Recently, we have developed Image Scanning Microscopy (ISM) that doubles the resolution of a conventional confocal microscope by replacing the confocal pinhole with an imaging detector. Here, we describe theory and realization of a new fully optical non-linear ISM suitable for two-photon excited fluorescence and second-harmonic generation. It provides excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution compared to a conventional two-photon laser-scanning microscope. We demonstrate the performance using fixed and living specimen, as well as hydrogels. The modular design allows straight-forward implementation into existing microscopes.
We also present a cost-efficient FLIM-ISM detector providing super-resolved fluorescence lifetime images using two-photon excitation and can be implemented into any confocal microscope.
Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates.
We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging.
1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101.
2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53–54.
Time-resolved confocal microscopy is well established to image spectral and spatial properties of samples in biology and
material science. Atomic Force Microscopy (AFM) in addition enables to investigate properties which are not optically
addressable or are hidden by the diffraction limited optical resolution.
We present a straight forward combination of single molecule sensitive time-resolved confocal microscopy with different
commercially available AFMs. Besides an extra of information about for example a cell surface, the AFM tip can also be
used to manipulate the sample on a nanometer scale down to the single molecule level.
The combination of atomic force microscopy (AFM) with single-molecule-sensitive confocal fluorescence
microscopy enables a fascinating investigation into the structure, dynamics and interactions of single
biomolecules or their assemblies. AFM reveals the structure of macromolecular complexes with nanometer
resolution, while fluorescence can facilitate the identification of their constituent parts. In addition,
nanophotonic effects, such as fluorescence quenching or enhancement due to the AFM tip, can be used to
increase the optical resolution beyond the diffraction limit, thus enabling the identification of different
fluorescence labels within a macromolecular complex.
We present a novel setup consisting of two commercial, state-of-the-art microscopes. A sample scanning
atomic force microscope is mounted onto an objective scanning confocal fluorescence lifetime microscope.
The ability to move the sample and objective independently allows for precise alignment of AFM probe
and laser focus with an accuracy down to a few nanometers. Time correlated single photon counting
(TCSPC) gives us the opportunity to measure single-molecule fluorescence lifetimes. We will be able to
study molecular complexes in the vicinity of an AFM probe on a level that has yet to be achieved. With this
setup we simultaneously obtained single molecule sensitivity in the AFM topography and fluorescence
lifetime imaging of YOYO-1 stained lambda-DNA samples and we showed silicon tip induced single
molecule quenching on organic fluorophores.
The influence of metal surfaces and nanoparticles on the fluorescence emission of fluorophores in close proximity is of
particular interest for biophysical applications, near field optics and biosensing. For instance, the quenching of
fluorophores by gold nanoparticles can be used for the investigation of biomolecular conformational changes or
interactions and silver coated metal tips are potent scanning near field optical microscopy tips. Apart from the
quenching effects, nanoparticles are used for fluorescence enhancement in biosensor applications.
Here we use a setup combining total internal reflection fluorescence microscopy (TIRFM) with the piezo-controlled
nanometer-sensitive movement of an atomic force microscope (AFM) in order to measure and quantify the fluorescence
emission as a function of distance between single fluorophores and metal nanoparticles or tiny metal tips. By using
CdSe/ZnS nanocrystals as fluorophores and gold as metal we observed significant fluorescence quenching as well as
enhancement due to exciton-plasmon coupling. In the future, these experiments will be extended to metal nanoparticles
of different elements, alloys, sizes and shapes, giving insight into the related energy transfer processes and quenching
Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.