Enabling simultaneous imaging of super-resolution fluorescence microscopy and scanning probe microscopy for the life sciences

Abstract

Atomic Force Microscope (AFM) has been integrated with different fluorescence microscopy techniques to correlate the AFM nano-mechanical data with biological specificity of the sample, thereby significantly enhancing the relevance of the information derived from the combined microscopy approach. However, up until this day it has not been possible to acquire data with fluorescence optical sectioningand scanning probe microscopy techniques within nearby spatial volumes at the same time. This severely limits the utility of combining both image techniques as all dynamical information is lost. We have previously circumvented existing integration problems and demonstrated simultaneous operation of aperture correlation microscopy through a Differential Spinning Disk (DSD) approach and nanomechanical mapping AFM. Obtaining data with two microscopy techniques simultaneously namely removes the need to perform both measurements independently and the subsequent data averaging required to eliminate cell to cell variation in observed signals. Here, we report for the first time a hardware set-up capable of achieving simultaneous as well as spatially overlapping super-resolution Structured Illumination Microscopy (SIM) microscopy and atomic force microscopy. SIM illumination is particularly suitable to combine with AFM as it allows to avoid induced cantilever bending due to fluorescence excitation light, a significant problem when integrating AFM with other super-resolution fluorescence microscopy schemes. We demonstrate system performance with sub-resolution fluorescent beads and report imaging simultaneous combined data of CRISPR/Cas9 genome-edited human bone osteosarcoma epithelial cells (U2Os line).