A. Materials and Methods
Our work uses an established imaging procedure published by Kalatsky and Stryker[12
]. Animal imaging was performed at the University of California, San Francisco (UCSF) according to a protocol approved by the UCSF Institutional Animal Care and Use Committee. The experimental setup is shown in . An anesthetized C57BL6 wild-type mouse is given a visual stimulus from a computer monitor consisting of a horizontal white stripe on a grey background (50% contrast.) Images are obtained by illuminating the visual cortex at 610 nm, 690 nm, 750 nm, 775 nm, or 850 nm. Light from a tungsten lamp is filtered at a given wavelength using interference filters with a FWHM bandwidth of 10 nm and delivered via an optical fiber. Images without a craniotomy (but with scalp removed) are taken first, then a small section of skull above the visual cortex is removed and images taken again. The stimulus is delocalizes the signals, making them more difficult to detect and maps more difficult to resolve. This work seeks to swept repeatedly in elevation across the visual field at a frequency of 0.125 Hz for 90 cycles (), with a DALSA 1M30 CCD camera capturing images at 30 frames per second. The stimulus is then swept in the opposite direction and images taken. Signals recorded from the two sweeps are then subtracted to remove shifts caused by hemodynamic delay.
Imaging setup. A C57BL6 mouse is placed under an illuminating fiber and CCD camera. Illumination is provided by a tungsten lamp. A cover slip supported by agarose gel presents a flat surface for imaging.
Visual stimulus. A horizontal white line is swept repeatedly in elevation across a black background with a period of eight seconds.
After the images are recorded, a Fourier transform in time is performed for each pixel, and the signal is filtered for components at the sweep frequency and normalized to improve the signal to noise ratio (.) The result is two maps, one of signal amplitude, indicating the relative intensity change, and one of phase, corresponding to the position of the stimulus within the visual field. Animals are euthanized after the entire set of maps is taken.
Image processing. A pixel-by-pixel FFT is used to select only the component at the stimulus frequency, thereby filtering out noise from other physiological and physical processes.
B. Results and Discussion
The maps obtained without craniotomy (i.e. through the skull) and with craniotomy are shown in and , respectively. The lower maps show amplitude, with darker regions indicating stronger activity. The primary visual cortex is located in the dark region shown in the amplitude maps. The upper maps show phase, with similar colors indicating similar phase, thus highlighting regions that are active at the same time. The functional organization of the visual cortex can be seen clearly in the phase maps, where well-defined areas of similar color indicate that neurons responsible for a given area of the visual field are grouped together. shows some disorganization in the maps taken at 690 nm and 750 nm, which we believe are due to fluctuations in the level of anesthesia. Despite this, we can still draw the conclusion that maps obtained through skull are more diffuse than those taken through craniotomies, but still show distinct features of cortical function and can be used for neuroscience research.
Fig. 7 Images taken without craniotomy (i.e. through skull.) Signal-to background decreases and maps become more diffuse as wavelength increases. Signal is measured in areas denoted by red circles and background level is measured over areas denoted by blue circles. (more ...)
Images taken with craniotomy. Signal is stronger and maps more sharply defined than without craniotomy. Signal is measured in areas denoted by red circles and background level is measured over areas denoted by blue circles.
Each set of maps from left to right in and was taken at progressively longer wavelengths. It is evident from the fading black region in the amplitude maps that the signal-to-background ratio decreases as wavelength moves from visible to NIR. shows that this is due to degradation in detected signal level rather than an increase in background.
Fig. 9 Signal-to-background analysis. Decreasing signal-to-background ratio is due to decreasing signal rather than increasing background. The increase at 850 nm in the craniotomy case is attributed to a slight decline in effectiveness of anesthesia. Error bars (more ...)
In the study with craniotomy, the signal level at 850 nm increases, which is inconsistent with the trend observed in the study without craniotomy. We believe this is due to a slight decline in the effectiveness of the anesthetic toward the end of the imaging session, which caused a stronger response. This multi-wavelength, no-craniotomy/craniotomy study required an imaging session nearly eight hours long. Typical single-wavelength imaging sessions last less than two hours (including time required for image processing.)
In addition to declining signal-to-background ratio, the spatial character of the signal becomes delocalized, as shown by degradation in the definition of the phase map with increasing wavelength. Decreased signal-to-background ratio and increasing delocalization are consistent with reduced absorption. Photons experience many scattering events and intermingle with those from neighboring regions of the brain, leading to a lower detected signal and more diffuse phase maps.