![]() Result 2 - Acquire 3D super-resolved images combining dSTORM and astigmatic lens.Īcquire live Super-resolved Images (resolution Up to 100 nm)Īndor´s cameras offer an integrated licence for SRRF (Super-resolution radial fluctuations). Result 1 - Resolution down to 20 nm with dSTORM. Dragonfly also has a motorised astigmatic lens that creates a calibrated asymmetric distortion of the single-molecule PSF, which varies with axial defocus. Result 2 - Detect two independent channels simultaneously without compromising speed or resolution.Īcquire images with a resolution of 50-100 nmĭragonfly supports dSTORM. Result 1 - Detect ultrafast events with acquisition speeds up to 400 frames per second. The internal beam splitters allow Dragonfly to acquire two channels simultaneously on two independent detectors (cameras). Dragonfly is at least ten times faster than point scanning confocal systems. The EMCCD and sCMOS detectors in the Dragonfly allow very low light imaging and high acquisition speeds. Mitochondria Imaging Solution: Dragonfly and Andor´s high QE cameras SRRF-stream is compatible with live-cell imaging, offering the advantage of high-speed super-resolved live imaging. Importantly any modality can be combined with the super-resolution technique SRRF (super-resolution radial fluctuations). Imaging modalities such as confocal spinning disk, dSTORM super-resolution (resolution ~ 20 nm) are possible with the Dragonfly, and each of these imaging modalities has advantages for mitochondria studies. Ultra-fast low-light imaging is possible with Dragonfly and Andor´s cameras such as the back-illuminated Sona or iXon EMCCD series. Highly sensitive cameras allow the detection of very dim signals with high Quantum Efficiency. ![]() Using Dragonfly with SRRF-stream, scientists can acquire live-cell super-resolved images of mitochondria.ĭragonfly is the complete solution to image live mitochondria due to its speed, sensitivity and resolution. An increase in resolution can be observed between the widefield, confocal and confocal with SRRF stream images. All these requirements render most currently available Super-resolution techniques incompatible with live-cell imaging.įigure 1 – Images of live cells stained with Mito Tracker acquired in widefield, confocal and confocal + SRRF stream. In most cases, sample preparation is complicated, and there is also the requirement of specific fluorophores. Live cell Super-resolution - Super-resolution techniques that go beyond the diffraction limit of the light microscope generally require the acquisition of a vast number of frames (on the order of the 10 3 to 10 4 images) and /or imaging with high light intensities.The diffraction limit of light microscopes is around 200 nm therefore, there is the need to use techniques that overcome the diffraction limit, i.e. Resolution - The resolution required to visualise mitochondrial trafficking lies within the 50-100 nm window in XY.Speed - The visualisation of mitochondria dynamics and calcium signalling requires an instrument that can acquire more than one channel at once, and due to the fast nature of the event, the microscope will need to acquire at very fast speeds.The equipment will need to acquire at fast speeds and with high imaging resolution. Even for fixed mitochondria, high spatial resolution is an essential requirement due to their size and complexity.Ī camera-based confocal system is a better solution for live mitochondria imaging that does not compromise cell viability due to phototoxicity. ![]() In fact, to achieve the most comprehensive picture of the spatial and temporal mitochondria dynamics, it is necessary to acquire the image data in 4 dimensions (3D + time). Images must be obtained with high temporal and spatial resolution. ![]() When imaging live mitochondria, besides the critical challenges of live imaging, including phototoxicity and photobleaching, other problems need to be overcome. All these characteristics pose obstacles for imaging live mitochondria for studying their dynamic behaviour (fission and fusion) as well as the underlying rapid signalling processes that lead to fusion or fission. Moreover, mitochondria are extremely dynamic organelles that divide, fuse, change shape and travel along the intracellular space. Besides the membrane architecture mitochondria are small, with a size near the resolution limit of the light microscopes. Microscopy in mitochondria is demanding, these organelles have a complicated inner architecture with two membranes, a smooth outer membrane and an extremely tortuous inner membrane. Defects in mitochondria dynamics are associated with acute syndromes such as neurodegenerative disorders, cardiovascular disorders and neurometabolic diseases. Mitochondria provide cell energy and thus, are essential organelles of the eukaryotic cell. Overcoming Challenges in Imaging Dynamic Processes ![]()
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