Patel Shwetal, Pittman Roland N
Department of Chemical Engineering, University of Delaware, Newark, 19716, USA.
Microvasc Res. 2002 Sep;64(2):198-206. doi: 10.1006/mvre.2002.2416.
In the microspectrophotometric method to measure hemoglobin concentration and oxygen saturation in microvessels it is recommended that a low numerical aperture (NA) condenser be employed to ensure that the recorded image is a true projection of the object. However, this tenet has never been rigorously justified. In this study, the microspectrophotometric method is evaluated using the theory of three-dimensional image formation by a light microscope for a wide range of NA. The results of the calculations show that for measurements for hemoglobin concentration, the recorded image is close to the true projection only when the size of the microvessel is large compared to the degree of smearing ( proportional, variant 1/NA) but small compared to the degree of defocus ( proportional, variant NA(2)). These opposing tendencies lead to an optimum NA for which the errors are minimum. This optimum NA is a function of the size of the microvessel and the manner in which the hemoglobin concentration is distributed within the lumen. For measurements of oxygen saturation, the recorded image is the true projection as long as the measurements are made in regions near the microvessel centerline. For measurements made in regions away from the centerline, good agreement was obtained only when the distribution of oxygen saturation was uniform. Reconstruction of the axisymmetric profiles from the recorded projections showed that the errors in the projections cause the recovered profiles to deviate from the true profiles. These deviations are directly related to the extent by which the recorded projections deviate from the true projection.
在用于测量微血管中血红蛋白浓度和氧饱和度的显微分光光度法中，建议采用低数值孔径（NA）聚光镜，以确保所记录的图像是物体的真实投影。然而，这一原则从未得到过严格论证。在本研究中，利用光学显微镜三维成像理论，针对大范围的数值孔径对显微分光光度法进行了评估。计算结果表明，对于血红蛋白浓度的测量，只有当微血管的尺寸相对于模糊程度（成比例，变量1/NA）较大但相对于散焦程度（成比例，变量NA²）较小时，所记录的图像才接近真实投影。这些相反的趋势导致了一个误差最小的最佳数值孔径。这个最佳数值孔径是微血管尺寸以及血红蛋白浓度在管腔内分布方式的函数。对于氧饱和度的测量，只要在微血管中心线附近的区域进行测量，所记录的图像就是真实投影。对于在远离中心线的区域进行的测量，只有当氧饱和度分布均匀时才能获得良好的一致性。从记录的投影重建轴对称轮廓表明，投影中的误差会导致恢复的轮廓偏离真实轮廓。这些偏差与记录的投影偏离真实投影的程度直接相关。
