Fluorescence microscopy providing highest resolution
Developing high resolution imaging techniques based on fluorescence microscopy is a highly topical field of research (Nobel Prize in Chemistry in 2014). Our research focusses on further developing techniques, which provide highest lateral resolution, three dimensional imaging as well as confocal laser scanning microscopy.
High lateral resolution (SIM, dSTORM)
In structured illumination microscopy (SIM) high spatial frequencies of the sample (finer object structures) are down-modulated into the transfer band of the objective by applying a stripe shaped illumination pattern. Hence structured illumination improves the resolution. The research work, regarding SIM focuses on:
- Multicolor SIM with up to three different excitation wavelengths
- Fast SIM with a recording rate of up to 700 images per second
- Multifocal SIM for simultaneous detection of several focal planes
- Nonlinear SIM to further enhance the lateral resolution
- Polarization coded SIM for improving the optical sectioning
- Multiphoton SIM for achieving highest resolution deep inside strongly scattering tissue
A second approach in order to enhance the resolution is direct stochastic optical reconstruction microscopy (dSTORM). In dSTORM a series of images is recorded. At each image only a small fraction (statistically determined) of all fluorophores is emitting light. Due to the sparse emission pattern, the emission centers can be determined with very high accuracy. The high resolution image is subsequently reconstructed by superposing all frames of the series.
Currently we aim for combining this method with automated multiple staining and image recording steps in order to develop a systematically working high resolution analysis tool.
Three Dimensional Imaging
- Light sheet microscopy enables the large volume microscopic analysis of strongly scattering samples. Our currently existing light sheet microscope is to be extended to be suited for clinical applications.
- Holoscopic methods allows very fast recording of three dimensional sample information.
Laser Scanning Microscopy
The third pillar of our research on fluorescence microscopy is laser scanning microscopy (LSM). There we focus on two approaches for enhancing the resolution provided by typical confocal techniques:
- Optical photon reassignment microscopy (OPRA)
- Image inversion interferometry (UZI)
Alternative Microscopic Approaches
We work on different approaches in order to develop alternative microscopic methods. On the one hand they complement the methods from fluorescence microscopy. On the other hand they aim for extending known as well as developing new techniques.
- Development of a low cost 3D printed confocal microscope: A confocal microscope with manufacturing costs below 100 Euro allows a wide distribution of those devices e.g. for educational purposes. It might as well serve to support laggard regions.
- Raman spectroscopic imaging: Highly functional imaging can be accomplished by exploiting Raman scattered light. We aim to combine this technique with different research topics, such as SIM or light sheet microscopy.
- Spin imaging: Nuclear spin induced magnetic field or temperature maps can be resolved in the sub nanometer range by microwave excitation of nitrogen vacancies in Nano-diamonds and simultaneous recording of the corresponding fluorescence. We currently establish this approach for imaging and analysis of single molecules
- Fluorescence up-conversion microscopy: Specific marking of biologically relevant molecules with nano-fluorescence converters allows highly localized generation of UV light. This can be used to trigger chemical reactions on only a few molecules, activate biological processes highly localized or – as known form dSTORM – determine the molecule position on the sub 100 nanometer scale.
Development of Novel Algorithms for Image Processing
Particularly high resolution microscopy requires a complex post record image processing. Thus our research also focusses on new and further development of theoretical approaches for image processing as well as their implementation:
- SIM reconstruction: We follow different approaches for analyzing SIM data:
- Fast image reconstruction for previous determined experimental parameters
- Image reconstruction without exact knowledge of selected crucial parameters (e.g. the OTF)
- Development of algorithms in order to correct aberrations caused by the detection system or sample movement. Thus, our systems can be widely applied and are particularly suitable for live cell imaging.
- Neuronal networks: We develop novel, optimized, neuronal network based algorithms in order reconstruct multi-dimensional fluorescence and OCT data.
- Parallelization: Graphic chip (GPU) based parallelization of our algorithms allows very fast data analysis. This enables for instance “On-the-fly” reconstruction of SIM data. We developed a special Cuda library for MatLab (CudaMat) to implement the parallelization.
High Sensitive Detection of Smallest Losses in Optical Material and Coatings
The main issue is the detection of light absorption in optical materials and coatings with sub-ppm sensitivity. In order to achieve this high sensitivity, we have developed the LID technique (LID… laser induced deflection, patent-registered), which is based on the photo thermal effect. The LID possesses an independent absolute calibration, which is superior compared to other photo thermal methods. Combining the LID technique with laser induced fluorescence (LIF) helps to investigate a multitude of questions in material science, such as investigating the interaction between laser irradiation and optical materials, as well as dielectric optical coatings.
We apply the Cavity Ring-Down (CRD) technique in order to determine smallest optical losses. This enables for instance the precise determination of highest mirror reflectance (R>> 99.9%). In combination with the LID method we are also able to assign the losses to their particular origin (scattering, absorption).
Apart from scientific projects, our multitude of available laser sources/wavelengths allows offering a broad range of measurement services for optical material and coating characterization.
Following the institute’s guiding idea “From Ideas To Instruments”, both, the LID – as well as the CRD technique have been transferred recently intocommercial prototypes/systems – partially based on an external partner.