The project has realized breakthroughs and novel technology within multiple different fields. In particular, new insights have been developed in (i) smart genetically-encoded fluorophores, (ii) smart biosensors, (iii) novel optical instrumentation, and (iv) new data analysis methodologies for advanced imaging.
A key goal was the development of new light-controllable fluorophores for bioimaging. Key questions remained not only regarding the achievable performance, but also how their spectroscopic properties related to the chemical structures of the probe. Accordingly, we developed a range of new labels and performed an extensive structural investigation of their properties, resulting in a publication in Angewandte Chemie. This allowed us to obtain detailed structural insights by developing new data analysis, presenting a more unified picture of their ‘smart’ photochromic behavior than was previously possible.
We then incorporated these labels into smart biosensors. Fluorescent biosensors are the only way to monitor the cell chemistry within live cells, though they do not easily permit high-resolution imaging. We therefore incorporated smart fluorophores into a FRET-sensor for kinase activity and also created a light-controllable version of calcium sensor. We then showed that our ‘smart’ FRET sensor could used to simultaneously measure multiple biosensors that are spectrally indistinguishable. Such FRET-based multiplexing holds the potential for revealing much more complex information on the cellular biochemistry and is an area that we are now further exploring. This study was published in Nature Communications.
We then showed that the Ca2+ sensor could be used to realize quantitative biosensing using a novel approach based on photochromism. In this way we could show that absolute calcium levels could be determined within live cells showing calcium oscillations. We furthermore developed a novel approach showing how slower but fully quantitative measurements can be combined to deliver fully quantitative measurements that are both faster and may also have (much) less phototoxicity. This work was also published in Nature Communications.
We then explored the possibility of including also fluorophores that are not genetically-encoded, taking advantage of chemigenetic labels. We developed a strategy to create chemigenetic FRET sensors, which was published in ACS Sensors. Beyond their intrinsic usefulness in offering a higher spectral versatility, I expect that this type of probe will allow to create further imaging development by offering good sensing properties as well as direct access to the favorable optical properties of synthetic dyes.
Another key advance is in the development of new instrumentation. We developed an optical device that allows for simultaneous multicolor imaging with a single camera and access to the full field of view. The device does this by engineering the point-spread function (PSF) of the imaging, in such a way that each emitter appears as two spots where the relative orientation of the spots reports on the color of the emitter. This enables simultaneous imaging in single-molecule measurements, which are often characterized by slow acquisitions of the individual color channels. We have furthermore acquired a patent on this device. The concept itself is currently being revised for resubmission to Nature Methods, where it was favorably reviewed.
Two instrumentation projects are currently progressing upon initial results realized as part of the ERC. One of these is a PSF-engineering device similar to the previously described, except that it separates emitters based on the excitation wavelength. This enables much faster ratiometric imaging for biosensors as well as easier multiplexing of single-molecule fluorescence measurements.
The last optical instrumentation development is on a novel approach for fast 3D imaging with high (single-molecule) sensitivity. Our novel design directly addresses a gap in the current instrumentation, by offering fast imaging performance that is compatible with the high-throughput sample preparations. This system is currently in a fairly early stage, though we have already obtained IP protection on the basic design.
In terms of data analysis, we have continued to advance (amongst others) the SOFI imaging methodology, developing a full theoretical model of the imaging (published in Optics Express) as well as a methodology to quantitatively evaluate the suitability of particular fluorophores and imaging conditions for this superresolution imaging modality (published in Biomedical Optics Express). We have also developed new analysis algorithms and software in support of the work described above, detailed in more information in the relevant publications.
These are the highlights of the research performed thus far. Other work has been realized as well with the support of this project, and is best appreciated from the list of publications obtained.
