No abstract

Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

SummaryHighly optically active nonlinear bio-photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through secondharmonic generation (SHG) interactions, behaving similarly to man-made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third-harmonic generation, and multiphoton-induced fluorescence. With no energy release during harmonic generation processes, the nonlinear-photonic-crystal-like SHG activity is useful for investigating the dynamics of structure-function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.

The aim of this study was to distinguish basal cell carcinoma (BCC) from actinic keratosis (AK) and Bowen's disease (BD) by fluorescence lifetimes of hematoxylin and eosin (H&E) and phasor analysis. Pseudocolor images of average fluorescence lifetime (τm) exhibited more contrast than conventional bright field and/or fluorescence images of H&E-stained sections. The mean values (μ) of τm distribution (τm) in three layers of skin were first explored for comparison with the corresponding layers of AK, BD, and BCC. Moreover, analysis of the H&E fluorescence lifetimes in the phasor space was performed by observing clusters in specific regions of the phasor plot. Various structures in the skin were distinguished. Comparisons of phase distributions from the corresponding layers of skin resulted in quantitative separation and calculation of distinctive parameters including coordinate values, diagonal slopes, and phasor areas. The combination of fluorescence lifetime imaging microscopy (FLIM) and phasor approach (phasor-FLIM) provides a simple method for histopathology analysis and can significantly improve the accuracy of bright field H&E diagnosis. We therefore believe that phasor-FLIM is an aided tool with the potential to provide rapid confirmation of diagnostic criteria and classification of histological types of skin neoplasms.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications. In principle, STED super resolution is obtained by stimulated emission partially inhibiting the spontaneous emission in the periphery of a diffraction-limited area. However, very high depletion laser power is generally necessary for the enhancement of imaging resolution, which is harmful to live biological specimens due to its high phototoxicity and photo-bleaching effects. Therefore, further improving the STED resolution at a lower depletion power level has recently attracted increasing interest from researchers in various fields. In this work, a phasor plot approach combined with fluorescence lifetime imaging microscopy (FLIM) is used to resolve the abovementioned problem based on a long- and short-lifetime criterion. Firstly, the time-resolved data obtained by STED-FLIM is converted to the frequency domain via a phasor approach. Next, partial data is extracted according to the information on the phase and amplitude for resolution improvement. Then, fluorescent microspheres (100 nm in diameter) are observed under different depletion powers, resulting in a series of improved resolution through phasor plots. Finally, this method is applied to image human Nup153 in fixed HeLa cells, providing a 86 nm higher resolution than that in traditional STED imaging at a depletion power of 20 mW.