Abstract

Emerging visualization technologies are playing an increasingly important role in the study of numerous aspects of cell biology, capturing processes at the level of whole organisms down to single molecules. While developments in probes and microscopes are dramatically expanding the areas of productive imaging, there are still significant roadblocks. Primary challenges include 1) fluorophore bleed-through, which limits the number of fluorophores that can be simultaneously imagined, 2) imaging speeds that are too slow, and 3) labeling densities that are too low for deciphering fine subcellular architecture. Here, I will discuss new imaging methods that can overcome these roadblocks, focusing on their potential for clarifying subcellular organelle dynamics. To surmount fluorophore bleed-through, we combined excitation-based spectral unmixing and lattice light sheet microscopy to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lysosomes, peroxisomes and lipid droplets) simultaneously within cells. This allowed us to track these organelles through time and analyze their inter-organelle contacts. To increase temporal resolution during imaging, we employed total internal reflection fluorescence combined with structured illumination microscopy to visualize organelle dynamics at very high temporal-spatial resolution. Examining the ER, we observed that many peripheral ER sheets seen using diffraction-limited imaging are actually highly perforated structures comprised of tightly latticed groups of dynamic tubules. Within the latticed ER tubule meshwork, subdiffractionlimited holes were observed (~150-250 nm diameter) having transient lifespans (~250 msec). Viewed at higher resolution using lattice light sheet microscopy combined with point accumulation for nanoscale topology (PAINT), the peripheral ER sheets represented a complex meshwork of tightly cross-linked ER tubules. I discuss possible roles this complex ER structural organization has for diverse cellular functions.