Simultaneous 3D super-resolution fluorescence microscopy and atomic force microscopy: combined SIM and AFM platform for cell imaging

Abstract

Correlating data from dierent microscopy techniques holds the potential to discover new facets of signaling events in cellular biology. Here we report for the rst time a hardware set-up capable of achieving simultaneous imaging of spatially correlated super-resolution uorescence microscopy and atomic force microscopy, a feat only obtained until now by uorescence microscopy set-ups with spatial resolution restricted to the Abbe resolution limit. We hereby remove the need to perform independent measurement and subsequent data averaging required to eliminate cell-to-cell variation in observed signals.We detail system integration, demonstrate system performance and report imaging of sub-resolution uorescent beads and genome-engineered human bone osteosarcoma epithelial cells.

Correlative AFM and Scanning Microlens Microscopy for Time‐Efficient Multiscale Imaging

“…Recently, super‐resolution OM, including stochastic optical reconstruction microscopy (STORM), [ 17 ] photoactivated localization microscopy (PALM), [ 18 ] and stimulated emission depletion (STED), [ 19,20 ] has allowed the optical diffraction limit to be exceeded. Although these super‐resolution optical microscopes have significantly improved the optical imaging resolution with some correlation imaging methods being proposed, [ 21–23 ] these technologies are based on fluorescence labeling methods, [ 24 ] so they are generally limited to life sciences applications rather than to other label‐free, real‐time nanoimaging applications. More recently, coupling dielectric microsphere arrays with traditional optical microscopes has enabled super‐resolution imaging than can resolve 50 nm features under white light illumination by transforming evanescent waves into propagating waves.…”

The Value of Watching How Materials Grow: A Multimodal Case Study on Halide Perovskites

“…The term multimodal is used to describe the simultaneous data collection from complementary techniques, also referred to as multiprobe and correlative characterizations. While these measurements are applicable to many research domains including soft matter, polymers, organic and inorganic semiconductors, [ 15–18 ] halide perovskites are chosen as a model system to delineate advances in synthesis science and establishing relationships by coupling functional properties.…”

Correlative Analysis of Intra‐ Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification

“…(2006), [ 2a ] we implemented a PIFOC system into our device, which enabled a piezo‐controlled positioning of the objective's focal plane, synchronized to the vertical movement of the 100 µm Z‐stage. In addition, with the integration of a high numerical objective whose application is already well‐established in the context of correlative SR‐AFM microscopy, [ 11,12,25 ] we achieved a high‐resolution visualization of cell detachment and corresponding unbinding processes (Figure 2d–f). However, although these components are already widely used in microscopic apparatus, it was the first time to be integrated into a FluidFM‐based microscope.…”

“…[ 9 ] However, none of these well‐established techniques alone can address and encompass the complexity of cell adhesion. Therefore, the complementary combination of AFM with diverse optical methods such as Total Internal Reflection FL (TIRFM), [ 10 ] Super‐Resolution Structured Illumination Microscopy (SR‐SIM), [ 11 ] or d STORM [ 12 ] has taken over an increasingly important role in the understanding of cellular biomechanics in the last decades. Nevertheless, the data acquisition of these correlative methods is mostly performed subsequentially and superimposed afterwards due to interference of AFM cantilever and fluorescence excitation light.…”

“…Nevertheless, the data acquisition of these correlative methods is mostly performed subsequentially and superimposed afterwards due to interference of AFM cantilever and fluorescence excitation light. [ 8,11,13 ] However, recently, Fernandes et al. (2020) realized a correlative and truly simultaneously operational scheme of AFM Force Spectroscopy (AFM‐FS) and confocal Fluorescence Lifetime Imaging Microscopy (FLIM) and therefore, took the development of novel multifunctional devices to the next level.…”