A VERSATILE 2-PI-TILTING DEVICE FOR FLUORESCENCE MICROSCOPES

A tilting device for biological specimens (rotation angle up to 2 pi), especially fluorescence-lablelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adapter for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser fluorescence microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor. The three-dimensional (3-D) image-forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that in spite of aberration effects, the optical arrangement used allows a gain in the 3-D resolution by tilting the object. A low-cost, fully automated 3-D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black-and-white CCD camera. The system was operated by a personal computer. The online visualization ('movie') of rotating objects indicates the feasibility of the system.