2.2. In Vitro PAFFC Analysis of CTCs and CTPs

In vitro PAFFC was used to verify in vivo data because there is a higher sensitivity in vitro due to low light attenuation, autofluorescence, absorption background, well-controlled flow parameters, and sample dilution to exclude overlapping peaks from closely located objects. Specifically, in vitro analysis of CTPs and CTCs was performed in 50 and 100 µm (i.d.) square quartz capillary tubes in phosphate-buffered saline (PBS) or blood. The flow velocity of 1–5 mm/s was controlled with a syringe pump (Figure 1(b)). To ensure that only one cell/particle is in the detection volume at a time, the solution of cells or particles were diluted properly. PA and fluorescence signals were detected through the capillary walls using the integrated PAFFC system as described above. PA and fluorescence traces were analyzed to identify a transient increase in PA or fluorescence signal amplitudes during a short time window (typically ~0.5 ms). The 2D scatter plots are constructed as PA signal amplitude versus fluorescence intensity with each object presented as a single dot.