STED imaging of green fluorescent nanodiamonds containing nitrogen-vacancy-nitrogen centers

We report Stimulated Emission Depletion (STED) imaging of green fluorescent nanodiamonds containing Nitrogen-Vacancy-Nitrogen (NVN) centers with a resolution of 70 nm using a commercial microscope. Nanodiamonds have been demonstrated to have the potential to be excellent cellular biomarkers thanks to their low toxicity and nonbleaching fluorescence, and are especially appealing for superresolution imaging technique like STED microscopy. However, only red fluorescent nanodiamonds containing Nitrogen-Vacancy (NV) centers have been used with STED microscopy so far. The existence of only one color nonbleaching center limits the possible observations, for instance it complicates spatial correlation studies with STED. To provide a nonbleaching probe in a different color, we characterize here the optical properties of the NVN defect for STED imaging. We demonstrate STED imaging of the green fluorescent nanodiamonds containing NVN centers, opening the door for long term two-color STED observation. Furthermore we exemplify the use of green nanodiamonds as a second color nonbleaching STED biomarker by imaging 70 nm fluorescent crystals up taken into HeLa cells. (C) 2015 Optical Society of America

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...

EPFL2016

Compact lensless digital holography

Manon Rostykus

Microscopy is of high interest for biology since it allows imaging features that are too small to be seen with naked eyes. However, cells are mostly transparent to visible and infrared light which makes it difficult to see with a traditional microscope. To do so, phase imaging was invented by F. Zernike, who received the Nobel prize in 1953 for this invention. It provides intensity contrast to visualize transparent samples such as those found in biology without any staining. Among the different implementations of phase imaging, digital holographic microscopy (DHM) is a well-known phase method which provides a quantitative value of the phase generated by the sample under test. Implementations of DHMs without the help of any lens element (so called lensless) offer the added advantage to provide large field of views (severalmm2 compared to several hundred ¹m2) and more compact setups than traditional DHMs which have high quality microscope objectives. The lateral resolution is limited by the pixel size of the camera. However, several techniques have been developed to obtain subpixel resolution with lensless setups, hence giving a resolution smaller than the pixel size. In this thesis, two lensless transmission DHMs are presented using a side illumination technique in order to further reduce the device size and keep a visual access to the sample. The first one operates in an in-line configuration: the most compact but using time-consuming image post-processing. The second one is also lensless and uses an off-axis configuration allowing quasi-real time imaging but less compact. Their practical use is described and images of transparent (phase only) samples are shown. Super-resolution (subpixel) using several subpixel shifted holograms was then investigated for the in-line configuration and demonstrated on absorptive samples. Since those microscopes are compact and made out of off-the-shelf low priced elements, they could be broadly spread in schools for educational purposes. They could also be implemented in cells incubators, allowing visualization inside processes at low cost and multiplication of the number of measurements made at the same time.

EPFL2018

Nanoscopy in nonlinear scanning fluorescence imaging systems

Grégoire Pierre Jean Laporte

EPFL2016

Compact lensless digital holography

Manon Rostykus

EPFL2018