Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe

Abstract

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.

Squaraine Dyes for Photovoltaic and Biomedical Applications

“…SQs are attractive materials in biomedical fields due to their molecular transformation in biological environment where typically applied in cell, [ 107 ] tissue, [ 108 ] and organs [ 109 ] for tumor and cancer treatments. The SQs can be functionalized diversely by designing molecules with respect to the applications, for example, direct capture of targeted ions and polar molecules, that results in color change in range of visible and fluorescent range and electrical response.…”

Stimulated emission depletion microscopy for biological imaging in four dimensions: A review

“…The typical STED resolution ranges from 30 to 70 nm, as recorded with the commercial STED systems, such as Leica TCS SP8 STED (Gemmink et al, 2018; Masch et al, 2018; Thompson et al, 2017; C. Wang et al, 2019; Yang et al, 2020) or Abberior's STED systems (Bergstrand et al, 2019; Carravilla et al, 2019; Kamper, Ta, Jensen, Hell, & Jakobs, 2018; Soliman, Göttfert, Rosewich, Thoms, & Gärtner, 2018; Stephan, Roesch, Riedel, & Jakobs, 2019), as well as several custom‐built STED systems. Abberior STEDYCON can achieve resolution <40 nm, when using 640 nm as excitation beam and 775 nm as STED beam, 100× 1.45 NA objective.…”

“…When pixel number of images are fixed, temporal resolution is influenced by the velocity of the piezo stage or the galvanometer. For commercial STED microscopy such as Leica TCS SP8, temporal resolution (imaging time for taking one image) can be 0.5–10 s (Kamper et al, 2018; Spahn et al, 2018; Thompson et al, 2017; C. Wang, Taki, et al, 2019; Yang et al, 2020). A quad scanning STED microscope (Abberior Instruments) can achieve 5 s temporal resolution and 70 nm lateral resolution (Stephan et al, 2019).…”

Tools and approaches for analyzing the role of mitochondria in health, development and disease using human cerebral organoids

“…However, tools providing super-resolution are currently accessible only in a few specialized labs and are characterized by a low depth of penetration along the z-axis (up to 100 nm). For these reasons, the little literature there is on the topic focuses mainly on traditional cell cultures (Jakobs et al, 2020;Wang et al, 2019;Yang et al, 2020). To adapt super-resolution microscopy to image thick samples, several groups have attempted to use NIR emission saturation nanoscopy to visualize transcription factors and nanoparticles within intact cancer spheroids (Bon et al, 2018;Liu et al, 2020).…”