Technology

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. 

labThe 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 multi-parametric FCS experiments, ultra-fast TCSPC and, recently, we upgraded our HDIM system with a novel and more photon-efficient design.


Milestones

Over the last few years, we have achieved quite a few “firsts”:

  • 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, unpublished)
  • 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)

pipeline_tech


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.

Impact | The detection system we demonstrate is compact (not larger than a textbook), USB connected and interfacable with most existing microscopes. Despite its simplicity, this technology enables to gather more information at higher acquisition speeds or dynamic ranges and it is currently limited only by the typical constraints of a prototype. This system integrates elegantly with the most recent advances in data analysis, permitting users to analyse complex dataset with no a priori information and minimal input. For all these reasons, CMOS detectors of this kind, promise to impact biochemical imaging techniques (including tissue imaging) enabling very sophisticated assays yet at lower overall costs and requiring less specialized know-how.
A inear array of smart pixels for fast and simple biochemical and medical imaging
(a) The design of the spectral FLIM system, comprising a Ti:Sapphire laser, coupling optics, a laser scanning confocal microscope and a simple direct vision spectrograph based on the linear array of SPADs. Photon counts (b), true-colour (c) and fluorescence lifetime (d) images of unstained liver tissue excised from a tumorigenic murine model. Blind unmixing performed with the use of phasors (e-g) exhibits maximum image contrast within a single image; panel (h) shows the fractional intensities of the unmixed components. Fractional intensities are displayed in blue (B), green (G), red (corresponding to a dominant second harmonic signal, R) corresponding to the pure phasors marked with the same letters in (E-G). Scale bar: 20µm. 

Confocal spectropolarimetry

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).

spectropolarimetry (HDIM-1G)
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

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.

You can also read our patent application or check the wonderful work that PCO have done to make this prototype performing at high standards and commercially available.

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).

in-pixel FLIMI 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.

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