Estimation of the RBC speed based on the measured RBC-passage time through the excitation focal volume (i.e. ‘RBC-passage’ or ‘point-scan’ method) requires a knowledge of the RBC longitudinal size. In this work, we assumed a constant RBC longitudinal size (6 µm) (Unekawa et al., 2010) when estimating the RBC speed (please see Materials and methods section for details). However, the RBC longitudinal size may vary with capillary diameter, RBC speed and hematocrit (Chaigneau et al., 2003). To better understand this limitation, we performed line-scan measurements in 58 capillaries in two awake C57BL/6 mice (3–5 months old, female, 20–25 g, Charles River Laboratories). The cranial window was prepared following the same protocol as described in the Materials and methods section. The blood plasma was labeled by dextran-conjugated Sulforhodamine-B (0.1 ml at 5% W/V in saline, Sigma R9379). The line-scans (2-s-long acquisition in each capillary with 2 kHz line-scan rate) were performed within the cortical depth of 0–200 µm. The RBC speed was calculated from the line-scan images with the procedures described in Kleinfeld et al. (1998). In addition, we extracted the fluorescence intensity time courses from the same parallel line-scan images, and then the RBC speed was calculated by using the procedures described in the Methods section (i.e. by the ‘point-scan’ or ‘RBC-passage’ method). Furthermore, we investigated the dependence of RBC longitudinal size on capillary diameter, RBC speed, and linear-density. (a) The capillary RBC speed values estimated simultaneously by both the line-scan and point-scan methods. The RBC speed values obtained by the two methods in each capillary (green circles) are connected by green lines. Boxplots of the line-scan and point-scan RBC-speed values indicate the median values, the 1st and 3rd quartiles, and the maximum and minimum values. (b) and (c) Correlation between the line-scan and point-scan RBC speed values from the same n = 58 capillaries used in a), grouped by the capillary diameter: 2–3 µm (b) and 3–5 µm (c). In panels b and c, we also presented the corresponding linear regression lines (orange), as well as their slopes and coefficients of determination (R2). (d-f) Dependence of the RBC longitudinal size on capillary diameter (d), RBC speed (e), and line-density (f). For each of the 58 capillaries, we estimated the capillary diameter by fitting the transversal intensity profile to a Gaussian model. The diameters were calculated as the full width at half maximum of the Gaussian profiles. The RBC line-density and longitudinal size were calculated by following the procedures described in Chaigneau et al. (2003). By averaging over all the RBCs identified in each capillary and then across the 58 capillaries, we obtained the mean RBC longitudinal size (6.9 ± 3.0 µm; Mean ±STD). The mean RBC longitudinal size in the capillaries with smaller diameter (2–3 µm; panel d) was just slightly (statistically not significantly) larger than in the capillaries with larger diameter (4–6 µm). This result can be expected as the RBCs in the smaller capillaries may be squeezed to a greater extent. We further observed that the RBC longitudinal size increased with RBC speed (e), where the RBC longitudinal size in the fastest group of capillaries (1–1.5 mm/s) was statistically significantly larger than in the other two lower speed groups. Finally, the capillaries with lower-line-density had more elongated RBC size than the capillaries with median- and higher-line-density (f), although the difference was not statistically significant. The statistical comparison in d) was conducted using Student’s t-test. The statistical comparisons in e) and f) were conducted using ANOVA followed by a Tukey-HSD post-hoc test. The asterisk symbol indicates p<0.05. Data from 58 capillaries were used for the analysis in a and (d-f) and data from 25 and 19 capillaries were used for the analysis in b and c, respectively. In addition, we analyzed the temporal fluctuation of the RBC longitudinal size during the 2-s-long acquisition. For each capillary, we computed the standard deviation (STD) and coefficient of variance (CV) of the RBC longitudinal size from the individual RBC measurements acquired during the 2-s-long acquisition. Then, we obtained the mean STD (2.3 ± 0.6 µm) and CV (0.4 ± 0.1) values averaging over the 58 capillaries. Based on presented results, RBC longitudinal size may be different from capillary to capillary as a function of capillary diameter, RBC speed, and line-density, and may vary over time within the same capillaries. The RBC longitudinal size was especially large at high RBC speed (e). In addition, the average temporal fluctuation of the RBC longitudinal size was moderate (STD = 2.3 ± 0.6 µm). Finally, pairwise comparisons between the RBC speed values obtained by the two methods (a–c) reveal variability, especially at the high RBC speed. Therefore, instantaneous RBC speed obtained by the point-scan method may have larger measurement error, which needs to be considered when interpreting the data. On the other hand, the small difference between the mean RBC speed obtained in the paired measurements (a) did not reach statistical significance (Student’s t-test), and the linear regression slopes in panels b and c are reasonably close to 1. Therefore, for the purpose of providing mean values for group comparison, the RBC speed measurements based on the point-scan method, while limited by assuming the constant RBC longitudinal length, may still be reasonably accurate. Please note that regarding the extreme values of the estimated RBC longitudinal size (d–f), they were calculated as the product of the fitted temporal width of the shadows and the RBC speed, both of which were extracted from the line-scan images. Measurements of both these parameters have limitations. The line-scan method is less accurate for high RBC speed, while estimation of the shadow width sometimes may be influenced by the stacked RBCs, and the fluorescence intensity time courses may be noisier when acquired with the line-scan method (due to shorter dwelling time per time-point) than with the point-scan method.