Periodic Reporting for period 2 - Switch2See (Genetically encoded reporters and sensors for whole animal imaging using photo-modulated Optoacoustics)
Periodo di rendicontazione: 2023-03-01 al 2024-08-31
One of the grant challenges for biological research is to visualize the localization and function of cells in live mammals with high resolution; the same is true for visualizing the distribution of the chemicals that drive life. Such imaging on the level of organs, tissues or the whole animal is indispensable to understand the “bigger picture”. Current whole animal imaging techniques are either limited in the richness of detail (resolution), their ability to capture a whole tissue in a holistic fashion (volume-of-view / penetration depth), the availability of labels for unperturbed and targeted imaging of specific cells or chemical distributions and finally their potential to work in vivo.
A methodology that can overcome those limitations can be an indispensable driver to advance our fundamental understanding of organismal functioning and advance bio-medical research necessary to counter pathophysiologies. In this regard, the tumor micro-environment (TME) is a prime example of a pathophysiological state characterized by a complex and highly interdependent mosaic. High resolution imaging (resolving single cells or small populations) of immune cell infiltration with respect to concentrations of specific chemicals in the tumor, for example, is crucial in understanding the disease and develop therapeutic strategies. The same is true for numerous other questions in immunology, developmental- or neurobiology. All are characterized by the necessity to resolve spatial and temporal patterns of cell populations and chemical distributions on the level of whole tissues or the animal in vivo.
The project aims to advance an in vivo imaging modality that combines high resolution, large volume of views and availability of genetically encoded labels. The project builds on opto- or photoacoustic imaging; a young methodology that affords deep penetration in tissue at comparably high resolution by detecting ultrasound signals upon illumination. The technique already proofed to be valuable in research and clinical imaging by visualizing for example the vessel infrastructure whose aberrations are indicative of many pathophysiologies. To date optoacoustic imaging however lacks a successful way to employ targeted labels alike fluorescent proteins in fluorescence microscopy (Nobel Prize 2008). We seek to implement such genetically encoded labels tailored for optoacoustic imaging and with this enable the use of optoacoustic as a method for high resolution in vivo research imaging of labeled cells and molecular distribution allowing us to see the “bigger picture”. This goal will be achieved by relying on protein-engineered photoswitching proteins. This class of proteins can be switched between an ON and OFF state by illumination with two different wavelengths. Accordingly, alternating illumination results in the modulation of the optoacoustic signal. Thus, the modulating signal of cells expressing a photoswitchable reporter can be used to discriminate those cells from the non-modulating tissue background in vivo – rendering the latter virtually invisible – hence, Switch2See.
A methodology that can overcome those limitations can be an indispensable driver to advance our fundamental understanding of organismal functioning and advance bio-medical research necessary to counter pathophysiologies. In this regard, the tumor micro-environment (TME) is a prime example of a pathophysiological state characterized by a complex and highly interdependent mosaic. High resolution imaging (resolving single cells or small populations) of immune cell infiltration with respect to concentrations of specific chemicals in the tumor, for example, is crucial in understanding the disease and develop therapeutic strategies. The same is true for numerous other questions in immunology, developmental- or neurobiology. All are characterized by the necessity to resolve spatial and temporal patterns of cell populations and chemical distributions on the level of whole tissues or the animal in vivo.
The project aims to advance an in vivo imaging modality that combines high resolution, large volume of views and availability of genetically encoded labels. The project builds on opto- or photoacoustic imaging; a young methodology that affords deep penetration in tissue at comparably high resolution by detecting ultrasound signals upon illumination. The technique already proofed to be valuable in research and clinical imaging by visualizing for example the vessel infrastructure whose aberrations are indicative of many pathophysiologies. To date optoacoustic imaging however lacks a successful way to employ targeted labels alike fluorescent proteins in fluorescence microscopy (Nobel Prize 2008). We seek to implement such genetically encoded labels tailored for optoacoustic imaging and with this enable the use of optoacoustic as a method for high resolution in vivo research imaging of labeled cells and molecular distribution allowing us to see the “bigger picture”. This goal will be achieved by relying on protein-engineered photoswitching proteins. This class of proteins can be switched between an ON and OFF state by illumination with two different wavelengths. Accordingly, alternating illumination results in the modulation of the optoacoustic signal. Thus, the modulating signal of cells expressing a photoswitchable reporter can be used to discriminate those cells from the non-modulating tissue background in vivo – rendering the latter virtually invisible – hence, Switch2See.
The aim at this stage of the project was twofold, establish methodologies needed for the future developments and initiate the development of photoswitching proteins tailored for optoacoustic imaging.
Despite advances in protein engineering, it is still not possible to fully rational predict which amino acids in a protein must be changed to arrive to a desired characteristic. Due to this, research employs an approach which, next to rational changes, relies on random alteration of the amino acids in the protein of interest and identification of beneficial changes by screening (directed evolution). Towards this end a screening methodology is required that allows to efficiently probe large numbers of proteins variants for their desired characteristics –- in this case photoswitching of the optoacoustic signal and dependence on binding of molecules of interest. We implemented a plate-based screening system with a throughput of ~1000 variants per hour and are advancing a microfluidic system. The latter is based on the detection of so-called surface-acoustic waves which allow a detection of the optoacoustic signal directly in the microfluidic chip.
The second important methodological area was the development of robust ways to establish the so-called ground-truth, i.e. “absolute” information on the presence of labeled cells in the organism at a given position. Such ground-truth is provided by a method that allows to detect the label relatively unambiguously ex vivo after the imaging with the novel in vivo imaging technology of Switch2See. A critical aspect was the co-registration, i.e. the alignment of the two imaging datasets in a reliable and routine fashion.
Simultaneously, the actual protein engineering of reporters and small molecule / ion sensors begun with the first lead candidates of Switch2See capable sensors.
Despite advances in protein engineering, it is still not possible to fully rational predict which amino acids in a protein must be changed to arrive to a desired characteristic. Due to this, research employs an approach which, next to rational changes, relies on random alteration of the amino acids in the protein of interest and identification of beneficial changes by screening (directed evolution). Towards this end a screening methodology is required that allows to efficiently probe large numbers of proteins variants for their desired characteristics –- in this case photoswitching of the optoacoustic signal and dependence on binding of molecules of interest. We implemented a plate-based screening system with a throughput of ~1000 variants per hour and are advancing a microfluidic system. The latter is based on the detection of so-called surface-acoustic waves which allow a detection of the optoacoustic signal directly in the microfluidic chip.
The second important methodological area was the development of robust ways to establish the so-called ground-truth, i.e. “absolute” information on the presence of labeled cells in the organism at a given position. Such ground-truth is provided by a method that allows to detect the label relatively unambiguously ex vivo after the imaging with the novel in vivo imaging technology of Switch2See. A critical aspect was the co-registration, i.e. the alignment of the two imaging datasets in a reliable and routine fashion.
Simultaneously, the actual protein engineering of reporters and small molecule / ion sensors begun with the first lead candidates of Switch2See capable sensors.
We developed screening methodology that allows to interrogate the photoswitching characteristics of a high number of photoswitching protein variants in a short time. Along those developments we established the first readout of optoacoustic signals through surface acoustic waves from cells in flow (in collaboration with colleagues from the Leibniz institute Dresden, Germany). Surface acoustic waves are commonly known from actuation and are, for example, used to position cells. Here we use the acoustic wave emitters – so called interdigital transducers – as receivers which allows us to record the optoacoustic signal of single cells in flow after hit by a laser pulse. Compared to conventional detection with standard optoacoustic detection approaches (bulk wave ultrasound transducers) this arrangement has likely a higher sensitivity due to the ultrasound wave not needing to “leave” the microfluidic chip and it potentially allows also easier exploitation due to the affordability and scalability of fully microfluidic based technologies. Along those lines we envision use of such technology beyond our screening efforts of Switch2See in for example diagnostics.
Further, we developed a robust co-registration pipeline which allows to eventually compare results obtained in optoacoustic imaging (not only of the photoswitching labels used in Switch2See) in a quantitative spatial manner and aims at advancing the reproducibility and comparability of developments in the overall field of optoacoustic label imaging. Such is essential to arrive to a mature technology that allows the practitioner to reliably choose a certain approach and be able to judge it against comparable technologies.
At the end of the action Switch2See aims at allowing to image cells specifically in the whole mouse at high resolution for bio (- medical) research. It will enable longitudinal studies on chemical distributions at high resolution. With this we will take the first steps towards routine application of photoswitchable reporters and sensors in optoacoustic. The universality of the concept and the relationship to fluorescent proteins will spark a similar revolution as fluorescent proteins did for fluorescence imaging (more reporter, sensors, tools, instrumentation), altering the way how we study live animals.
Further, we developed a robust co-registration pipeline which allows to eventually compare results obtained in optoacoustic imaging (not only of the photoswitching labels used in Switch2See) in a quantitative spatial manner and aims at advancing the reproducibility and comparability of developments in the overall field of optoacoustic label imaging. Such is essential to arrive to a mature technology that allows the practitioner to reliably choose a certain approach and be able to judge it against comparable technologies.
At the end of the action Switch2See aims at allowing to image cells specifically in the whole mouse at high resolution for bio (- medical) research. It will enable longitudinal studies on chemical distributions at high resolution. With this we will take the first steps towards routine application of photoswitchable reporters and sensors in optoacoustic. The universality of the concept and the relationship to fluorescent proteins will spark a similar revolution as fluorescent proteins did for fluorescence imaging (more reporter, sensors, tools, instrumentation), altering the way how we study live animals.