The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking

Magnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells. Cells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. Using the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 × 1016 19F atoms) were detected with 19F MRI, with SNR > 5. MPI has the potential to be more sensitive than 19F MRI for cell tracking. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

Sensitivity for MPI. MPI directly detects superparamagnetic iron oxide nanoparticles (SPIONs), which are used as cell labeling agents. Strong magnetic gradients (T/m) are used to localize SPIONs by creating a field-free region (FFR), and oscillating excitation fields (mT) are applied to alter the magnetization of SPIONs present in the FFR. The FFR is traversed across the imaging field of view and the change in SPION magnetization is detected by a receive coil. The resulting image has positive contrast, and the signal is directly related to the amount of SPION and cell number.
The type of SPION and the amount of SPION taken up by cells are two major factors that determine the sensitivity of MPI for cell tracking. Optimal SPIONs for MPI will strongly magnetize with magnetic fields outside the FFR and experience fast relaxation rates within the FFR. Monodisperse, single core SPIONs with core sizes of ~ 25 nm have been considered ideal 4,5 . Cell labeling by SPIONs is typically conducted through co-culture and is dependent on endocytosis. Carbohydrate coatings, such as carboxydextran, increase the interactions of SPIONs with cell membranes, similarly, transfection agents can be used to coat SPIONs to enhance their incorporation to cells.
The most commonly used SPION for MPI is ferucarbotran (VivoTrax™, Magnetic Insight Inc., Alameda, USA), which is repurposed from the original use as an MRI contrast agent (Resovist®, Bayers Healthcare). Ferucarbotran has a carboxydextran coat and is a polydisperse agent; some nanoparticles have a core size of 24 nm (30%) and the majority have 5 nm cores 6 . The in vitro detection limit using ferucarbotran has been estimated to be approximately 1000 embryonic stem cells (27 pg/cell) 7 . SPIONs designed specifically for MPI are being investigated and show improved performance; in vivo, Wang et al. (2020) demonstrated detection of 2500 bone mesenchymal stem cells with cubic nanoparticles (29 pg/cell) 8 . Beyond this, detection limits for SPION-labeled cells have not been carefully studied. Sensitivity for 19 F MRI. For 19 F MRI, cells can be labeled with perfluorocarbon agents such as perfluoropolyether (PFPE) nanoemulsions. Since there is little endogenous 19 F in biological tissues, these cells can be visualized with high specificity. The signal intensity of these images is directly linear to the number of 19 F atoms and cell number. The sensitivity of 19 F MRI cell tracking is impacted by 19 F cellular loading. PFPE are formulated into nanoemulsions for safe and effective labeling of cells; clinical-grade PFPE agents are available (CS-1000, CelSense Inc.) and have been used in humans 9 . The amount of 19 F uptake is different for various cell types due to differences in cell size and endocytic ability 10 .
MRI hardware and imaging parameters also play a major role in determining 19 F sensitivity. Higher magnetic field strengths improve detectability of cells, additionally, the use of specialized coils is integral. For example, our group has previously demonstrated comparable signal detection at 9.4 T using a birdcage coil compared to 3 T using a surface coil 11 . 19 F cellular detection limits using various field strengths, hardware, and sequences have been reviewed by Srinivas et al. 10 . Notably, there were no reported studies at field strengths ≤ 3 T. The translation of cellular MRI techniques to the clinic will require the use of human MRI systems at clinical field strengths. Our group has demonstrated a cellular detection limit of 25,000 PFPE-labeled macrophages in vitro at 3 T 11 . In the first human clinical trial at 3 T, an in vivo cellular detection limit between 1 and 10 million dendritic cells was demonstrated 9 . The sensitivity of 19 F MRI for detection of MSC and breast cancer cells at 3 T has not been evaluated.
MPI and 19 F MRI have similar characteristics for cell tracking (positive contrast and quantitation), and MPI is expected to be more sensitive for cell tracking, however, this has not been carefully compared. The objective of this study is to assess the in vitro and in vivo cellular sensitivity of MPI and 19 F MRI for MSC and breast cancer cells.
Cell labeling. 2 × 10 6 4T1 cells or MSCs were seeded for labeling in T75 cm 2 flasks. After 24 h, 2.5 mg/mL PFPE nanoemulsion (Cell Sense, Celsense Inc., Pittsburgh, PA, USA) was added to 10 mL complete media and left to co-incubate overnight 12,13 . Alternatively, cells were labeled with 55 μg Fe/mL ferucarbotran (Vivotrax, Magnetic Insight Inc., Alameda, CA, USA) using transfection agents in a protocol described by Thu et al. 14 Briefly, 60 μL protamine sulfate (stock 10 mg/mL) was added to 2.5 mL of serum-free medium, and in a second tube, 20 μL heparin (stock 1000 U/mL) and 90 μL ferucarbotran (stock 5.5 mg/mL) was added to 2.5 mL serumfree medium. These two tubes were individually vortexed, then combined. After adhered 4T1 cells or MSC were washed in PBS, this labeling mix was added to the cells. 4 h later, 5 mL of complete media was added to cells and left to co-incubate overnight.
Evaluation of cell labeling. A cytospin of 100 × 10 3 cells was prepared for all labeled cells which were fixed in 3:1 methanol:acetic acid. Iron-labeled cells were stained with Perl's Prussian blue (PPB) to identify iron in cells 15 . 19 F-labeled cells were labeled with nuclear fast red to assess for PFPE nanodroplets 11 . Microscopy of these slides was conducted using the EVOS imaging system (M7000, Thermo Fischer Scientific). The mean intracellular 19 F content in MSC was measured by NMR using a Varian Inova 400 spectrometer (Varian Inc, Paulo Alto, USA) and methods previously described 11 . Preparation of cell pellets for imaging. PFPE-labeled cells or ferucarbotran-labeled cells were washed 3 times in phosphate-buffered saline (PBS) and collected in triplicate samples prepared in a dilution series: 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 (× 10 3 ) cells. Ferucarbotran-labeled cells were pelleted in 50 μL PBS for MPI. 19 F-labeled cells were pelleted by centrifugation and covered in 50 μL agarose. This process was repeated two more times to produce a total of 9 replicates for each cell number and cell type (4T1 or MSC), with each cell labeling agent (PFPE or ferucarbotran).

MPI of ferucarbotran-labeled cells.
All MPI was conducted on a MOMENTUM™ system (Magnetic Insight Inc.) (Fig. 1A). A small well was secured to the imaging bed where a PCR tube containing the cell www.nature.com/scientificreports/ pellet could be placed. This allowed for imaging to occur in the identical location and for the user to easily transfer cell samples. Each cell pellet was imaged individually with the following parameters: field of view (FOV) = 6 cm × 6 cm, gradient strength = 3.0 T/m, dual-channel acquisition (X and Z), excitation amplitude = 22 mT (X-channel) and 26 mT (Z-channel), imaging time = 1.5 min. Cell pellets were imaged in descending order of cell number. Once the cell pellet was undetected, 2D imaging with 8 averages (imaging time = 11.8 min) and 3D imaging was conducted for all 9 replicates, in attempt to improve MPI sensitivity. 3D images combine 35 projections in a FOV = 6 × 6 × 6 cm (imaging time = 22.8 min). If cells were detected in these longer scans, fewer cells were imaged until undetected in 2D (8 averages) and 3D.
19 F MRI of perfluorocarbon-labeled cells. 19  In preparation for MPI, 4 mice were fasted for 12 h, by removing food pellets from their cages, and a piece of ex-lax® (regular strength stimulant laxative) was added to the cage. Additionally, cotton bedding, which has been shown to produce MPI signal (unpublished results) was replaced with corn bedding. The goal of these measures was to minimize background signal associated with the iron in the mouse digestive system.
To investigate differences in sensitivity in vivo, 1 × 10 5 ferucarbotran-labeled MSC were injected to NSG mice by subcutaneous, intraperitoneal, or intravenous injection. A fourth mouse received 2 × 10 6 ferucarbotran-labeled MSC by subcutaneous injection. Each mouse was imaged with MPI with a FOV = 12 cm × 6 cm × 6 cm in 2D (2.2 min). All other imaging parameters are described above. MPI was conducted immediately before injections, to measure background signal, and immediately after injections. MPI signal for each mouse was calculated as the difference between pre-injection signal (background) and post-injection signal. MPI signal from cells in vivo was compared to MPI signal from cell pellets.
Similarly, 2 × 10 6 PFPE-labeled MSC were injected to NSG mice by subcutaneous or intraperitoneal injection. A third mouse received fewer PFPE-labeled cells (1 × 10 5 ) by subcutaneous administration. Following cell  www.nature.com/scientificreports/ administration, 19 F MRI was conducted for each mouse by placing the surface coil directly above the injection site to maximize sensitivity (shown in Fig. 7E); the mouse receiving cells by subcutaneous was prone for imaging and the mouse receiving cells by intraperitoneal injection was supine. Imaging parameters are the same as listed above, however with a FOV = 60 × 30 mm with matrix size = 60 × 30 (1 × 1 × 1 mm 3 resolution), and 200 excitations (imaging time = 35.5 min). In vivo 19 F signal was quantified (described below) and compared to signal detected from cell pellets, which were included alongside the mouse.
Image analysis. For MPI signal calibration, an additional 8 samples of ferucarbotran (5.5 mg/mL) were imaged with identical parameters in a dilution series in PBS: 33, 22, 11, 5.5, 2.25, 1.38, 0.68, 0.34 μg ferucarbotran. A linear relationship was found between iron mass and MPI signal (Fig. 1C) and the equation of the line was used to calculate associated iron content from each cell sample. 2D MPI of the empty sample holder was conducted at the beginning (0 h), middle (3 h), and end (6 h) of six imaging sessions. The standard deviation of background noise (SD noise ) was measured in these 18 images. To quantify signal from ferucarbotran-labeled cells in pellets and in vivo, a threshold of 5 times the average background SD noise was used to mask lower amplitude signal and yield a reliable measurement of MPI signal. This imaging criteria is based on MPI signal with SNR > 5 (Rose Criterion) 17 . Total MPI signal was calculated as mean MPI signal * volume of ROI (mm 2 or mm 3 ).
Delineation of 19 F signal was conducted using a similar method as MPI (5*SD noise ). Background SD noise of 19 F signal for each 3D image was measured by drawing an ROI in background noise of one image slice. Due to Rician distribution observed in background signal noise, 19 F signal between 5*SD noise and 8*SD noise was corrected using a factor of 0.655, as described by Bouchlaka et al. (2016) 18 (Fig. S2). Total 19 F signal was calculated as mean 19 F signal * volume of ROI. Two reference phantoms containing 3.33 × 10 16 19 F spins/μL were included in the imaging field of view for calibration (Fig. 1D). 19 F content from cell pellets was calculated as: We defined MPI and 19 F MRI in vitro cell detection limits as the minimum number of MSC and 4T1 cells detected with SNR > 5. Thus, cells with signal below the 5*SD noise criteria were considered undetected. The amount of ferucarbotran or PFPE associated with the lowest cell number was calculated as iron mass per cell * number of cells or 19 F atoms per cell * number of cells, respectively. Statistical analysis. All statistical analysis were performed using GraphPad Prism version 9. Linear regression was performed for MPI calibration (known iron mass vs. measured MPI signal) to determine the calibration equation. This line is forced through the origin, under the assumption that background MPI signal, without a sample of iron, has an average of 0. Pearson's correlation was conducted for MPI (number of cells vs. measured MPI signal) and 19 F MRI (number of cells vs. measured 19 F signal). Analysis of co-variance (ANCOVA) was used to evaluate whether the MPI sensitivity (slope) was significantly different for ferucarbotran-labeled 4T1 cells and MSC (number of cells vs. measured MPI signal). Analysis of variance (ANOVA) was used to analyse differences between MPI signal measured from each cell number and again for 19 F signal measured from each cell number. A p-value of 0.05 was used to determine statistical significance, unless otherwise indicated.

Results
MPI cellular sensitivity. The detection of ferucarbotran-labeled cells using 2D MPI (imaging time = 1.5 min) is shown in Fig. 2A (4T1 cells) and Fig. 3A (MSC). MPI signal measured from samples of 8000-1,024,000 4T1 cells and 4000-1,024,000 MSC had SNR > 5. The iron mass significantly increased with cell number for both cell types ( Figs. 2A, 3A). Importantly, the standard deviation of background noise, measured three times over six imaging sessions, showed no significant differences (Fig. S3).
MPI signal (and the associated iron content) was strongly correlated with cell number for ferucarbotranlabeled 4T1 cells and MSC (Fig. 5A). The slope of the line was significantly steeper (factor of 2.07) for MSC compared to 4T1 cells (P < 0.0001). Enhanced ferucarbotran labeling was measured in MSC (19.09 ± 2.50 pg Fe/cell) compared to 4T1 cells (9.22 ± 1.42 pg Fe/cell), which can be visualized in PPB-stained cells (Fig. 5B).

In vivo sensitivity of MPI and 19 F MRI. A comparison between MPI signal from a cell pellet and cells
in vivo was conducted with different injection routes (Fig. 6A). These MSC were labeled with 28.9 ± 3.4 pg iron/ cell. Compared to a pellet of 2 × 10 6 ferucarbotran-labeled MSC, MPI signal was only reduced by 5% with subcutaneous injection of these cells (Fig. 6 B,C). Quantification revealed the iron mass measured from the cell pellet (52.98 μg) was similar to what was measured after subcutaneous injection (50.21 μg). However, for 1 × 10 5 MSC, in vivo MPI showed a reduction in MPI signal measured from MSC injected subcutaneously (49%), intraperitoneal (53%), and intravenous (15%), compared to signal from a pellet of 1 × 10 5 MSC (Fig. 6 D,E). For 1 × 10 5 MSC, the measured iron content was 3.13 μg in the cell pellet, compared to 1.52 μg after subcutaneous injec- www.nature.com/scientificreports/ tion, 1.66 μg after intraperitoneal injection, and 0.48 μg after intravenous injection. Therefore, the iron content measured from 1 × 10 5 cells in vivo was reduced compared to cells in the pellet, despite being the same number of cells. After mouse fasting, the background in vivo MPI signal from the mouse digestive system was 25.8 ± 10.0 arbitrary units (A.U.) (shown Fig. 6F). This background signal was accounted for in each mouse by signal subtraction, prior to calculation of MPI signal and iron mass measured from cells. The detection of 2 × 10 6 19 F-labeled MSC in vivo was compared to MSC in a pellet (Fig. 7). Reduced 19 F signal (72%) was detected from MSC following subcutaneous injection (Fig. 7A, D). After intraperitoneal injection, the same number of cells were dispersed and appeared as lower intensity 19 F signal (Fig. 7B), however higher 19 F signal was measured from these cells compared to the pellet by 6.65 times (Fig. 7D). 1 × 10 5 MSC administered subcutaneously were undetected as this cell number is below the detection limit (Fig. 7C).

Discussion
In this study, we began with an evaluation of in vitro sensitivity for MPI and 19 F MRI of cells using ferucarbotran and PFPE nanoemulsions as labeling agents (respectively). Overall, fewer MSC were detected using MPI (4000) compared to 19 F MRI (256,000) using the same imaging time (1.5 min per cell pellet). Compared to ferucarbotran-labeled MSC, more 4T1 cells were required for MPI detection (8000) as a result of lower cell uptake of ferucarbotran. These limits were defined with imaging criteria SNR > 5 and tested with 9 replicates to provide confidence.
These measurements of lowest cell number detected with MPI and 19 F MRI are reasonable based on previous reports. As described earlier, Zheng et al. 7 achieved detection of approximately 1000 ferucarbotran-labeled human embryonic stem cells (27 ng n/cell, 27 ng) with SNR > 5. Our MSC detection limit was higher (4000 cells  www.nature.com/scientificreports/ or 76 n g) and this is resulting from differences in MPI systems and acquisition. The amount of iron we detected (76 ng) is consistent with findings by Liu et al. 19 , where 64 ng ferucarbotran could be detected with mean SNR of 3.6, using another MOMENTUM MPI system and the same 2D imaging parameters. With longer imaging times in 3D, we demonstrated a minimum of 1000 ferucarbotran-labeled MSC (19 n g) could be visualized with SNR > 3. To the best of our knowledge, this is the lowest report of ferucarbotran detected from labeled cells. This study was the first to measure MPI detection limits for iron-loaded cancer cells (in 3D, as few as 4000 4T1 cells were detected, or 37 n g iron).
Cell labeling is fundamental in determining cellular sensitivity for both MPI and 19 F MRI. We previously discussed factors that impact labeling efficiency with SPIONs 20 . In this study, we observed enhanced MPI detectability of MSCs compared to breast cancer cells, owing to increased endocytosis of SPION. Likewise, 4T1 cells did not label with PFPE sufficiently for 19 F MRI detection. Increased uptake of SPION/PFPE is expected in phagocytic cells and cells which have a larger cytoplasmic volume 21 , such as macrophages and dendritic cells. Although MSC is considered a non-phagocytic cell type 22 , enhanced labeling compared to breast cancer cells has been observed with SPIONs 15 and PFPE 10 . The cell labeling protocols used in this study use commonly used concentrations of ferucarbotran and PFPE for cell labeling and have been well established. We, and other groups, have not observed compromises in cell viability or function at these labeling concentrations, even in delicate cell types 12,13,23 -27 . In this study, we use transfection agents to facilitate uptake of ferucarbotran. It is expected that SPION surface modifications will be improve labeling efficiency without transfection agents 8,28 . Further improvements to PFPE nanoemulsions will enhance 19 F cellular sensitivity, such as incorporation of paramagnetic agents 29,30 , or the addition of surface modifications to enhance the uptake of PFPE nanoemulsions 31 .
We recognize it is impossible to directly compare cell detection with MPI and 19 F MRI due to their inherent differences, including structural configurations and imaging parameters. For this study, we attempted to optimize unique aspects of each modality in favor of sensitivity. MPI of cells was conducted using weak gradients  19 F signal measured from 2 × 10 6 cells injected subcutaneous (SQ) was reduced compared to signal measured from the cell pellet, however, elevated 19 F signal was measured from cells following intraperitoneal (IP) injection. (E) The dual-tuned surface coil is approximately the same size as a mouse and is placed directly over the injection site for imaging. www.nature.com/scientificreports/ (3 T/m) to increase the size of the FFR and enhance sensitivity. For FFL (line) MPI, the use of weak gradients (3 T/m) compared to stronger gradients (e.g. 6 T/m) expands the volume of FFR. This leads to enhancement in sensitivity, at the cost of reduced resolution. It is also expected that signal averaging would improve sensitivity, however this has not been studied for MPI. In 2D, a significant reduction in background noise was measured with 8 averages compared to 1 average, however, this did not improve cell detection with MPI, as we defined it. 3D imaging using 35 projections did offer improvement in sensitivity for both ferucarbotran-labeled MSC (detection of 2000 cells) and breast cancer cells (4000 cells). Lastly, optimization of excitation amplitudes may lead to improved cell sensitivity; in this study we used 22 mT (X-channel) and 26 mT (Z-channel) by default. 19 F MRI of PFPE-labeled cells was conducted at a clinical field strength (3 T). The implementation of the surface coil and optimized 3D bSSFP sequence is crucial to enable 19 F cell tracking at 3 T. The theoretical optimal flip angle for 19 F at 3 T is 72° and our investigation showed highest SNR was produced for flip angles between 60-80°. However, transmit/receive surface coils provide non-uniform sensitivity, due to spatial variations in applied energy and flip angle 32,33 . For this reason, cell pellets were imaged directly in the center of the coil to maximize sensitivity. Likewise, the surface coil was placed directly above the region of interest for in vivo 19 F imaging. High signal averaging was used for 19 F image acquisition (115) to improve SNR. Longer imaging times with 345 signal averages enabled detection of 3 additional pellets of 256 × 10 3 cells and 3 of 9 pellets of 128 × 10 3 PFPE-labeled MSC.
After assessing in vitro cell detection limits of MPI and 19 F MRI, a preliminary assessment of in vivo detection factors was conducted. It has previously been shown that there is no attenuation of MPI signal from biological tissue 34,35 . In agreement, following subcutaneous injection of 2 × 10 6 MSC, we measured only a small reduction in cell detection with MPI (5%). However, for a lower cell number (1 × 10 5 ), there was reduction of signal measured in vivo compared to the pellet (by 47-85%, depending on injection route). Here we recognize that the dispersion of cells from the injection site reduces the cell density per voxel, leading some cells to fall below the intravoxel detection limit. MPI detection of MSC was most reduced after intravenous injection (85% reduction). It is expected that cells administered intravenously would be most disperse as they circulate through the venous circulation before accumulating in the lung capillaries (shown in Fig. 6A). Similarly, previous work by Wang et al. (2020) 8 showed that 1 × 10 5 ferucarbotran-labeled stem cells could be detected with MPI in vivo following subcutaneous injection but not following intravenous injection. In our study, we could achieve detection of 1 × 10 5 cells after intravenous injection, which can be attributed to the choice of gradient strength (3.0 T/m vs. 5.7 T/m).
Likewise for 19 F MRI, dispersion of PFPE-labeled cells (1 × 10 6 ) in patients was previously reported to limit the detectability of these cells following administration 9 . In our study, 19 F signal detected from a cell pellet of 2 × 10 6 PFPE-labeled MSC was higher than 19 F signal from the same number of cells injected subcutaneously. Conversely, 19 F signal measured from cells injected to the intraperitoneal space was overestimated. This result could be explained by the large quantification region, as 19 F density per imaging voxel was 3.79 × 10 16 19 F/mm 3 for cells in a pellet, compared to 2.03 × 10 16 19 F/mm 3

for cells in vivo.
For both MPI and MRI, there are other important considerations which reduce detectability of cells in vivo. For MPI, this includes increased background signal associated with mouse digestion 36 . While we accounted for background signal in the mouse using signal subtraction, this technique is not permissible for longitudinal cell tracking studies. Background signal is variable across mice and day-to-day. In our experience, this can be reduced by mouse fasting, however, the amount of signal is unpredictable. Ultimately this background signal may obscure detection of cells, especially in low cell numbers 37 , as it is challenging (at this time, impossible) to distinguish the signal associated with cells from background. Second, there is some evidence that Brownian relaxation of SPION in different tissue environments may be altered, leading to reduced MPI sensitivity 38 -40 . Brownian motion refers to the physical rotation of SPIONs; this motion is reduced in tissues with increased stiffness (e.g. muscle) which leads to increased Brownian relaxation times (thus, lower sensitivity and resolution). Ongoing work aims to determine whether this plays a role in the detection of SPION-labeled cells. Another consideration affecting 19 F detection of PFPE-cells in vivo is the coil filling factor. The volume of a mouse is much larger than the volume of a cell pellet, thus SNR for detection of cells in vivo is expected to be reduced due to increased image noise. Lastly, mouse breathing motion can lead to blurring of signal which will reduce the maximum signal intensity associated with cells and could potentially render cells undetected by MRI and MPI.

Conclusion
MPI has the potential to be more sensitive than 19 F MRI for cell tracking. In this study, commonly used cell labeling agents, ferucarbotran and perfluoropolyether nanoemulsions, were used to label MSC and 4T1 cells to assess cellular sensitivity of MPI and 19 F MRI, respectively. Fewer MSC (4000 cells, 76 ng iron) were detected with MPI than 19 F MRI (256,000 cells, 9.01 × 10 16 19 F atoms), using the same scan time. Furthermore, reduced ferucarbotran labeling was observed in 4T1 breast cancer cells compared to MSC, leading to a detection limit of 8000 breast cancer cells (74 ng iron). With longer imaging times, as few as 2000 MSC (38 ng ferucarbotran) and 4000 breast cancer cells (37 ng ferucarbotran) were detected with MPI and 128,000 MSC (4.51 × 10 16 19 F atoms) were detected with 19 F MRI with SNR > 5. Determination of these detection thresholds in vitro is useful to anticipate the minimum number of cells that are required for detection in vivo. However, we demonstrated that there are several factors in vivo which led to reduced detectability of cells, particularly the effect of cell dispersion which reduces cell density per imaging voxel. There is no doubt that cellular sensitivity for these modalities will continue to improve with further developments. It is essential to understand and improve cellular sensitivity to advance imaging of cellular therapeutics.