An example from our laboratory involves collecting flow cytometry data from a population of tumor cells. Figure 1
show maps representing two different approaches to the experiment. The goal is to determine how a fluorescent signal changes over time as a tumor population proliferates, or in biophotonics terms, how cell division leads to the reduction or dilution of a fluorescent readout over time.
The principal difference between these two experiments is that one represents a continuously sampled culture set up at the start of the experiment (i.e., t = 0) and then sampled on five subsequent days (continuous experiment); while the other (staggered experiment) represents a time course with a staggered start so that all the samples are analyzed at a single time point (an endpoint assay). In both cases, the cell culture is sampled after 1, 2, 3, 4, and 5 days of growth. Either approach allows the biologist to adequately monitor the growth parameters of the cultures, and each allows users to logistically setup their experiments. Also in both scenarios, the nanoparticle readout (per cell), acquired by flow cytometry, becomes attenuated over time; in other words as the cells have an opportunity to go through more division after 5 days each cell will have less signal compared to day 1. The staggered approach is classically used when the perturbing agent to be added is a small molecule or 'drug', where delivery to the cell is assumed to be invariant each time. It might also be preferred if it is important for data acquisition to occur on the same day for convenience or to control for variability in the instrument.
However there is a fundamental flaw in the staggered approach from the physics/biophotonics perspective. This arises because 'particulate' labelling is innately variable and as a result the effective cellular labeling is not constant. This presents unreliability of the nanoparticle signal leading to a misinterpretation of the cellular system growth. The mathematical assumption of a conserved readout over time becomes computationally buried in this variability of labeling. Since in this study it is essential to determine the inheritance of a nanoparticle signal as a result of cell division, a continuous sampling approach allows the mathematical modeler to apply the assumption of signal conservation (since loading variability is no longer a problem); in other words the sum of nanoparticle signal at 144h = the signal at 24h. It became clear that for the mathematical modeler to appropriately use these datasets (FCS files in this case) for this purpose, details and provenance of the experiment set-up was critical. This book provides the environment for ensuring that communication is unambiguous and accessible across the technical and discipline environments.