The image to the left is of an oligodendrocyte labeled with Alexa488-phalloidin to label the actin filaments ;(image courtesy of Brad Zuchero in the Barres lab and Andrew Olson, Neuroscience Microscopy Service). It was imaged on a Deltavision OMX microscope located in the Neuroscience Microscopy Service (NMS) in the basement of SIM1. This microscope can acquire three dimensional super-resolution images by using a structured illumination (SI) pattern produced by a grating. The grating is mounted on a piezo-actuated rotating stage, which automates the acquisition of multiple images using different SI patterns, which are then combined to produce the super-resolution image. This microscope has three sCMOS cameras, a 60X 1.4NA objective for SI and widefield microscopy and a second 60X objective for total internal reflection fluorescence (TIRF) microscopy. It has excitation lasers for SI and TIRF that emit lines at 405, 488, and 568nm. It also has a solid-state illuminator for widefield microscopy with 405, 445, 488, 514, 568, and 642nm excitation wavelengths.
There is a second Deltavision OMX microscope located in the Cell Sciences Imaging Facility (CSIF) in the basement of the Beckman Center. It generates the SI pattern using a very fast “BLAZE” electro-optical device. SI images are acquired using a 100X 1.4NA lens and three emCCD cameras. SI uses lasers with lines at 405, 488, 514, 568, and 642nm. This microscope can also do TIRF using 60X and 100X TIRF lenses and ultra-fast widefield imaging using a 60X 1.3NA silicon lens. These can be done on live cells, since it has CO2 and temperature control of the stage. Widefield imaging uses the same type of solid-state illuminator as the NMS instrument. Potential applications of the ultra-fast widefield live cell imaging capability include observing rapid changes in intracellular signaling pathways, such as changes in calcium ion concentration.
A recent overview of super-resolution microscopy can be found here.
As with all School of Medicine scientific service centers, CSIF and NMS are open to all Stanford University researchers (some training is required to use the microscopes). We look forward to seeing your journal cover-quality SIM micrographs.