The conventional optical microscope naturally has the capability of tomography, so obtaining 3D images requires Z-axis scanning, which inevitably increases the acquisition time. A long acquisition time can cause a series of problems, such as photobleaching to the fluorophore, phototoxicity to the biological samples and motion blur for in vivo imaging [1], [2], [3], [4], [5]. Light-sheet microscope has a natural advantage in fast 3D imaging, however, it requires the use of two orthogonal objectives to perform excitation and detection tasks, complex systems, and higher demands on the sample [6], [7], [8]. To solve this problem, two-photon microscope with Bessel and Airy beams were used and achieved fast volume imaging [9], [10], [11], [12], [13], [14]. With the characteristic of elongated focal length and non-diffracting property, the fluorescence signal along the axis can be captured at once within the effective focal length, and more structural information were covered within a frame volumetric image, which significantly improved the frame rate of volume imaging, skipped the axial scanning and avoided the extra noise and the possible time attenuation. However, the 3D structure is projected to a 2D image is usually indistinguishable, especially those that overlap along the axis. Identifying this information effectively is a huge challenge.

FLIM is used to measure fluorescence lifetime changes of the microenvironment and reveal more biological information. Unlike ordinary fluorescence microscopes that only provide information of fluorescence intensity, FLIM technology can obtain not only fluorescence intensity information, but also functional information (such as changes in the microenvironment). Since the fluorescence lifetime is an inherent characteristic of fluorescent probes, it is only related to changes in the microenvironment, and has nothing to do with the intensity of the laser and the concentration of the probes. Therefore, FLIM can give a quantitative distribution of microenvironmental parameters [15], [16], [17], [18]. The phasor plot is a common method for FLIM data analysis, which can transform collected data from the time domain to the frequency domain to avoid some problems resulting from exponential analysis and provide an approach for the graphical global view of fluorescence decay at each pixel [19], [20], [21]. Here, we developed a new method to separate multiple layers of information in volume images by using the phasor plot analysis. This method not only effectively solves the indistinguishable information in volume images, but also can be applied to multi-color volume imaging.