STED and STORM Superresolution Imaging of Primary Cilia

Abstract

The characteristic lengths of molecular arrangement in primary cilia are below the diffraction limit of light, challenging structural and functional studies of ciliary proteins. Superresolution microscopy can reach up to a 20 nm resolution, significantly improving the ability to map molecules in primary cilia. Here we describe detailed experimental procedure of STED microscopy imaging and dSTORM imaging, two of the most powerful superresolution imaging techniques. Specifically, we emphasize the use of these two methods on imaging proteins in primary cilia.

Keywords: Ciliary protein; Diffraction limit; Fluorophore; Primary cilium; STED; STORM; Superresolution microscopy.

Fig. 1

Schematic diagrams of a dSTORM system and a CW STED system in our lab. (a) The dSTORM system consists of three excitation laser lines (637, 561, and 488 nm) and one activation laser (405 nm). A high-speed EMCCD captures single-molecule emission over 5000–20,000 frames to reconstruct a superresolution image. (b) In the STED microscope, two excitation lasers (447 and 491 nm) are chosen for Oregon Green 488 and BD V500 whose emission fluorescence can both be depleted with a 592-nm laser. The light pattern of the depletion laser is converted to a doughnut-shaped distribution through a spiral phase plate. Fluorescent signals from a sample are detected pixel-by-pixel by an avalanche photodiode (APD), a single photon counting module. SMF single-mode polarization-maintaining fiber, MMF multimode fiber, DC dichroic mirror

Fig. 2

Cilia grown at different orientations and with different bending characteristics. Most of are bent to a certain degree, basal body may not be aligned with me ciliary axoneme (middle). Some cilia are not parallel to the surface of coverslip, challenging to focus the entire structure (right). One has to search a horizontal cilium (left) so that its orientation is ideal for Side-view imaging

Fig. 3

Achieving isolated single emitters for good dSTORM images. Emitter overlapping should be avoided for a better imaging (upper). The overlapping signals from multiple emitters results in mislocalization (lower). In this case, the turn-on frequency should be reduced to meet the requirement of separable single-molecule detections per frame

Fig. 4

Tuning the power of 405-nm laser line to control the activation efficiency of dyes. When seeing the reduction of number of molecules being localized through the imaging process, one should increase of the power of 405-nm laser to maintain an efficient imaging acquisition, minimizing the required number of frames for reconstructing the whole image. Ideally, number of molecule localization should be a constant during the acquisition

Fig. 5

Chromatic aberration between two imaging channels compensated by calibrating distance deviation of fiducial markers between two channels. The localization error between two channels should be less than optical resolution to ensure an effective superresolution imaging

Fig. 6

The spectral concept behind the two-color STED system using excitation lasers (447 and 491 nm) and one depletion laser (592 nm). In addition to exciting V500, the 447-nm laser can also excite Oregon Green 488 slightly, causing minor cross talk between channels. Therefore, suitable detection windows to separate the signal of V500 from mat of Oregon Green 488 are necessary. The cross talk can be further reduced by assigning the protein with brighter signals to the V500 channel