van der Ploeg M, Vossepoel A M, Bosman F T, van Duijn P
Histochemistry. 1977 Apr 4;51(4):269-91. doi: 10.1007/BF00494363.
Principles and techniques are discussed for measuring with high topological resolution local emission in fluorescing objects, using photographic negatives. Determination of fluorescence intensities is only possible when an unequivocal relation between the original local fluorescence emission intensities of the object, and the transmittances or densities recorded in the microfluorophotograph is known. This relation is formulated in the theoretical part. From this relation it can be concluded that the recorded intensities can be measured optimally when the optical density values produced by the fluorescence emission fall in the range of the linear portion of the Hurter and Driffield curve. In order to obtain this situation, a uniform low-level pre-exposure of the film emulsion to (white) light is carried out prior to the actual fluorescence emission exposure. This pre-exposure acts to elevate the signal exposure to the linear (steeper) part of the H.D. curve. Inhomogeneity of the excitation beam in the object field, or differences in film emulsion response to the light exposure, will result in erroneous optical densities recorded in the photographic negative. Correction for such artifacts could be obtained by addition of a low concentration of fluorophore to the mounting medium of the microscopic preparation. The overall fluorescent background produced in this way, enabled calibration of local fluorescence intensities in different parts of one fluorophotographic negative, and also of the intensities in different negatives taken from one microscopic preparation. The validity of this approach was checked by comparing data obtained from several photographic negatives of the same quinacrine-stained metaphase, taken with different exposure times to imitate fluctuations in excitation illumination, after conversion of the scanning data into emission intensity values with an alogarithm based on the proposed theoretical relation. In another experiment, fluorescence emission intensities of Feulgen-stained chromosomes which had been measured with a cytofluorometer, were compared with results obtained by conversion of the scanning data measured in the fluorophotographic negatives of the same metaphases. Both types of experiment confirmed the applicability of the procedure described.
讨论了使用照相底片以高拓扑分辨率测量荧光物体中局部发射的原理和技术。只有当物体的原始局部荧光发射强度与显微荧光照片中记录的透光率或密度之间存在明确关系时，才能确定荧光强度。这种关系在理论部分进行了阐述。由此关系可以得出，当荧光发射产生的光密度值落在赫特和德里菲尔德曲线的线性部分范围内时，记录的强度可以得到最佳测量。为了实现这种情况，在实际荧光发射曝光之前，对胶片乳剂进行均匀的低水平（白光）预曝光。这种预曝光的作用是将信号曝光提升到H.D.曲线的线性（更陡）部分。物场中激发光束的不均匀性，或胶片乳剂对光曝光的响应差异，将导致照相负片中记录的光密度出现误差。可以通过向显微制片的封固介质中添加低浓度的荧光团来校正此类伪影。以这种方式产生的整体荧光背景，能够校准一张荧光照片负片不同部分的局部荧光强度，以及从同一显微制片拍摄的不同负片中的强度。通过比较从同一喹吖因染色中期的几张照相负片获得的数据来检验这种方法的有效性，这些负片在不同曝光时间下拍摄以模拟激发照明的波动，在根据所提出的理论关系用对数将扫描数据转换为发射强度值之后。在另一个实验中，将用细胞荧光计测量的福尔根染色染色体的荧光发射强度，与通过转换同一中期荧光照片负片中测量的扫描数据获得的结果进行比较。这两种类型的实验都证实了所述程序的适用性。
