Systematic Tuning of Rhodamine Spirocyclization for Super-resolution Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a nonfluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, and an adjustment of the equilibrium poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows one to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell-stimulated emission depletion (STED) microscopy or a spontaneously blinking dye for single-molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

Single Molecule Sensing

Claudio Dellagiacoma

Living organisms consist of cells, the elementary components of which are proteins providing cellular structures and functionality. Nowadays, proteins of the size of one to a few tens of nanometers can be efficiently monitored by fluorescence microscopy and/or spectroscopy reaching the necessary single molecule sensitivity. However, for a more complete understanding of biological processes, the combined investigation of both cellular structures and function at the single molecule level is essential. This work extends the possibilities of existing single molecule investigation methods. The goal was to conceive a functional super-resolution imaging technique by joining the strong concepts from state-of-the-art microscopy and spectroscopy and was approached in three steps. As a first step, confocal fluorescence correlation spectroscopy (FCS) was applied in nanochannels to create sub-diffraction sampling volumes. The resulting enhanced sensitivity to the surface effects depending on ionic surface layers led to a modified protein diffusion measured with FCS. Triplet state lifetime and population analysis have confirmed the model developed for the protein diffusion as a function of the ionic concentration. In the second step, stochastic optical reconstruction microscopy (STORM) was implemented and enhanced by merging with a recent labeling technique called SNAP-tag. As a result, the labeling technique for bright and switchable organic fluorescent dyes attached by a short linker of only ≈ 2.5 nm to the target molecule enabled a localization precision down to ≈ 10 nm. To meet the stringent photo-physical requirements imposed by STORM, toxic imaging buffers based on thiols have to be used. It was found that these requirements could be relaxed by super-resolution optical fluctuation imaging (SOFI) making a step forward to live-cell super-resolution investigations, which introduced the third step. In the third step, SOFI was characterized providing the basis for combining functional and structural super-resolution imaging techniques. The comparison of STORM and SOFI revealed the strengths of each technique. Under ideal blinking conditions with ultra-stable long-lived dark states, STORM outperforms SOFI. However, if the emitters are not well isolated, SOFI achieves a higher effective resolution than STORM and even without mislocalization artifacts. This led onto a novel imaging technique derived from SOFI: Cumulant microscopy merges structural super-resolution with functional imaging. Simulations and measurements showed that photo-physical parameters can be extracted from the emitters serving as nano-sensors. In conclusion, concepts of FCS have been merged with an improved super-resolution technique leading to a novel functional super-resolution imaging technique, which is compatible with living cells.

EPFL2011

Advanced Image Processing for Biology, and the Open Bio Image Alliance (OBIA)

Ricard Delgado Gonzalo, Daniel Sage, Michaël Unser

The field of biological imaging has evolved considerably during the past decade as a result of recent (r)evolutions in fluorescence labeling and optical microscopy. Bioimage informatics has been identified as a top priority to cope with the ever-increasing amount of microscopy data. The challenges and opportunities for researchers in image and signal processing are manyfold. They span the areas of mathematical imaging, with problems such as denoising, 3-D deconvolution and super-resolution localization, as well as image analysis for the segmentation, detection and recognition of biological structures in 3-D. The dynamic aspect of the data requires the development of novel algorithms for tracking fluorescent particles and analyzing high-throughput microscopy data (labeling of cells, phenotyping, extraction of gene expression profiles). A crucial aspect of bioimage informatics is making image analysis tools available to biologists so that they can be applied to real data and used on a routine basis. Developers may benefit from open-source frameworks and international initiative such as OBIA for easying-up this process and creating collaboration networks with biologists.

EUSIPCO2013

Nanoscopy in nonlinear scanning fluorescence imaging systems

Grégoire Pierre Jean Laporte

In the last 30 years, superresolution in optical microscopy has been a major field of research. During this time, different techniques have been created to break the diffraction limit in order to make observations at a nanometric scale. Given that optical microscopy is non-invasive, those superresolution methods pave the way for a better understanding of biological mechanism at a molecular level. Most of those methods are based on a nonlinear interaction between the excitation light intensity and the sample response (often fluorescent signal). In the same time, nanodiamonds containing fluorescent defects have been proven to be a choice probe for superresolution nanos-copy since they exhibit a strong and stable fluorescent signal even under high light intensities exposure (often required to obtain nonlinear photoresponse). Nanodiamonds containing Nitrogen Vacancy (NV) defects that exhibit a red fluorescent signal had been previously shown to be a viable biomarker for STED superresolved image. First, we demonstrated that green fluorescent nanodia-monds containing Nitrogen-Vacancy-Nitrogen (NVN) defects can be used with a Stimulated Emission Depletion (STED) superreso-lution microscope. Then, we implemented a STED microscope in our lab and compared the properties of NVN and NV centers for STED imaging. We conclude that even if nanodiamonds with NVN defects are less intense, they can be used as a second color nonbleaching biomarker. To illustrate the potential use of green nanodiamonds as bio-compatible probe, we superresolved them internalized into a cell with STED microscopy. Second, we tried to work on one of the main limitation in STED nanoscopy: the lack of information in the axial direction within a single scan. We combined our home made STED microscope with a Double Helix phase mask that modifies the detection point spread function in order to obtain axial localization of the superresolved emitters. We achieved three dimensional localization of nanometric fluorescent emitters but we note that photobleaching was the main limitation of this approach with organic dyes. We discussed different solutions to limit the photobleaching and their feasibility. We also worked on a different superresolution technique that we named Computational Nonlinear Saturated (CNS) microscopy. We showed that with digital post treatment of the acquired data, a nonlinear photoresponse can be harnessed to any scanning microscope equipped with a camera detector to enhance the resolution. We demonstrated that increasing the excitation power and inducing fluorescence saturation, it is possible to break the diffraction limit in a conventional confocal microscope (after data post-treatment). However, with this method, we did not obtain a gain in resolution as high as with other superresolution tech-niques involving fluorescence saturation, such as saturated structured illumination microscopy. To understand the origin of this limitation, we carried out simulation to investigate the performance of CNS microscopy in noisy environments compared with wide field techniques. We propose alternative implementation and quantify the possible resolution gain with simulations. Finally, we demonstrated how a technique, initially created for optical microscopy, can be adapted to lensless endoscopic imaging...