Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2–3.6-fold compared to the corresponding conventional images.
Structured illumination microscopy suffers from the need of sophisticated instrumentation and precise calibration. This makes structured illumination microscopes costly and skill-dependent. We present a novel approach to realize super-resolution structured illumination microscopy using an alignment non-critical illumination system and a reconstruction algorithm that does not need illumination information. The optical system is designed to encode higher order frequency components of the specimen by projecting PSF-modulated binary patterns for illuminating the sample plane, which do not have clean Fourier peaks conventionally used in structured illumination microscopy. These patterns fold high frequency content of sample into the measurements in an obfuscated manner, which are de-obfuscated using multiple signal classification algorithm. This algorithm eliminates the need of clean peaks in illumination and the knowledge of illumination patterns, which makes instrumentation simple and flexible for use with a variety of microscope objective lenses. We present a variety of experimental results on beads and cell samples to demonstrate resolution enhancement by a factor of 2.6 to 3.4 times, which is better than the enhancement supported by the conventional linear structure illumination microscopy where the same objective lens is used for structured illumination as well as collection of light. We show that the same system can be used in SIM configuration with different collection objective lenses without any careful re-calibration or realignment, thereby supporting a range of resolutions with the same system.
Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers improved axial resolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while fluorescence from each illuminated plane in the sample is mapped onto a line on the camera with a multifocus optical element. A synchronized rolling shutter readout realizes optical sectioning. We describe the technique theoretically and verify its optical sectioning and resolution improvement capabilities. We demonstrate a prototype system with a multifocus beam splitter cascade and record monolayers of endothelial cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue and visualize the distribution of mitochondria at high axial resolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 µm deep into the tissue.
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