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.

Scanning ion conductance microscopy revealed cisplatin-induced morphological changes related to apoptosis in single adenocarcinoma cells

“…We acknowledge that other methods of defining cell width include using the full width at half maximum (FWHM) across a line over the cell, as reported by Cao et al , Steifert et al , and Bednarska et al , 27,64–66 while using the longest distance of a cell for length measurement has been described by Yoda et al (2022). 28 We have chosen not to use FWHM method to avoid potential loss of information in the cell body, especially given our interest in comparing specific parts of the cell body before and after apoptosis. The width measurement adopted in our studies has been demonstrated by Osiro et al (2012).…”

“…Atomic force microscopy (AFM) is a label-free and well-established scanning probe microscopy (SPM) technique that offers high resolution imaging of the 3D topography of single cells, 27,28 but due to the likelihood of contact between the AFM tip and the cell membrane, imaging mammalian cell lines is challenging, as the cantilever tip can deform the cell membrane and only information about the cytoskeleton can be obtained. 29,30 SICM is a non-contact, non-invasive, and label-free SPM technique that uses a sharp nanopipette for studying the 3D topography of live cells in aqueous cell culture media under physiological conditions and with nanometer resolution.…”