3.1.

Experimental Setup

The mesoscopic epifluorescence tomography (MEFT) system employs standard optoelectronic components (Fig. 1). The beam of a diode laser-pumped all-solid-state laser with an output power of 5 mW at a wavelength of λ = 473 nm (CNI, Changchun, China) is reflected off a dichroic mirror (Thorlabs, Newton, New Jersey) and scanned over the object's surface using x/z-moving stages (Standa, Vilnius, Lithuania) with a travel range of 25 mm in each direction, to create arbitrary excitation points. Fluorescence emission reflected back into the optical pathway is transmitted through the dichroic mirror due to its longer wavelength, filtered through a long pass color filter (455 nm, Schneider Optik, Bad Kreuznach, Germany) and subsequently collected by a 12-bit 3-CCD camera with a frame rate of 51.5 Hz (Hamamatsu Photonics, Hamamatsu City, Japan) using a 35 mm wide angle lens (Carl Zeiss, Oberkochen, Germany). Acquired images are corrected for dark noise before feeding them into the reconstruction algorithm. To reduce required data storage and computation time, a region of interest in the images is chosen outside of which image data is discarded.

Fig. 1

Schematic setup of the mesoscopic epifluorescence tomography system.

During the ex vivo experiments, the imaged specimen is fixed in an imaging chamber, which slightly compresses the animal using an adjustable glass plate, similar to chambers used in other in vivo tomographic imaging systems.²¹ The chamber guarantees that the animal remains stable and immobilized, without restraining breathing. In addition, using an imaging chamber allows transfer of the animal between imaging systems, e.g., to perform MEFT and x-ray tomography without body movement, making different measurements comparable to each other. Furthermore, the compression creates a defined flat imaging surface on the skin necessary for the currently used reconstruction algorithm.

3.2.

Study Preparation

3.3.

Experimental Setting

3.3.1.

Phantom study

The region of interest was defined as a square with side lengths of 5000 μm (see Fig. 2). A grid of 10×10 source positions with a spacing of 500 μm [indicated by blue dots in Fig. 2(V1)] was placed on the RoI [indicated by the gray square in Fig. 2(V1)]. The measured data volume thus consisted of 100 images at the intrinsic CCD resolution of 1024×1344 pixels. For the second series of measurements, we added further sources outside of the RoI [see Fig. 2(V2)] and accordingly acquired 196 images. Figure 2(V3) indicates the third setting, where the outer grid was refined by positioning additional sources with a spacing of 250 μm (red circles) and thus yielded a data volume of 296 images. Figure 2(V4) shows the complementary situation, where the inner source grid was refined. This led to a data set of 185 images. To reduce the amount of data fed into the reconstruction process, images were reduced by a factor of ten using bicubic interpolation, and a region of interest was chosen inside the images. The resulting image pixels were used as detector positions in the reconstruction algorithm. Numbers of iterations between 50 and 90 were automatically chosen as described above, refer to Sec. 2. Depending on the detected signals, exposure times were set between 4 ms for a tube depth of 200 μm and 40 ms for a tube depth of 2200 μm. However, the exposure time was held constant for all measurements at one tube depth. To acquire purely planar epifluorescence images as control data, an expanded laser beam was used for image acquisition. Beam widening was achieved using a light diffuser (Luminit, Torrance, California) and led to a spot full width at half maximum of 24.7 mm.

Fig. 2

Source grid arrangements applied in the phantom studies. The defined RoI is marked by a gray square and the blue dots indicate the commonly utilized sources (see V1). V2 shows the measurement series, where additional source positions (red circles) have been placed outside the RoI. V3 and V4 demonstrate the refinement of sources (red circles) that have been embedded in the outer and inner grid. (Color online only.)

3.4.

Validation of the Ex Vivo Results

To validate the reconstructed depth of the tube, we sectioned the imaged tissue and subsequently performed white light and fluorescence imaging using a modified cryoslicer system.²² For each measurement, the respective field of view (FOV) of the camera was marked on the skin of each mouse. Directly after the MEFT imaging session, the mice were frozen within the imaging chamber at −20°C to ensure that the geometry would not change between MEFT imaging and cryoslicing. Subsequently, the mice were removed from the chamber, and the tissue regions within the FOV of the measurements were embedded in optimal cutting temperature compound (OCT) and refrozen to −80°C. Afterwards, the tissue samples were cut in slices at a thickness of 250 μm, during which a white light image as well as a filtered fluorescence image was acquired for each slice. From the images, the depth and position of the fluorescent tube could be accurately extracted as shown in Fig. 6d. Furthermore, we used the cryosection images to create a surface plot of the abdomen of mouse 1 and the fluorescent tube that was inserted in its right leg. To get an impression of the perspective of the camera, the merged surface plots are shown in Fig. 3. Additionally the RoI and the anatomical orientations are indicated.

Fig. 3

Merged surefaceplots of the abdomen and inserted fluorescent tube (green) shown in the perspective of the camera (mouse 1, right leg). Additionally the region of interest and the anatomical orientations are indicated. (Color online only.)

4.1.

Phantom Studies

The reconstruction results for the phantom study are shown in Fig. 4 for each tube depth (rows in the figure) and source grid arrangement (columns in the figure). In the first row, the epifluorescence images and their line profiles, which were detected orthogonal to the middle of the fluorescent tube, are shown. The full width at half maximum (FWHM) in the middle of the tube at a depth of 200 μm was detected as FWHM_{200 μm} = 246 μm and increased up to a FWHM_{2200 μm} = 780 μm measured for a depth of 2200 μm. Slices of the reconstructed fluorescence distribution, following the center of the tube, are presented in the rows of the figure. Data acquisition and reconstruction performed with the source grid arrangement that is shown in Fig. 2(V1) lead to significantly improved reconstructions of shape and dimension of the tube (V1, FWHM_{200 μm} = 147 μm and FWHM_{2200 μm} = 290 μm) as compared to the raw epifluorescence images. The results [Fig. 2(V2), FWHM_{200 μm} = 154 μm and FWHM_{2200 μm} = 284 μm] of the enlarged source grid arrangement [Fig. 2(V2)] are comparable to the previous row Fig. 2(V1), but the boundary artifacts have been removed. However, in the case of imaging a limited area, e.g., the leg, some source-detector pairs of the enlarged source grid arrangement [Fig. 2(V2)] might be positioned outside of the tissue, which would lead to additional artefacts. One possible solution to overcome this limitation would be to refine the peripheral grid by placing additional sources within the region of interest instead of outside the RoI, as shown in [Fig. 2(V3)]. However, the accuracy of reconstructions in fact deteriorated as boundary values of the RoI were imprecisely reconstructed (FWHM_{200 μm} = 150 μm and FWHM_{2200 μm} = 209 μm). This behaviour can be explained easily as for sources and detectors located on the periphery, measurements are always fully attributed to the region of interest, but not to effects from outside this area. Therefore, the increased amount of measurements that detect signals coming from outside the RoI led to an increase in boundary artifacts. That finding led to the fourth arrangement [Fig. 2(V4)], where the inner source grid has been refined. Due to the additional sources in the inner volume, this area received a stronger weight during the reconstruction process. When reconstructing the tube at a depth of 200 μm, this source grid refinement has a negative impact, since the boundary areas are imprecisely reconstructed. However, the deeper the tube is covered in the surrounding material, the higher is the benefit of the source grid arrangement V4 [see Fig. 2(V4)] on the accuracy of the reconstructed shape and fluorescence distribution (FWHM_{200 μm} = 149 μm and FWHM_{2200 μm} = 267 μm).

Fig. 4

The results of the phantom study are exposed for each tube depth (rows) and source grid arrangement (columns) in this table. In the first row the epifluorescence images and their line profiles, which were detected orthogonally to the middle of the fluorescent tube, are shown. Slices of the reconstructed tube, following the center of the reconstructed fluorescence distribution, are presented in the rows V1-V4 utilizing the source grid arrangement V1-V4.

Due to the improved source grid arrangements, the data acquisition and reconstruction process are more time consuming. Data acquisition took t = 4 to 5 sec per source position, which led to a total acquisition time for one complete data set of t_V1 = 9 min (V1) up to t_V3 = 24 min (V3). The reconstruction time for the enlarged volume [Fig. 2(V2)] or additional measurements [Figs. 2(V3) and 2(V4)] was measured as t_V1 = 9 min (V1), t_V2 = 31 min [Fig. 2(V2)], t_V3 = 26 min [Fig. 2(V3)] and t_V4 = 17 min [Fig. 2(V4)] per iteration.

To validate the phantom study using different source grid arrangements, a more quantitative analysis is presented in Fig. 5. The fluorescence distributions at a depth of 1200 μm along the tube are shown in Fig. 5a. The solid line indicates the fluorescence signal at the center of the tube in the epifluorescence measurement. Accordingly, the reconstructed fluorescence concentrations using the source grid arrangement V1 (dashed curve), V2 (dotted line), V3 (line with diamonds), and V4 (line with circles) are presented. It can be seen that the variation of the detected fluorescence distributions measured in the epifluorescence image is negligible. This is due to the constant tube depth and homogeneous optical properties of the surrounding material. The source grid arrangement V1 leads to a steep drop of reconstructed fluorescence intensity at the grid boundaries to less than 60% of the maximum intensity. Utilizing the source grid arrangement V2 provides the best result, as the reconstructed fluorescence distribution is continuously more than 94% of the maximum. V3 yields a drop at the center of the tube to less than 80% of the maximum fluorescence distribution. This effect is due to the refined and thus higher weighted boundaries. The fluorescence distribution given by the source grid arrangement V4, drops to 90% at the boundaries. This effect is due to the refined and accordingly higher weighted inner volume. However, the benefit of the source grid arrangement V4 increases for deeper tube locations, as shown in Fig. 4. In Fig. 5b, the actual tube diameter (line with dot markers) and the reconstructed values are plotted. The FWHM of the observed epifluorescence signal versus the tube depth is presented as a solid line. To obtain values of the reconstructed diameter using the source grid arrangements V1 (dashed line), V2 (dotted line), V3 (line with diamonds), and V4 (line with circles), the FWHM of the reconstructed tube was determined in the x- and z-direction for each slice along the tube (y-direction). The voxel values were averaged to obtain the signal values for each source depth. The FWHM in the planar image is, on average, more than three times larger than the actual tube diameter. However, the FWHMs of the reconstructions correspond well with the actual values, where the source grid arrangement V1 leads to the largest error at a depth of 2200 μm. In Fig. 5c, the actual depth of the tube (solid line with dot markers) is plotted versus the average depth of the reconstructed fluorescence distribution. The reconstructed tube depths using the source grid arrangement V1 (dashed line), V2 (dotted line), V3 (line with diamonds), and V4 (line with circles) lead to similar results, where the localization error rises with increasing depth. The maximum error of 9% is obtained at a depth of 2200 μm for the reconstruction using the source grid arrangement V1.

Fig. 5

Validation of MEFT phantom imaging results. (a) Fluorescence distribution in a depth of 1200 μm along the tube. The solid line depicts the relative fluorescence intensity in the epifluorescence imaging along the center of the tube. The reconstructed fluorescence concentration using the source grid arrangement V1 (dashed line), V2 (dotted line), V3 (line with diamonds), and V4 (line with circles) are presented. All curves were normalized to their respective maximum value. (b) Plot of the FWHM of the reconstructed tube diameter for different tube depths. The horizontal line with dot markers depicts the actual tube diameter of 150 μm. The solid line indicates the tube diameter in the epifluorescence image. To obtain the values of the reconstructed diameter using the source grid arrangement V1 (dashed line), V2 (dotted line), V3 (line with diamonds), and V4 (line with circles) the FWHM of the reconstructed tube was determined in the x- and z-direction for each slice along the tube (y-direction). The voxel values were averaged to obtain the signal values for each source depth. (c) Reconstructed depth in comparison to actual depth (solid line with dot markers). The dashed line indicates the source grid arrangement V1, the dotted line, V2, the line with diamonds, V3, and the line with circles, V4.

4.2.

Ex Vivo Studies

The results of the ex vivo measurements of mouse 1 (right leg) are presented in Fig. 6. The epifluorescence measurement is shown in Fig. 6a. The intensity of the fluorescent signal strongly decays along the y-direction, corresponding to the increasing depth at which the fluorescent tube was located. In comparison, Fig. 6b depicts an angular slice through the reconstructed volume, following the center of the reconstructed fluorescence distribution. Although some slight variation in intensity can be observed, it occurs independently of depth and, overall, the signals within the reconstructed tube are more homogeneous than in Fig. 6a and the tube has a focused appearance. To visualize the location of the fluorescent tube in the reconstructed 3D volume stack, a surface plot of the reconstructed fluorescence distribution is presented in Fig. 6c. The colorbar in the grayscale images was normalized to the maximum intensity in the images.

Fig. 6

MEFT ex vivo imaging results. (a) Epifluorescence intensity distribution. (b) Angular slice of reconstructed fluorescence distribution along the depth of the tube center. (c) Three-dimensional rendering of the reconstructed fluorescent tube. The box depicts the dimension and localization of the reconstructed tube. (d) White light cryoslice image where the tube reached a depth of 640 μm. The rectangular inset shows a magnification of the area of the fluorescent tube, where the green false color shows an overlay of the fluorescence distribution detected in the cryoslicer. Additionally the tail bone (Tb), the tibia (Ti), the colon (Co), the ovary (Ov) and the bladder (Bl) are indicated.

As an example, the image of the cryoslicer section that was taken at 2500 μm in the y-direction [Fig. 6b] is shown in Fig. 6d. The field of view (FOV) and the position of the glass plate are indicated. It can be observed that the skin surface, although compressed by the glass plate, was not perfectly flat. Skin folds within the field of view, however, were less than 250 μm in depth. The rectangle marks the area where the fluorescent tube was located. The corresponding region is enlarged, featuring (in green) the overlay of the fluorescence as detected in the cryoslicer. Additionally, the tibia (Ti), the tail bone (Tb), the colon (Co), the ovary (Ov) and the bladder (Bl) are indicated to get a sense of the tube location inside the mouse.

To validate the reconstruction of mouse 1 (right leg) obtained by MEFT imaging in a more quantitative manner, different analyses are presented in Fig. 7. The fluorescence signal or estimated concentration along the tube (y-direction) is shown in Fig. 7a. The solid line shows the fluorescence signal from the center of the tube in the epifluorescence measurement. The dashed curve depicts the MEFT-reconstructed fluorescence concentrations along the tube. To obtain these values, at first the FWHM of the reconstructed tube was determined in the x- and z-directions for each slice along the y-axis. Within this area, voxel values were averaged to obtain the signal values in the figure. Again, the intensities in the epifluorescence image are strongly correlated with inclusion depth, while the reconstructed concentrations relate well to the true situation. Figure 7b depicts the FWHM of the observed signals for the planar epifluorescence image (solid line) as well as for the x- (dotted line) and z-axis (dashed line) in the 3D volume stack in comparison to the actual diameter of the tube (solid line with dots). While the FWHM in the planar image is, on average, more than three times larger even though the tube is located less than 1 mm underneath the skin, the FWHM in the reconstruction corresponds well with the true values. Along the x-axis, the average diameter is reconstructed as 231 μm, while the reconstruction along the z-direction has a greater accuracy (170 μm). In Fig. 7c, the depth of the tube as obtained by image analysis on the cryoslicer images (solid line with dots) is plotted versus the average depth in the reconstructed volume (dashed line). With increasing depth, the localization error increases accordingly, but remains well below 10% up to a depth of 1000 μm.

Fig. 7

Validation of MEFT ex vivo imaging results. (a) Fluorescence distribution along the tube length (y-direction). The solid line depicts the relative fluorescence intensity in the epifluorescence imaging along the center of the tube. The dashed curve shows the corresponding reconstructed fluorescence concentrations. Both curves were normalized to their respective maximum values. (b) Plot of the reconstructed tube diameter (FWHM) for different depths of the tube. The horizontal line with dot markers depicts the actual tube diameter of 150 μm. The solid line indicates the tube diameter in the epifluorescence image. The dashed curve shows the reconstructed diameter in z-direction and the dotted in x-direction. (c) Reconstructed depth in comparison to actual depth. The solid line with dots indicates the actual and the dashed curve the reconstructed depths.

The three additional measurements, where the extra fluorescent tubes have been analyzed, show similar results. Mouse 1 (left leg) showed a strong correspondence between the tube depth obtained in the cryoslicer images and the reconstructed depth (error < 5%). However, in this case the maximum tube depth in the RoI was 850 μm. Mouse 2 (right leg) presented a nearly constant tube depth in the RoI of 500 μm to 550 μm in the cryoslicer images as well as in the reconstructions (error < 5%). For the RoI of Mouse 2 (left leg), the tube depth obtained in the cryoslicer images was measured between 300 μm and 900 μm. Again, we found the localization error increases with depth, but remains between 2% and 10%. Additionally, we calculated the contrast to noise ratio CNR = 12401, which is an adequate value for reconstruction purposes.¹ The leakage of excitation light alternated between 150 and 200 photon counts. These values are negligible compared to the background levels that are subtracted.