A schematic of the FT system shown in (a) and the constructed system is illustrated in (b). Removable filter slides allow users to easily install different filter sets as necessary for imaging a range of fluorophores, as depicted in the top left of (b). The bottom left part of (b) illustrates the modified mouse bed, holding a mouse phantom, along with a close-up view of the rotating gantry housing the five fiber coupled optical focusers, C1–C5.
The block diagram of the system hardware is shown in (a). LABVIEW based control of the laser, TCSPC instrumentation and motors controlling the gantry, linear stage, and filter slides allow full automation of the data acquisition. The fan-beam detection geometry is shown in (b). Here, a single excitation source position is used and the diffuse signals at the surface of the specimen are collected using focalized detection. The detected signals are then separated, and directed to two sets of PMTs dedicated to Fl and Tr signals at each fiber channel.
A photograph of the alignment jig is shown in (a). The jig fits precisely into the gantry allowing the optics to be radially aligned by maximizing the light signal through a pinhole, as shown in (b). Mechanical alignment is depicted in (c) and is performed to ensure the stage translates through the center of the gantry, allowing the source/detector information to be placed virtually, given the microCT contour information.
Plots of the 635 nm pulsed diode laser’s intensity (a) and temporal FWHM (b) are shown as a function of the driver’s pumping capability.
In (a), the setup used to assess PMT linearity is shown. By using a collimated source and injecting it orthogonally into the center of a 25 mm cylindrical phantom, a diffuse uniform source is obtained and the same optical power was sampled by all channels simultaneously, allowing the relative sensitivities to be examined. PMT linearity over the entire dynamic range was also examined by examining the count rate as a function of input optical power (b). Plots of the dark count rate as a function of integration time are shown in (c) and the PMT time linearity response is shown in (d).
In (a) the relative intensity of each detection channel was calculated over the entire dynamic range of the TCPSC system using the experimental setup in Fig. 5(a). The results were used to determine the useful dynamic range and determine the relative SFs (b). The SF results were then used in calibrating raw data files.
In (a), the data calibration process was analyzed by taking the Fl-to-Tr ratio measurements of the calibrated relative intensity data. Using the experimental setup depicted in Fig. 5(a), a Fl-to-Tr ratio of one can be expected for well calibrated data. The validity of absolute intensity data values were also examined in (b) and determined to be problematic, based on an observed 11% standard deviation in the data that should be calibrated to the same value. The Fl-to-Tr data type appeared more resistant to errors from possible misalignment of the stage with respect to the gantry’s center of rotation, as shown in (c).
The position of a 25 mm phantom was systematically adjusted between the proper working distance and ±10 mm out of focus. The percent deviation in calibrated intensity relative to the proper working distance is shown in (a) and the Fl-to-Tr ratio results are highlighted in (b).
A cross sectional image of the 25 mm diameter resin phantom is shown in (a). System linearity was examined by filling the target region with a range of protoporphyrin IX concentrations with Intralipid to match the phantom. The recovered Fl yield values , from tomography were as shown in (b). The corresponding images are shown without prior information (top) and with (bottom) priors in (c). In (d) a profile plot is shown for the reconstruction of the highest concentration, showing the cross section vertically through the inhomogeneity, for the two cases of with and without prior information in the reconstruction.
An image of the mouse phantom is shown in (a), with an anatomical image obtained from the microCT shown in (b) in the plane of FT imaging. The superimposition of the microCT and corresponding Fl image is shown in (c) with diffuse tomography and in (d) with the use of spatial prior information from the scan.
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