A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with...

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.