New horizons in biology and medicine are opened by enabling technologies that allow a deeper understanding of the molecular mechanisms that regulate cellular function and dysfunction. We specialize in the development of novel biophysical/biochemical imaging tools including the quantitative detection of emission spectra, fluorescence lifetime and polarization and the integration of all these modalities. Our work relies on encoding the biology of interest in the photophysics of fluorescent probes and the optimal engineering of instrumentation to decode the biochemical signatures we can read-out by fluorescence. Therefore, one part of our work often results in complex assays and instruments tailored to achieve a specific measure of biological interest. However, we are interested in helping others to adopt our approach. For this reason, we also work to render our techniques more user-friendly and cost-effective. Cost-effective systems are critical to permit the implementation of new tools for biological studies in non-specialist laboratories, drug discovery and diagnostics.
Our motto: innovating technology to push the boundaries of biology.
The biophotonics lab at the MRC Cancer Unit. On the left, a cluster of detectors with motorized optomechanics that can be easily reconfigured by software to perform a very broad range of biophysical imaging and spectroscopic techniques. These include fluorescence lifetime imaging microscopy (or FLIM, to detect protein-protein interactions or to read out biosensors), time-resolved anisotropy (or rFLIM, to detect homo-dimerization and interactions), spectral FLIM (or SLIM, to multiplex biochemical interactions) and spectrally and polarization resolved FLIM, a technique we have introduced and named Hyper-dimensional imaging microscopy (HDIM) to maximize biochemical resolving power in fluorescence microscopy or contrast in tumour imaging. On the right: smart pixel arrays in CMOS technology (collaboration with the IRIS group of David Stoppa at FBK, Italy) for fast and efficient SLIM/HDIM. The system illustrated here comprises a Ti:Sapphire laser for two-photon microscopy, a pulse-picker, a Leica SP5 with NDD detectors and the cluster of detectors just described. In addition to these techniques, we can perform ultra-fast TCSPC (ELIS) and, recently, we upgraded our HDIM system with a novel and more photon-efficient design.
Technology-projects at MRC Cancer Unit
We have developed a number of microscopy platform currently operating at the Unit:
- Hyper-Dimensional Imaging Microscopy (HDIM) | a Leica SP5 two-photon microscope capable to resolve energy, polarizations state and arrival time (by TCSPC) of each photon in a 2D/3D image. Used to validate theoretical studies on microscopy resolution, to test multi-parametric tumour imaging and multiplexing capabilities in fluorescence microscopy [see Biophysical Journal paper or blog post] [K. Suhling’s commentary] [bioRxiv pre-print].
- ELIS | a detection system based on the Leica SP5 two-photon microscope dedicated to fast multi-colour TCSPC. When built this was the fastest TSCPC-based FLIM system, now rivalled by commercial technologies such as the FALCON (Leica) and RapidFLIM (PicoQuant) systems. This fast FLIM is based on multi-hit TDCs [see Methods and Applications in Fluorescence manuscript]. It was developed for fast multiplexing of FRET pairs (NyxBits&NyxSense) showcased in this pre-print [BIORXIV/2018/427237].
- Optogenetics | We have developed a special incubator (OptoFarm) that host high power LEDs to culture cells engineered with optogenetic tools. Also, we have a double-deck Nikon microscope equipped with the Mosaic Duet (Andor), TuCam (Andor) and two EMCCD for fast seFRET and optogenetic studies. This platform has been recently integrated with microfluidics.
- ATLAS.ONE | ATLAS.ONE is a new platform currently still under development. It is aimed to be a fast FLIM wide-field system integrating structured illumination microscopy and microfluidics. In time, this should evolve as high throughput microscopy. The first planned applications are related to cancer metabolisms.
- ATLAS.TWO | ATLAS.TWO is a new platform that is just in the very early phases of development. It is going to be a complex development where we will attempt to integrate high-throughput technologies with biochemical imaging and optogenetics with a light-sheet microscope. Watch this space for news.
- OncoLive SPIM | Just funded, this is going to be a SPIM system broadly modelled around the OpenSPIM platform but with some relevant differences. We will use this system for imaging of organoids and light dose painting to seed mutations with a light-inducible CRE recombinase. We will soon update a dedicated website for this project.
Over the last few years, we have achieved quite a few “firsts”:
- (Fast) FLIM-based direct imaging of a three-node biochemical network (~2015, pre-print in 2018)
- Fast spectrally-resolved FLIM with an array of smart CMOS pixels (~2012)
- Hyper-dimensional imaging microscopy (HDIM): the first fully resolved (spectra, polarization and lifetime) images of biological samples (~2010, pre-print in 2018)
- Confocal spectropolarimetry (~2009)
- Unsupervised high-throughput FLIM for high content screening (~2007)
- Single-shot parallel fast wide-field FLIM with a solid state CMOS detector (~2005)
Fast and simple spectral FLIM for biochemical and medical imaging
Collaborative work with David Stoppa at the Fondazione Bruno Kessler in Trento; project outcomes published in 2015 in Optics Express
Abstract | Spectrally resolved fluorescence lifetime imaging microscopy (FLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of spectral FLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art spectral FLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.
This work was carried out in the laboratories of Prof. Hans Gerritsen, Prof. Clemens Kaminski and Prof. Ashok Venkitaraman; project outcome published in 2011 in Optics Express
Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. In this paper, we demonstrated a novel and simple architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner. This sensing platform achieves excellent versatility of use at comparatively low costs. We demonstrate the novel optical design with example images of plant cells and of mammalian cells expressing fluorescent proteins undergoing energy transfer.
This system is very efficient and, for those interest in fluorescence anisotropy or to analyze spectrally dependent birefringence in materials may find this architecture quite useful. For biological applications, this system would permit to detect homo-FRET, multiplexed over the visible spectrum or to detect changes in fluorescence of environmentally sensitive probes. This is probably one of the most sensitive and simple technique I have developed and the only reason I am currently not using it is that I wanted to allocate my resources on different more ambitious projects. If you are interested to implement this technique, I can share know-how and software (in Matlab).
To me, this represents the foundational milestone of multiplexed techniques I am currently developing and I often refer to this system as HDIM-1G (first generation HDIM), albeit the detector I used is an EM-CCD and, therefore, not time-resolved.
Unsupervised FLIM for high throughput imaging of biochemical events
This work was carried out in the laboratory of Prof. Fred Wouters; Click to see paper published in Molecular and Cellular Proteomics (F1000 recommended) in 2007
Proteomics and Cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Foerster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. We demonstrated that the high content offered by these techniques can be combined with the high-throughput levels offered by automation of a fluorescence lifetime imaging microscope setup, capable of unsupervised operation and image analysis. Systems and software dedicated to Image Cytometry for Analysis and Sorting represent important emerging tools for the field of proteomics, interactomics and cellomics.
high content screening by FLIM
Fast wide-field FLIM
This work was carried out in the laboratory of Prof. Fred Wouters in collaboration with CSEM (now spun-off to MESA imaging); project outcomes published in Optics Express in 2005 and in Journal of Biomedical Optics in 2006.
Until a few years ago, FD-FLIM systems were limited by the use of expensive technologies and specialized instrumentation, by limited spatial resolution and acquisition throughput and limited capability to resolve heterogeneous systems. We developed methods of analysis and novel technologies to overcome those limitations and to foster the engineering of the new generation of sensing technologies.
Fast and cost-effective system: Solid state technologies for sensing (CMOS and CCD) and sample excitation (LED and laser diodes) were combined in the first cost-effective (~12kEUR + microscope) prototype of an FD imaging system capable of full-field imaging with a single exposure. This technology may replace in the near future the obsolete multi-channel plates used in intensified camera providing fast and efficient FLIM systems. Indeed, PCO is commercializing the first of these cameras (PCO.FLIM) which specs should be good enough for lifetime imaging of biological samples at qualities at least matching those of MCPs.
However, technology is not sufficient to make of wide-field FLIM the technique that we wished: simple, efficient and fast. For this reason, we have developed theoretical frameworks (see Theory section of this website) to optimize it and reach high throughputs and photon-efficiencies (see also papers published in JOSA-A and Biophysical Journal).
I believe that all these development signed a transition in the community demonstrating for the first time that solid-state technologies developed originally for time-of-flight ranging applications were compatible and mature enough to be redeveloped and applied to lifetime imaging within the life sciences. You can read my thoughts on this topic in this communication to Remote Sensing.