Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide ~300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells with a superresolution technique. The low saturation intensity and high photostability of MitoESq-635 make it ideal for long-term, high-resolution (stimulated emission depletion) STED nanoscopy. We performed time-lapse imaging of the mitochondrial inner membrane over 50 min (3.9 s per frame, with 71.5 s dark recovery) in living HeLa cells with a resolution of 35.2 nm. The forms of the cristae during mitochondrial fusion and fission can be clearly observed. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes with nanoscale resolution for an extended period of time.

These authors contributed equally to this workMitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide ~300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells at superresolution. The low saturation intensity and high photostability make it ideal for long-term, high-resolution STED nanoscopy. We demonstrate the time-lapsed imaging of the mitochondrial inner membrane over 50 minutes in living HeLa cells at 35.2 nm resolution for the first time. The forms of the cristae during mitochondrial fusion and fission can be clearly resolved. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes at nanoscale resolution for long periods of time with minimal phototoxicity.

Stimulated emission depletion (STED) nanoscopy plays a key role in achieving sub-50 nm high spatial resolution for subcellular live-cell imaging. To avoid re-excitation, the STED wavelength has to be tuned at the red tail of the emission spectrum of fluorescent probes, leading to high depletion laser power that might damage the cell viability and functionality. Herein, with the highly emissive silica-coated core–shell organic nanoparticles (CSONPs) enabling a giant Stokes shift of 150 nm, ultralow power STED is achieved by shifting the STED wavelength to the emission maximum at 660 nm. The stimulated emission cross section is increased by ∼20-fold compared to that at the emission red tail. The measured saturation intensity and lateral resolution of our CSONP are 0.0085 MW cm–2 and 25 nm, respectively. More importantly, long-term (>3 min) dynamic super-resolution imaging of the lysosomal fusion–fission processes in living cells is performed with a resolution of 37 nm.

Stimulated emission depletion (STED) microscopy allows high lateral and axial resolution, long term imaging in living cells. Here we review recent technical advances in STED microscopy, with emphasis on resolution and measurement range of XYZt four dimensions. Different STED technical advances and novel STED probes are discussed with their respective application in biological subcellular imaging. This review may serve as a practical guide for choosing a suitable approach to the advanced STED super‐resolution imaging.

Multiphoton microscopy (MPM) plays important role in biological imaging for its low scattering nature, yet it typically requires high illumination intensity. Although time-stretch of the ultrashort pulse can achieve ultrahigh speed scanning and deep penetration, the near-infrared illumination yields a compromised resolution because of its long wavelength. Here, by combining structured illumination with up-conversion materials, a multiphoton up-conversion time-encoded structured illumination microscopy (MUTE-SIM) with the scanning rate of 50 MHz is developed, which overcomes the limitation on the resolution. The resolution limit of near-infrared light is surpassed by a factor of 223.3% with low illumination intensity. This imaging strategy provides an ultrafast, low intensity, super-resolution MPM approach imaging, which has great potential in deep-tissue with high spatial resolution.

STimulated Emission Depletion (STED) microscopy attains super‐resolution in biological imaging beyond the diffraction limit. Here, we give a concise protocol to construct a dual‐pulse STED setup with one super‐continuum laser. Moreover, a flexible and dismountable Bessel modulation module is introduced for potential 2D‐stack STED imaging. Experiments and notices are introduced in detail, with discussion on some important check‐points for STED, such as detector saturation. Finally, the results validate the system working.

Using an all-fiber mode selective coupler (MSC) at the visible band, here we experimentally demonstrate a generating and wavelength multiplexing scheme for the cylindrical vector (CV) and vortex beams (VBs). The proposed MSCs act as efficient mode converters to produce spectrally insensitive high-order modes (HOMs) at the wavelength ranging from 450 to 980 nm, which have broad operation bandwidth (more than 7 nm), high mode conversion efficiency (94%), and purity (98%), and low insert loss (below 0.5 dB). By adjusting the polarization state and the phase shift of linear polarization (LP)11 mode respectively, the donut-shaped CVs and circular-polarization VBs are achieved. The focused intensity distribution of the donut beam on the cross- and axial-sections is monitored by using a confocal system. The all-fiber solution of producing and multiplexing HOMs opens a new route for stimulated emission depletion microscopy applications.

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