Abstract
Three-dimensional fluorescence lifetime microscopy is achieved by combining wide-field fluorescence lifetime imaging with a remote optical refocusing method. As required for some applications in dynamic research for physics, chemistry, or biology, it is thereby not necessary to move the sample, i.e., the specimen is not disturbed during measurement. Using a fluorescent microsphere the performance of the system has been tested successfully with respect to three-dimensional fluorescence lifetime microscopy as well as time-resolved fluorescence spectroscopy.
Highlights
In recent years, fluorescence lifetime imaging microscopy (FLIM) has been experiencing a rapid increase of attention, because it can provide local environment information of a fluorophore but does not depend on fluorophore concentration
Three-dimensional fluorescence lifetime microscopy is achieved by combining wide-field fluorescence lifetime imaging with a remote optical refocusing method
As required for some applications in dynamic research for physics, chemistry, or biology, it is thereby not necessary to move the sample, i.e., the specimen is not disturbed during measurement
Summary
Fluorescence lifetime imaging microscopy (FLIM) has been experiencing a rapid increase of attention, because it can provide local environment information of a fluorophore but does not depend on fluorophore concentration. In normal WF-FLIM setups, just like in most FLIM systems, optical sections are obtained by mechanically changing the distance between the specimen and the objective lens to refocus at different depths. In this paper a three dimensional WF-FLIM system is achieved by combining wide-field FLIM with a remote optical refocusing system that does not require mechanical movement of the specimen for three dimensional imaging [17,18]. We test this system using a fluorescent microsphere as sample. The results show that the design has been implemented successfully
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