Optimal slice thickness for improved accuracy of quantitative analysis of fluorescent cell and microsphere distribution in cryo-images

Cryo-imaging has been effectively used to study the biodistribution of fluorescent cells or microspheres in animal models. Sequential slice-by-slice fluorescent imaging enables detection of fluorescent cells or microspheres for corresponding quantification of their distribution in tissue. However, if slices are too thin, there will be data overload and excessive scan times. If slices are too thick, then cells can be missed. In this study, we developed a model for detection of fluorescent cells or microspheres to aid optimal slice thickness determination. Key factors include: section thickness (X), fluorescent cell intensity (I(fluo)), effective tissue attenuation coefficient (μ(T)), and a detection threshold (T). The model suggests an optimal slice thickness value that provides near-ideal sensitivity while minimizing scan time. The model also suggests a correction method to compensate for missed cells in the case that image data were acquired with overly large slice thickness. This approach allows cryo-imaging operators to use larger slice thickness to expedite the scan time without significant loss of cell count. We validated the model using real data from two independent studies: fluorescent microspheres in a pig heart and fluorescently labeled stem cells in a mouse model. Results show that slice thickness and detection sensitivity relationships from simulations and real data were well-matched with 99% correlation and 2% root-mean-square (RMS) error. We also discussed the detection characteristics in situations where key assumptions of the model were not met such as fluorescence intensity variation and spatial distribution. Finally, we show that with proper settings, cryo-imaging can provide accurate quantification of the fluorescent cell biodistribution with remarkably high recovery ratios (number of detections/delivery). As cryo-imaging technology has been used in many biological applications, our optimal slice thickness determination and data correction methods can play a crucial role in further advancing its usability and reliability.