Compact laser scanning confocal microscope

Abstract

Within the framework of the General Support Technology Program of the European Space Agency (ESA), a compact dedicated confocal laser scanning microscope has been developed for 3D fluorescence imaging of biological samples. The microscope permits normal confocal mode operation with excitation at 488nm and fluorescence lifetime imaging (FLIM) with excitation at 630nm and a time resolution of 200ps. Each fluorescence signal is detected by a dedicated photomultiplier tube. Proper optical signal separation and filtering is performed by a set of optical filters and dichroics. The software and hardware further include the specific imaging modes allowing for fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). In addition to this dual wavelength fluorescence imaging mode, the microscope includes transmission imaging capabilities via differential imaging contrast (DIC). Both fluorescence and DIC imaging can be acquired simultaneously. The source for the DIC is a near infrared LED. This choice permits the decoupling of DIC and fluorescence signals by a dichroic cold mirror. The opto-mechanical assembly is constructed on the two sides of a rigid 16mm thick aluminum base plate of dimensions 389 mm by 575.5 mm. The total volume under light and dust shielding removable covers is just 53 dm3, excluding PC and control electronics. In this paper, the design, performance and limitations of this compact confocal microscope are discussed. Illustrative examples of applications on biological samples are shown