Optical imaging of the spinal cord has made considerable advancements since the first published reports in mid-1990’s. These early experiments used calcium dyes that fluoresced with neural activity; the combination of the transparent larval zebrafish with confocal microscopy allowed for the in vivo
study of motor circuits of the spinal cord [1
]. While revolutionary for its ability to visualize the activity of neuronal populations interacting with each other, confocal microscopy is limited by its penetration depth and the use of exogenous voltage sensitive dyes. Other optical imaging methods utilized in the spinal cord include light transmittance assays in thin ex vivo
spinal cord slices [2
] and the use of optical reflectance signals from the dorsal surface of an in vivo
rodent model [3
]. More recently, laser speckle imaging was used in the setting of a rodent spinal cord injury model whereby the light reflectance was used to estimate the spinal cord vascular response to electrical stimulation before and after traumatic spinal cord injury [4
]. The drawback of each of these techniques is the lack of depth resolution into the spinal cord beyond approximately 300 μm.
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
]. 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
]. 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
]. 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.
Here, we provide the first report of depth resolved OCT of the spinal cord using an entirely endogenous contrast mechanism taking advantage of the speckle variance approach recently described by Mariampillai [16
]. We demonstrate methods to optimize bulk motion correction and image a 3D volume of both the rat and mouse spinal cord.