Live-Cell SOFI Correlation with SMLM and AFM Imaging

Abstract

Standard optical imaging is diffraction-limited and lacks the resolving power to visualize many of the organelles and proteins found within the cell. The advent of super-resolution techniques overcame this barrier, enabling observation of subcellular structures down to tens of nanometers in size; however these techniques require or are typically applied to fixed samples. This raises the question of how well a fixed-cell image represents the system prior to fixation. Here we present the addition of live-cell Super-Resolution Optical Fluctuation Imaging (SOFI) to a previously reported correlative process using Single Molecule Localization Microscopy (SMLM) and Atomic Force Microscopy (AFM). SOFI was used with fluorescent proteins and low laser power to observe cellular ultrastructure in live COS-7 cells. SOFI-SMLM-AFM of microtubules showed minimal changes to the microtubule network in the 20 min between live-cell SOFI and fixation. Microtubule diameters were also analyzed through all microscopies; SOFI found diameters of 249 ± 68 nm and SMLM was 71 ± 33 nm. AFM height measurements found microtubules to protrude 26 ± 13 nm above the surrounding cellular material. The correlation of SMLM and AFM was extended to two-color SMLM to image both microtubules and actin. Two target SOFI was performed with various fluorescent protein combinations. rsGreen1-rsKAME, rsGreen1-Dronpa, and ffDronpaF-rsKAME fluorescent protein combinations were determined to be suitable for two target SOFI imaging. This correlative application of super-resolution live-cell and fixed-cell imaging revealed minimal artifacts created for the imaged target structures through the sample preparation procedure and emphasizes the power of correlative microscopy.

Figure 1

New three step correlative pipeline with live-cell super-resolution added to the beginning of the previous method. Seeded cells are first transfected with a DNA plasmid, expressing a fluorescent protein within the living cell. Once enough fluorescent protein has been expressed, the coverslip is taken to be imaged. The cell can then be fixed and membrane (magenta) removed to ensure no major structural rearrangement from natural processes. Antibody labeling can be performed and the cell imaged again to determine the effects of permeabilization and fixation on the internal structures. Finally the sample is washed extensively with ultrapure water and dried in ambient conditions before being imaged on AFM to produce a topographical map. Figure adapted with permission from ref (7). Copyright 2022 IOP Publishing.

Figure 2

Same cell imaged under SOFI using pMAP4-N1-ffDronpa, SMLM with Alexa Fluor 647 and AFM. (a) shows the original SOFI image, (b) is the SMLM image of the same region after membrane removal and antibody labeling. (c) is the AFM image of the same region. (d) is the overlaid SOFI/SMLM image comparing the microtubule network. (e) shows the overlaid microtubule network from SMLM on the topographical map from AFM. (f,g,j,k) are a zoom in of a cluster of microtubules outlines with a green box. The green arrowhead indicates a microtubule that appears on SOFI but is not shown on SMLM or AFM. (h,i,l,m) are a zoom in of a microtubule intersection outlined with a purple box. The yellow arrowhead indicates another microtubule present in live-cell SOFI but not shown under either SMLM or AFM. Scale bars for (a–e) and (f–m) are 5 and 2 μm, respectively.

Figure 3

Widths of microtubules measured across the different imaging modes. The yellow lines in (a) (SOFI/SMLM) and (b) (SMLM/AFM) show the single microtubule widths measured in (c). (d) and (e) also show a microtubule with widths in (f). For SOFI and SMLM images, widths were calculated from fitting Gaussian curves to the intensity cross sections averaged over 100 nm sections and determining 2× their standard deviation. AFM widths were calculated from the measured height of the microtubule in relation to its surroundings. SOFI normalized intensity has been offset for clarity. Scale bar is 2 μm.

Figure 4

Two color correlative SMLM-AFM imaging of microtubules and actin. (a) shows the SMLM image of microtubules in green and labeled with anti-α-tubulin Alexa Fluor 532 while (b) shows the actin of the same region in orange labeled with Phalloidin Alexa Fluor 647. (c) is the AFM topographical map of the same region. (d) shows the two color SMLM image, with (a) and (b) overlaid. (e) has the multitarget SMLM image from (d) overlaid on the AFM topographical map (c), highlighting the microtubules (green) and actin (orange). Scale bars are 5 μm.

Figure 5

Multitarget SOFI of various structures with microtubules in magenta (faster blinking) and actin and mitochondria in green (slower blinking). (a–c) shows microtubules (pMAP4-N1-rsGreen1) (a), actin (pcDNA-lifeACT-rsKAME) (b), and the merged image (c), (d–f) shows microtubules (pMAP4-N1-rsGreen1) (d), mitochondria (pcDNA-DAKAP-Dronpa) (e), and the merged image (f). (g–i) shows microtubules (Tub-ffDronpaF) (g), actin (pcDNA-lifeACT-rsKAME) (h), and the merged image (i). Scale bars are 10 μm.