Cortical veins and arteries, as classified by principal component analysis of data collected during induced ischemia. Flow values are estimated from laser speckle flow index.
IntroductionA significant advantage of optical brain imaging over other imaging modalities like fMRI, PET, and CT is that optical systems are low cost, flexibility and small size. These properties allow for several applications, including multispectral tissue imaging and minimally invasive optical coherence tomography imaging.
(LSC) Imaging measures relative velocity of blood flow . The scattering of coherent light off tissue produces a random interference pattern known as speckle. The intensity variation of interference fringes is reduced by sampl movement as different patterns are superimposed. The speckle contrast ratio (CR) is a measure of fringe intensity which can be related to coherence time, a quantity inversely proportional to velocity. The absolute flow velocity i brain tissue can be obtained using sequential exposures of different duration . Intrinsic Optical Signal (IOS) Imaging measures reflectance changes from brain tissue resulting from neural activity . The reflection changes are due to changes in blood oxygenation and volume, which are a surrogate to neural activity. This technique is widely used in the evaluation of structure and function relationships, and brain plasticity . Non-coherent light is an ideal light source for tissue reflectance imaging, such as IOS as it avoids noise from scatter-based speckle patterns. A light emitting diode (LED) is a common low noise light source for such measurements. Previous studies have shown that increased noise values due to coherence effects in laser diodes mitigate the benefits of increased brightness they have over LEDs . The spectral linewidth ∆λ of a laser can be increased by operating the laser in a “multimode” regime, where multiple transverse modes are supported. The coherence length Lc can be reduced by increasing the spectral linewidth according to:
Dual modality imaging is a powerful approach for measuring brain hemodynamic properties. Previous research looked at dual modality Multispectral Reflectance (MSR) and LSC imaging using a beam splitter in conjunction with two light sources and two cameras . In this work we investigate an architecture utilizing rapid alternating light sources and a single camera. We constructed a dual imaging system, collected images of a rat cortex at varying depths illuminated by fast alternating LED and a VCSEL sources, and evaluated noise values. We aim to use a single VCSEL light source with alternating light pattern output for simultaneous IOS and LSC imaging, enabling simultaneous comparisons as well as co-registration between imaging techniques. This single source and detector architecture is also advantageous for implantable imaging devices. Potentially, these imaging devices will allow for monitoring of brain activity in freely behaving animals and then in continuous monitoring of patients.
Methods and Results
A schematic of the experimental setup is shown in Fig. 1a. The camera is used for imaging the cortex, while an LED (625 nm) or a VCSEL (670 nm) is used for illumination. Craniotomies were performed on anesthetized rat subjects, between the bregma and lambda ridges, revealing a 4-6 mm region of cortex for imaging. Petroleum jelly was used to create side walls around the opening, filled with agarose gel, and covered by a cover slip. All rat surgeries were conducted according to protocol approved by the local animal care committee. A CCD camera (Retiga 4000R, QImaging) collected 1024x1024 pixels, 12 bit images at 8 Hz. The imaged field of view was 7.6 x 7.6 mm. The camera was mounted on a vertical translation stage to adjust focal plane height. LED and VCSEL illuminated images of the cortex were taken in an alternating sequence. Control of the VCSEL current (Keithley 6221) allowed for varying operation modes. Focus was given to single mode operation, at 6 mA drive current, and to sweep mode, with a 1 kHz current sweep from 7 to 14.5 mA. Recorded images were processed to retrieve temporal and spatial contrast values. Spatial and temporal noise values were found to be highly correlated for VCSEL illumination. Resulting speckle contrast values can be used to calculate speckle correlation time, and relative flow velocities . Figs. 1b and 1c give examples of how the spatial contrast in single mode VCSEL operation can be interpreted as a vascular flow map. Fig. 1b shows blood vasculature on the brain surface, illuminated with a green LED (530 nm). Fig. 1c shows a spatial contrast map of the same region, with the camera focused 300 μm below the surface, illuminated by a VCSEL in a single mode operation. Dark regions are interpreted as relating to low contrast, “blurred out” speckle regions, which indicate movement of scattering elements. The VCSEL image reveals a network of vasculature 300 μm below the surface, even through static scattering elements of the brain surface.
Fig 1. Imaging system schematic. Images from both LED (odd frames) and VCSEL (even frames) illumination are collected on the same camera. Alternatively, the laser alone can be driven while switching power schemes between frames. b) Image of vasculature on the cortical surface, illuminated with a green LED. Slight bruising and blood spots are observed. c) Single mode spatial contrast map of the same region, focused 300 μm below the surface, averaged over 40 frames. Contrast values were taken over 5x5 pixel regions. Grey level values can be correlated with blood flow. The top left dark rim is due to petroleum jelly window shadowing. Scale bar values is shown in (standard deviation/mean) units.
Comparison of temporal noise values for VCSEL illumination in single mode, in sweep mode, and for the LED operation indicated that by applying the current sweep, coherence effects of a VCSEL laser can be reduced to produce noise levels similar to those of an LED (Fig. 2).
Fig. 2. Temporal speckle contrast images of rat cortex, at a focus 600 μm below surface. a) VCSEL illumination in a single mode operation, b) VCSEL illumination in a current sweep mode operation, c) LED illumination. Each images is produced from pixel-wise temporal noise values taken over 10 images at 8 Hz, averaged 40 times. Scale bars in (standard deviation/mean) units.
Fig. 2a shows that temporal contrast can also be used to produce flow topology maps based on speckle variation. In a single mode VCSEL illumination, we observe high noise values and vascular detail even at non-ideal focus plane for capturing blood vasculature. In current sweep VCSEL illumination (Fig. 2b) the vasculature details are washed away and noise values are reduced significantly compared to single mode illumination. In LED illumination we observe noise values comparable within a factor of 2 to current sweep VCSEL illumination mode, deviation/mean) units. and essentially no vascular details are visible. We evaluated Lc to estimate the contribution of reduced coherence to the significant noise reduction observed in current sweep VCSEL mode. To evaluate the change in Lc, ∆λ was measured for the laser in both current sweep and single mode, as well as for the LED. A factor of 7.3 decrease in coherence length was found between single mode (∆λ=0.12 nm, Lc= 3.74 mm) and current sweep mode (∆λ=0.88 nm, Lc= 0.51 mm). The LED, however, was still found to have a much lower coherence length (∆λ=20 nm, Lc= 0.02 mm) as compared to a VCSEL operating in current sweep mode. This observation can be partly related to brain tissue movement that assists in averaging speckle patterns from the tissue.
The temporal analysis as portrayed above implies that the reduced coherence noise values associated with current sweep and transverse VCSEL mode sweeps allow a VCSEL illumination to approach the noise performance of an LED. In particular, a VCSEL is more amenable to rapid variation of transverse beam shape, and mode characteristics than an edge emitting laser diode. VCSEL dimensions (~ 30 μm diameter, 2-20 μm gain profile diameter, 3-6 μm height) are much smaller. [Evan JQE 2004] Current changes also introduce thermal and polarization changes that support rapid transverse mode sweep. Utilizing this phenomena, a single laser illumination source under two power schema can support a dual modality imaging system. By taking advantage of the strong coherence effects of the VCSEL in single mode, speckle contrast images can be generated to reveal vasculature. The resulting contrast ratio maps can be converted to speckle correlation time maps, and through use of multiple exposures, this data can be used to directly quantify blood flow. Alternatively, by driving the VCSEL in current sweep mode, we can utilize the VCSEL for imaging as we would use a low noise LED source. As discussed here, IOS is one method by which we can apply VCSELs in measuring small reflectance changes associated with neural activity. Through trial averaging, even greater signal to noise ratios can be achieved for the measurement of sensory neural activation. A further potential application is in fluorescence imaging, where speckle reduction allows for uniform high spatial resolution excitation of a marker or dye.
We further assert that contrast imaging can be used as a way of isolating landmarks for real-time spatial co-registration. We see future applications of this approach in live animal studies, with implanted imaging sensors over longer time periods. The dual VCSEL illumination modalities (single mode/current sweep) as outlined here allow for the simultaneous production of flow velocity maps and reflectance images. By utilizing the vasculature landmarks, movement of a sensor with respect to the brain surface can be accounted for, leading to a stable imaging region of interest within which reflectance changes associated with cortical function may be recorded.
We have demonstrated that changing a VCSEL operation between single mode and current sweep mode can be used to manipulate speckle noise properties of a VCSEL source, allowing us to use a single light source to image a tissue sample via two different modalities. We show that the noise reduction associated with current sweep mode lower the temporal and spatial noise of the VCSEL to values comparable to that of a low noise LED. We attribute this to the combined effect of decreasing the coherence length of the source, along with spatial and polarization superposition of transverse modes that change in space and time, and brain movement. This allows the VCSEL to be implemented in IOS Imaging programs in place of an LED. Furthermore, we have shown the VCSEL in single mode to be effective for LCS imaging to image flow in blood vessels beneath surface vasculature. Future directions include investigation of multi-exposure speckle imaging to quantify absolute blood flow and IOS imaging.