3.1. Co-registration of functional OCT and OISI
illustrates the co-registered OISI and OCT imaging in the region of functional activation. The functional signal was computed as a ratio of the reflectance at each time point to the mean reflectance in the pre-stimulus period and is therefore representative of a percent signal change from the baseline. shows a representative ratio OISI image from the time window around the peak of maximal activation (t
= 4–6 s). The region of functional activation corresponding to forepaw stimulation is clearly identified. A decrease in the reflectance at 570 nm (denoted by the blue region) corresponds to increased concentration of haemoglobin. plots the time-course of the averaged fractional reflectivity changes in the region denoted by the dashed box in . The decreased signal intensity during the stimulation period arises from the stimulus-induced increase in blood volume absorbing more light. The time-course and maximum fractional signal changes (~0.03) agree with previously published results (Malonek et al., 1997
; Devor et al., 2005
; Dunn et al., 2005
Fig. 4 Representative results of OISI and OCT imaging of functional activation. (A) En face functional activation map from OISI with co-registered OCT scan indicated by the red arrow. (B) The averaged time-course for OISI over the region denoted by dashed box (more ...)
shows the functional OCT image from the time window around the peak of maximal activation (t = 4–6 s). The OCT scan location is shown over the region of activation indicated by the red arrow in . Similar to OISI fractional change map, the OCT fractional change map was computed by normalizing all time points to the baseline pre-stimulus period. The OCT functional map reveals a localized area of activation with both positive and negative changes in the cortex. The temporal sequences of activation for the representative regions of interest (ROI) in are plotted in . Size and shape of the boxed areas for time-course analysis were selected based upon visual perception of the boundaries of the localized change. The OCT functional signal time-courses reveal clear increases and decreases that deviate from baseline, reach a peak near the cessation of the stimulus, and then gradually return to baseline. The time-course of OCT signal changes correlates well with the co-registered OISI time-course as shown in . It is noted that the typical fractional changes in functional OCT (0.1–0.5) are larger than OISI, and the noise is also larger due to the enhanced sensitivity using OCT coherence detection. Nevertheless, the observed changes are statistically significant (p < 10−7). illustrates the map of p values showing the statistical significance of the fractional OCT changes from the baseline. In addition, the lateral line profiles of OISI signal changes and the depth-integrated OCT signal changes are plotted in , showing the spatial correspondence of OCT and OISI signals.
All five animals showed similar responses. Representative responses from different ROIs (one positive and one negative) for each animal are shown in .
Fig. 5 Representative responses from ROIs on five different animals. Each color represents the results from one animal. Both positive (solid line) and negative (dashed line) responses are presented. Although temporal location and intensity of the peak varied (more ...)
3.2. Spatial-temporal evolution of functional activation
shows the temporal sequence for the functional OCT images. The spatially distinct activation regions evolve during the stimulation period, reach the peak around the end of stimulation, and gradually fade back towards baseline during the recovery period.
Fig. 6 Spatial-temporal evolution of functional OCT signals in the rat cortex. A fractional change map demonstrates the presence of positive (warm colors) and negative (cool colors) changes in OCT signals during stimulation. Functional OCT images at each individual (more ...)
presents the results of functional OCT activation and control experiments. shows the localization of the activation area from OISI during the contralateral forepaw stimulation. Two OCT scans were denoted by arrows “1” and “2” with scan “1” imaging across the activation area and scan “2” imaging outside the region of activation. shows the functional OCT image at time window 4–6 s over the activation region denoted by arrow “1”. Localized regions of activation are visualized. Both positive and negative changes corresponding to the stimulation pattern are prominent. In contrast, as shown in , the functional OCT image at time window 4–6 s over the scan denoted by arrow “2” does not reveal any significant activation region. Small signal changes are observed which agree with the small changes in . shows the functional OCT image at a time window 4–6 s over the scan denoted by arrow “1”, but with the stimulation was performed on the ipsi-lateral forepaw. There is no distinct activation region observed in the OCT image. Paired t-test between (B and C), and (B and D) are performed. In both cases, the difference is statistically significant (p < 10−7).
Fig. 7 (A) Localization of activation area in OISI. Two OCT scans are denoted by arrows “1” and “2”. (B) Functional OCT image at time window 4–6 s over the activation region denoted by arrow “1” during (more ...)
3.3. Profile analysis for functional OCT signals
To further understand the characteristics of the positive and negative signals, detailed profile analyses were performed on the functional OCT images. illustrates the axial (x1 and x2) and transverse (z1 and z2) lines on OCT structural and functional images used to perform profile analysis. The functional OCT image is taken from the time window around the peak of maximal activation (t = 4–6 s). plot both differential functional signal (left panels) and the corresponding profiles at both baseline (pre-stimulation) and during the stimulation period (right panels) along the lines indicated by x1, x2, z1, and z2, respectively. First, distinct anatomical features such as the edges of the skull and the cortical surface can be appreciated in (denoted by the black arrows). The pre- and post-stimulation plots indicate that the signals from skull edge and cortical surface remain stable during the stimulation process. This confirms that the OCT signal changes are not from global motion artefacts. Second, there exist two distinct patterns for OCT signal changes. For scenario “1”, the identified peaks in the scattering intensities are either increased or decreased. This scenario is illustrated in the enlarged inset on the right panel of . For scenario “2”, the center of a scattering peak is shifted, as indicated by the enlarged inset on the right panel of . Both scenarios can result in positive and negative changes in functional OCT signals. A typical OCT A-scan profile might contain the combination of both patterns.
Fig. 8 Axial and transverse line profiles of functional OCT signals. (A) Position of the axial lines (x1 and x2) and transverse lines (z1 and z2) on OCT structural (left) and functional (right) images. (B) Left: Plot of differential functional signal along line (more ...)
3.4. Integrated OCT signals
In order to compare with OISI results, we analyzed the integrated OCT signals over the imaging region. In the OISI images (see ), the response curve is averaged over the region of interest (ROI) denoted by the dotted black box. This ROI is selected by the visual inspection of activation areas (blue color), in a way similar to the intensity threshold. In contrast, in the OCT cross-sectional images (see ), there are two prominent differences. First, both positive and negative signals are present. Second, compared to the single-location, wide-spread OISI activation region, the OCT activation regions are discrete and non-continuous local foci. Therefore, we applied a statistical significance threshold to select the activation ROI. Basically, for each pixel, the measurements in the baseline (0–1 s) and activation window (4–5 s) are collected as two groups (baseline and activation). A Student's t-test is performed on these two groups to test the null hypothesis that the activation signals and baseline signals are the same. Then the pixels with a significance level α < 0.05 are chosen since the null hypothesis is rejected for those pixels. shows an activation image where only the ROIs with significance level α < 0.001 are selected. In , the time course of the averaged positive signals, negative signals, and the summed signals are plotted. From the plot, both the positive and negative signals increase in magnitude during the activation period, and recover after the cessation of the stimulation. The temporal trend of signal variation agrees well with the stimulation pattern. The summed signal shows a net positive change during the activation, which indicates the increasing of overall OCT back-scattering signals. also shows the corresponding OISI time course indicating well-matched temporal responses.
Fig. 9 (A) The functional OCT image (red-blue color scale) at time window 4–6 s with significance level α < 0.001 superimposed with the OCT anatomical image (grey scale); (B) plots of positive, negative, and summation OCT signals at regions (more ...)
3.5. Depth-dependent activation pattern in OCT
Compared to depth-integrated OISI, OCT can resolve functional activation at different depths. The temporal properties of functional OCT time-courses at different depths (z
) were analyzed. shows a structural OCT image with two regions indicated by the red (a large vessel on the surface of cortex) and blue (in the cortex) boxes. The time-courses of functional OCT for these two regions are plotted in . The blue curve shows a time-course that corresponds well with the stimulation pattern. In contrast, the red curve indicated a time delay of approximately 2 s in both the onset and the peak of the response with respect to the stimulation pattern. Since the averaged OISI time-course matches well with the stimulation as indicated by , we choose the averaged OISI time-course as the reference and cross-correlated it with the functional OCT time-courses at each individual pixel:
−1) are series of functional OCT and OISI signals at each time point, respectively.
are the means of the corresponding series and d represents the delays (d
−1). To account for the change in magnitude of OISI signals and OCT signals for individual pixels, both data are first normalized for calculating the cross-correlation (i.e. the auto-correlation of the data series at delay 0 is equal to 1).
Fig. 10 (A) Structural OCT image with two regions indicated by red and blue boxes. (B) The time-courses of the functional OCT for these two regions. (C) Spatially-resolved time-lags overlaid with structural OCT image. Different colors indicate different time-lags (more ...)
The positive functional OCT signal trend will result in a negative correlation coefficient with OISI signal, and the negative functional OCT signal trend will give positive correlation coefficient. In the analysis, we take the absolute value of the correlation coefficient, since it indicates the degree of match between those two curves. We selected the pixels with correlation coefficients >0.7 (which have good correlation with the OISI signals) and calculated the shift of the correlation peak, which is an indication of time lag between the functional OCT and OISI time-courses. shows the spatially resolved time lags overlaid with the structural OCT image. Different colors indicate different time lags with respect to the averaged OISI time-course. Positive values indicate the OCT time courses are delayed compared to the OISI time course, while negative values indicate the OCT time courses precede the OISI time course. displays the histogram of the time lags. The majority of the cortex region shows the functional OCT response closely follows the OISI response (with time lag within ±1 s), while there are some regions near the surface of the cortex exhibiting delayed responses (with time lag >1 s). plots the averaged temporal response of all the pixels with different time lags, including [−1.5 s, −0.5 s), [−0.5 s, 0.5 s), [0.5 s, 1.5 s), [1.5 s, 2.5 s), and [2.5 s, 3.5 s). The temporal positions of their peak response from these time courses are different, indicating different dynamics.
shows another example of the time lag image of the functional OCT activation with respect to the OISI image. The averaged time lag for a given depth from the skull surface is plotted versus depth in . There exists a two-phase behaviour for the averaged time lag. In the first 200 μm of the cortex (corresponding to the region 100–300 μm from the skull surface), the averaged time lag decreases as the depth increases (characterized by slope S1). From 200 μm and deeper into the cortex (corresponding to >300 μm from the skull surface), the averaged time lag does not change significantly with the depth (characterized by slope S2). We analyzed the spatial-temporal correlation between OCT and OISI on 9 independent runs (each run include 60 stimulation trials) from 5 rats, and the distribution of S1 and S2 are indicated in . The averaged S1 = − 4.1 ± 1.3 ms/μm, while the averaged S2 = − 0.18 ± 0.35 ms/μm. There is statistically significant difference between S1 and S2 (p < 0.0001).
Fig. 11 (A) Structural OCT image overlaid with the time-lags with respect to the averaged OISI time-course at different activation regions. The time-lags are color-coded (unit: second). (B) Plot of the averaged time-lags at the same depth (from the skull surface) (more ...)