We have developed a new open-top selective plane illumination microscope (SPIM) compatible with microfluidic devices, multi-well plates, and other sample formats used in conventional inverted microscopy.Its key element is a water prism that compensates for the aberrations introduced when imaging at 45 degrees through a coverglass.We have demonstrated its unique high-content imaging capability by recording Drosophila embryo development in environmentally-controlled microfluidic channels and imaging zebrafish embryos in 96-well plates.We have also shown the imaging of C. elegans and moving Drosophila larvae on coverslips.
Selective plane illumination microscopy (SPIM) has emerged recently as a powerful tool in optically sectioning tissue samples. We have designed a new SPIM that allows samples to be mounted in a variety of common methods.
Super-resolution microscopy techniques are often extremely susceptible to sample drift due to their high spatial resolution and the long time needed for data acquisition. While several techniques for stabilizing against drift exist, many require complicated additional hardware or intrusive sample preparations. We introduce a method that requires no additional sample preparation, is simple to implement and simultaneously corrects for x, y and z drift.We use bright-field images of the specimen itself to calculate drift in all three dimensions: x, y and z. Bright-field images are acquired on an inexpensive CCD. By correlating each acquired bright-field image with an in-focus and two out-of-focus reference images we determine and actively correct for drift at rates of a few Hertz. This method can maintain stability to within 10 nm for x and y and 20 nm for z over several minutes.Our active drift stabilization system is capable of simultaneously compensating x, y and z drift through an image-based correlation method that requires no special sample treatment or extensive microscope modifications. While other techniques may provide better stability, especially for higher frequency drift, our method is easy to implement and widely applicable in terms of both sample type and microscopy technique.
Nonlinear structured illumination microscopy (NSIM) can extend the resolution beyond the 120 nm limit of linear SIM. By combining patterned depletion with rsEGFP2, we achieved 2D-NSIM imaging of live U2OS cells with 75 nm resolution.
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