Ultra-High Resolution 3D Imaging of Whole Cells

Abstract

SummaryFluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Visualization and characterization of individual type III protein secretion machines in live bacteria

“…S2). We grew the mEos3.2-PrgH-expressing bacteria under conditions that stimulate the expression of the type III secretion system and imaged them by SMSN (32,34). Superresolution images of these live bacteria showed the appearance of fluorescent clusters with a diameter of ∼45 nm ( Fig.…”

“…For SMSN or 4Pi-SMSN, bacteria were placed on agarose pads or on poly-D-lysine-coated glass-bottom microwell dishes, respectively. The 2D and 3D superresolution imaging was performed with a custom-built FPALM system as previously described (31,32,34,53). Cluster analysis was carried out using a DBSCAN clustering algorithm (54,55) and a wavelet algorithm (56) as described in SI Appendix, Materials and Methods.…”

Cytoskeletal control of B cell responses to antigens

“…To understand how signalling pathways interact with cytoskeletal structures to achieve specific mechanical and regulatory functions, future studies will likely benefit from improvements in these areas. Advances in optical microscopy have been making long strides towards three-dimensional high-resolution imaging and it will be exciting to see this new technology employed for studies of immune cells 76, [163][164][165] . Genetic manipulation is more accessible and precise than ever before, and new optogenetic and nanomechanical methods offer unprecedented temporal and spatial control of manipulation of live cells 166,167 .…”

ChromoTrace: Computational Reconstruction of 3D Chromosome Configurations for Super-Resolution Microscopy

“…However, since any given color 534 will have only a finite set of possible neighboring mistakes with associated error rates, a 535 substitution matrix of possible errors can be integrated into both the extension phase 536 and exploration phase of the suffix tree [26], changing the formulation of the problem into 537 a likelihood model of seeing the 3D position of probes given a certain path labeling. In 538 addition, recent advances in labeling techniques such as the 'Exchange-PAINT' method 539 now allow sequential hybridization and image capture, allowing to separate up to 10 540 pseudocolors based on a single dye in time [21]. This labeling technology requires long 541 super-resolution image acquisition times, but could massively increase the number of 542 probes available for the reconstruction algorithm.…”

“…Super-resolution microscopy can resolve unprecedentedly small volume elements 66 (approximately 20 x 20 x 20 nm [21]) inside the total nuclear volume (approximately 67 8 × 10 −6 µm 3 ), which will on average contain only up to 2 kb or a few nucleosomes. This 68 fundamental increase in information of the relative positioning of defined loci in the 69 genome can now be leveraged computationally.…”