In cancer and all biomedical researches, we always need a microscope with better imaging precision, speed, functionality, and depth, so that the diagnostics and treatments of cancer and other diseases can be accurate, fast, and noninvasive. However, conventional fluorescence microscopes cannot satisfy these needs due to physical limits on resolution, speed, information, and depth. In this work, we aim to overcome these limits using novel super-resolution microscopy and high-speed quantitative imaging techniques, both combined with multiphoton microscopy. First, we propose a super-resolution microscopy technique, termed stepwise optical saturation (SOS), to overcome the resolution limit. This technique is based on the principle of SOS, where M steps of raw fluorescence images are linearly combined to generate an image with a √M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. The method can be implemented easily and requires neither additional hardware nor complex post-processing. Second, we propose a high-speed quantitative microscopy technique, termed phase multiplexing (PM) fluorescence lifetime imaging microscopy (FLIM), to overcome the speed limit and obtain additional information. For each conventionally obtained scanning microscopy image, four PM images from four radio-frequency mixers are obtained simultaneously, whose linear combinations result in the intensity and lifetime (the fluorescence decay lifetime of excited fluorophores, providing additional information such as pH, ion concentration etc. about the microenvironment in biology) images of the sample. Because of the multiplexing, this technique is fundamentally faster than most state-of-the-art FLIM methods. With PM-FLIM, a typically slow and time-consuming fluorescence lifetime imaging process can become as fast as a conventional intensity imaging measurement. Finally, both techniques can be combined with multiphoton microscopy to overcome the depth limit of conventional fluorescence microscopes.
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