn of the spinal cord in response to somatosensory stimulation.