Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

Abstract

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

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

“…The tip scanning AFM design further enables a simultaneous acquisition of AFM and SIM images. We previously showed that for a simultaneous combination of an optical microscope equipped with a dierential spinning disc and AFM, there are certain system-specic noise sources, which stem either from the AFM cantilever bimorph distortion induced by the dierent thermal expansion coecients of cantilever material (Si/Si 3 N 4 ) and reective coatings (Au), or the pure mechanical noise transfer of the DSD system [8]. Our tests showed that there is no substantial dierence within the noise response with the 3D N-SIM illumination in place ( Figure 6).…”

“…To circumvent this problem and correctly ovarlay SIM data onto AFM images we use a software module (DirectOverlay, JPK BioAFM, Bruker Nano GmbH, Berlin, Germany) to calibrate the optical image. In this calibration process the cantilever is displaced to a set of predened coordinates in a 3 × 3 or a 5 × 5 grid pattern, registers an optical image at each position and a transform function between the AFM and the optical image is determined [8].…”

“…Current AFM techniques combining optical uorescence imaging normally perform successive measurements and then superimpose the images obtained from the separate acquisitions. Main reasons for this are: i) imaging in a simultaneous mode causes perturbation on the AFM cantilever operation due to uorescence excitation light, and ii) noise transfer from the optical microscope can disturb AFM measurements [7,8].…”

“…The atomic force microscope has been successfully integrated with dierent optical microscopy techniques [9,10], allowing to overcome some of its individual limitations when it comes to, for instance, sample penetration and biological specicity. In this regard, AFM has been combined with Confocal Laser Scanning Microscopy (CLSM) [11], Aperture Correlation Microscopy [8], Total Internal Reection Fluorescence Microscopy (TIRFM) [12], and Fluorescence Lifetime Imaging (FLIM) [13]. AFM has also been integrated with super-resolution microscopy schemes, namely Stimulated Emission Depletion (STED) [14,15], Photoactivated Localization Microscopy (PALM) [16] and Stochastic Optical Reconstruction Microscopy (STORM) [17,18].…”

“…In this context, we combine AFM with Structured Illumination Microscopy (SIM), a super-resolution technique that allows to surpass the diraction barrier by a factor of two in every spatial direction [20]. Contrary to other super-resolution modalities, SIM relies on a uorescent excitation light pattern favourable to avoid AFM cantilever disruption during simultaneous operation, as we reported previously on a novel system for synchronic uorescence optical sectioning microscopy through aperture correlation microscopy using a Dierential Spinning Disk (DSD) and AFM [8].…”