Maps of in vivo oxygen pressure with submillimetre resolution and nanomolar sensitivity enabled by Cherenkov-excited luminescence scanned imaging

Abstract

Low signal-to-noise ratios and limited imaging depths restrict the ability of optical-imaging modalities to detect and accurately quantify molecular emissions from tissue. Here, by using a scanning external X-ray beam from a clinical linear accelerator to induce Cherenkov excitation of luminescence in tissue, we demonstrate in vivo mapping of the oxygenation of tumours at depths of several millimetres, with submillimetre resolution and nanomolar sensitivity. This was achieved by scanning thin sheets of the X-ray beam orthogonally to the emission-detection plane, and by detecting the signal via a time-gated CCD camera synchronized to the radiation pulse. We also show with experiments using phantoms and with simulations that the performance of Cherenkov-excited luminescence scanned imaging (CELSI) is limited by beam size, scan geometry, probe concentration, radiation dose and tissue depth. CELSI might provide the highest sensitivity and resolution in the optical imaging of molecular tracers in vivo.

Figure 1.

A comparison of the excitation fluence loss with depth into tissue for (a) laser/LED excitation and (b) Cherenkov excitation generated within the tissue from a LINAC radiation beam. The diagram of the experimental setup is shown (c) with measurement devices. In (d) a raster scan approach to image measured by the ICCD is shown with gated emission detected by a spectrometer system. In (e) the absorption and (f) the luminescence emission spectra are shown for PtG4, with peak wavelengths labeled, and overlaid with the Cherenkov spectrum (blue) from single solution scans. The structure of PtG4 is illustrated in the Supplementary Information, Figure S2.

Figure 2.

The configuration of the radiation beam shape and region of the tissue where Cherenkov would be generated by a (a) single broad beam throughout the 3D volume of the animal, or (b) in a 2D sheet beam, or (c) in a 1D pencil beam. These geometric choices each affect the signal to noise possible with CELSI, and hence the sensitivity of other parameters such as minimum concentration and depth of imaging feasible and acquisition time. Images of the field light, Cherenkov emission on a phantom and the luminescence from a single square target in the phantom are shown in (d) for the geometries in (a)-(c), with duplicate images taken in separate imaging sessions showing nearly identical image quality. The contrast to noise ratio (CNR) of the target relative to the background is shown in (e) as measured by individual scans, where each data point represents an individual acquisition. The units for the color bars on images in (d) and inset in (e) are in detected photons/cm²/s and a 1cm scale bar is in the bottom right corner of each panel.

Figure 3.

The geometry of imaging camera relative to X-ray beam entrance position is shown in (a) an vertical (or epi-illumination) scan, and (b) a lateral sheet scan. Photographs of the LINAC-camera set up for (c) vertical and (d) orthogonal camera and LINAC beam, similar to schematics (a) and (b). Single frame images of PtG4 luminescence from one capillary within a broad tissue phantom are shown in (e) and (f) for these geometries, respectively, with color bars on the same scale for the two representing the full range available on the camera. These representative single images illustrate the high background and hence poorer contrast from the epi-illumination geometry, as seen from an individual snapshot. The signal to background values are 15% for (e) and 220% for (f) above the background level. The units for the color bars on images in (e) and (f) are in detected photons/cm²/s and 5mm scale bar is shown in the bottom right of each panel.

Figure 4.

Signal to background ratio measurements from PtG4 contained within a 1mm capillary with (a) different concentrations in the physiologically relevant range, (b) varying depth between the capillary wall to the surface of the phantom being imaged, and (c) varying radiation dose between 1 and 500 radiation pulses, with 1.67mGy/pulse. In each graph, the line scan raw data is inset. Each individual data point represents extraction of signal to background for a single scan of a test object, with varying conditions as shown in the x-axes. The red lines in each graph illustrate the linear trendline of the data on the log-log plots. In (d) the factors affecting CELSI signal strength are schematically illustrated as being reciprocal in their effect upon signal to background, illustrated by the red lines of constant value.

Figure 5.

Source and detector placement used for (a) the Epi-fluorescence tomography (Epi-FT), (b) the Full-FT, and (c) the lateral excitation CELSI with tomographic reconstruction. The blue arrows represent the source locations, and the red arrows denote the detector locations. Reconstructed luminescence distributions corresponding to the three geometries shown in Figure 2(a)-(c), for (d) the Epi-FT, (e) the Full-FT, and (f) the CELSI. Three angles of scanning for CELSI were examined as illustrated in (g) – (i), for horizonal, vertical and diagonal, respectively, and three test objects at different depths were used as a test field for this, with reconstructions as shown in (j) – (l). The combined set of all 3 scan geometries was used for the reconstructed image (m), showing the best preservation of the three objects space and intensities with 30 iterations in each reconstruction. These simulations were carried out once for each in individual graph shown.

Figure 6.

To establish the theoretical limits to spatial resolution, reconstructed results using CELSI tomography are shown in units of luminescent yield, ημ_af, in the colorbar, for two inclusions with varied edge-to-edge distance in different depths are shown, (a). The white scale bar in the bottom right is 30 mm. These simulations were carried out once for each location of the pair of 5mm diameter, 50 μM PtG4, inclusions. At each depth, the minimum resolvable distance was estimated and plotted in (b), for epi-illumination fluorescence tomography, diffuse fluorescence tomography, and CESLI tomography. Capillaries filled with PtG4 are shown (c) varying from 0.1mm diameter up to 1.0 mm diameter, and were embedded into a tissue equivalent phantom, and a sheet beam scanned laterally to extract the FWHM for each sized tube (d). Each data point reports a single measurement, and fitting was completed with r²>0.9 for each sample.

Figure 7.

The phantom experiments shown as measured once. (a) The luminescent yield images are overlaid on the top surface of the microCT image, in colorbar units of photons/cm²/s, and the reconstructed images shown with 3 orthogonal views, axial (b), sagittal (c) and transverse (d) with the colorbar reporting values for (b)-(d) in units of ημ_af. The in vivo imaging study is shown from a single animal, with an x-ray CT scan and a summed intensity projection image of the luminescence overlaid on the color-coded CT scan (e) in colorbar units of photons/cm²/s. Then CELSI tomographic data is overlaid on the high resolution microCT images for axial (f), sagittal (g) and 3D perspective (g) views of the reconstruction. The location of the two MDA-MB-231 tumors grown on the hind flanks are shown by arrows, with 50 nmol PtG4 injected into the tumor with a blue arrow, and 10 nmol PtG4 was injected into the tumor with the orange arrow, and the entire body of the animal was scanned and reconstructed with CELSI. The reconstructed image shows recovery of the high concentration injection while the lower concentration injection is not recovered, to illustrate the detectable limit is in the range of tens of nanomoles. (A full rotating 3D video of this mouse is available in the Supplementary Information, Section S.7)

Figure 8.

Nude mice with subcutaneous breast adenocarcinomas (MDA-MB-231) were injected with 50uL of 25uM PtG4 in each tumor and imaged. In (a) Maximum intensity projection (MIP) images of CELSI are shown with increasing delays after radiation pulse, with color bar in photons/cm²/s, and (b) the pixel-wise lifetimes (τ) estimated alive, and after sacrifice as a low pO₂ control. These were used to (c) calculate pO₂ maps via the Stern-Volmer relationship. In (d) box-plots show lifetime estimates for all tumors (n=8), and (e) shows the calculated pO₂ estimates for these (n=8). Measurement scans were acquired once for each animal. (A video of a single scan sequence is available in the Supplementary Information, Section S.7).