Abstract
ABSTRACTSuper-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a ‘top-down’ scaffolding event. Further, we identify an F-actin population – stable base clusters – that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals.
Highlights
Cells in living organisms exist in complex and packed environments with unique extracellular milieu next to cells of diverse morphologies and molecular profiles
Compared with conventional deconvolution methods, which utilize theoretical or measured point spread function (PSF) of the imaging system, blind deconvolution is superior because it adapts to the real PSF of the microscope and sample, which can be substantially different from theoretical or measured PSFs owing to instrument and sample variations
For deconvolution with stepwise optical saturation microscopy (DeSOS) microscopy performed on a confocal microscope, for example, if the raw images F1 and F2 are obtained with a laser power of I1=1 mW and I2=1.2 mW, respectively, the linear combination coefficients will be calculated as c1=1, c2=−I1/I2=−0.83 according to the saturation super-resolution (SOS) algorithm (Zhang et al, 2018), and the resulting super-resolution DeSOS image will be c1Decon[F1]+c2Decon[F2]=Decon[F1]−0.83Decon[F2] (Fig. 1A)
Summary
Cells in living organisms exist in complex and packed environments with unique extracellular milieu next to cells of diverse morphologies and molecular profiles. Optical techniques generally use extensive laser powers or exposure times within tissue, limiting longer-term imaging. Lattice light sheet microscopy was combined with optical imaging to provide super-resolution intravital protein organization over extended periods of time (Liu et al, 2018). Such optical techniques require the acquisition of expensive hardware. Expansion microscopy requires fixation and cannot be used in living animals (Chozinski et al, 2016; Freifeld et al, 2017) Together, these approaches, are limited in their ability to image cells in their native environment over extended periods of time in volume stacks
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