We have developed a range of endoscopes with the goal to better diagnose diseases such as cancer and osteoarthritis and to provide tools to study the impact of therapies for medical research, including as part of the drug discovery process. In particular, as we have developed FLIM technologies to study molecular interactions and to explore time-resolved autofluorescence readouts for label-free diagnosis of disease, we have worked towards FLIM endoscopy.


Wide-field FLIM endoscopy

Initially we implemented wide-field FLIM endoscopy with a rigid arthroscope at up to 29 fps [1] and we also implemented wide-field FLIM with flexible endoscopes incorporating fibre-optic bundles [2]. Initially we used frequency-doubled mode-locked Ti:Sapphire lasers to provide the ultrashort pulsed excitation for FLIM but subsequently we demonstrated wide-field FLIM endoscopy with a flexible optical fibre bundle endoscope using blue gain-switched diodes to provide picosecond excitation pulses [3].

Single-point fibre-optic probe fluorometers

For in vivo application, there is a trade-off between minimising light dose to the tissue (to avoid any phototoxicity) and acquiring sufficient photons to fit the fluorescence decay profiles to complex decay models. This can be addressed by binning all the image pixels or by using global fitting techniques. For the former approach, the instrumentation can be simplified if the goal is endoscopic time-resolved spectroscopy rather than imaging and we addressed this by developing a series of instruments utilising compact fibre-optic bundles incorporating excitation and detection fibres. These instruments are multidimensional fluorometers providing single-point measurements. The first such instrument utilised a compact frequency-tripled ultrafast Yb-doped glass fibre laser providing picosecond pulses at 355 nm and gain-switched diode laser providing picosecond pulses at 445 nm. This instrument was evaluated in vivo to diagnose skin cancer [4]. This instrument was also applied to explore studies of autofluorescence lifetime as a potential diagnostic readout for osteoarthritis through ex vivo studies of cartilage [5]. Subsequently a more compact version of this instrument utilising gain switch diode lasers at 375 nm and 435 nm was applied to ex vivo [6] and in vivo studies exploring autofluorescence lifetime as a readout of neoplasia in the human colon. A further version of this instrument that also incorporated while-light reflectance measurements was applied to in vivo preclinical studies of heart disease in a rat model [7] and to ex vivo studies of a Langendorff isolated-perfused rat heart model [8].

Confocal FLIM endomicroscopy

We also translated laser scanning confocal FLIM to endoscopy, developing a confocal FLIM endomicroscope based on the Mauna Kea Technologies CellVizio platform, to which we added a frequency-doubled mode-locked Ti:Sapphire laser to provide pulsed excitation and TCSPC detection [9]. We demonstrated that this provided comparable performance to our lasers canning confocal FLIM microscope in terms of lifetime determination and was able to realise confocal FLIM endomicroscopy of fluorescent-protein labelled cells at up to 10 fps. This instrument was applied in a preclinical study with colleagues at the Francis Crick Institute to quantify drug-target engagement in vivo in a murine cancer model utilising FLIM of FRET between the fluorescent anticancer (DNA-intercalating) drug, doxorubicin, and EGFP, which was expressed in the mouse histones [10].

Adaptive endoscopy

We were motivated to apply FLIM endoscopy to study autofluorescence in vivo for label-free studies of disease but the required excitation wavelengths also excite significant unwanted luminescence in the glass from which the optical fibre bundles in the endoscopes are made. This results in a problematic background that compromises FLIM data and reduces the dynamic range of the endoscope. Multiphoton microscopy avoids this issue through the longer excitation wavelengths used but instead can be compromised by the pulse broadening that results from self-phase modulation and group velocity dispersion in the optical fibre bundles. This can be minimised by reducing the peak intensity of the excitation pulses and to this end in 2008 we invented and patented the concept of spreading the excitation pulse energy over multiple cores of an optical fibre bundle optic bundle (or multiple modes in a multimode optical fibre) and utilised a proximal spatial light modulator (SLM) to control the relative phase of the distal light emerging from different cores or modes. We demonstrated that controlling the phase of the light from different cores enabled scanning and focussing of the distal light with no distal optical or scanning components [11]. Working with the University of Bath, we subsequently showed that we could enable multiphoton endoscopic imaging with dynamic correction of the phase control at up to 100 Hz [12] that enabled us to image through an optical fibre bundle undergoing motion that perturbs the relative phases in the fibre cores. We then showed that we could dynamically control the relative phases of the light from the fibre cores at >400 Hz using only proximal measurements [13], which is a key step towards ultracompact multiphoton endoscopes. We also developed a semi-random multicore fibre design that allowed a larger field of view to be imaged, and we also showed that multiphoton excitation allows the number of independent image resolution elements achieved to exceed the number of fibre cores [14].

Endoscopy references

  1. High-speed wide-field time-gated endoscopic fluorescence lifetime imaging
    J. Requejo-Isidro, J. McGinty, I. Munro, D.S. Elson, N. Galletly, M.J. Lever, M.A.A. Neil, G.W.H. Stamp and P.M.W. French
    Opt Lett, 29 (2004) 2249
  2. Towards the clinical application of time-domain fluorescence lifetime imaging
    I. Munro, J. McGinty, N. Galletly, J. Requejo-Isidro, P.M.P. Lanigan,D. S. Elson, C. Dunsby, M.A.A. Neil, M. J. Lever, G.W.H. Stamp and P.M.W. French,
    J Biomed Opt. 10 (2005) 051403
  3. A flexible wide-field FLIM endoscope utilising blue excitation light for label-free contrast of tissue
    Hugh Sparks, Sean Warren, Joana Guedes, Nagisa Yoshida , Nadia Guerra, Taran Tatla, Chris Dunsby, Paul French
    J Biophotonics 8 (2015) 168–178
  4. In vivo measurements of diffuse reflectance and time-resolved autofluorescence emission spectra of basal cell carcinomas,
    Alex J. Thompson, Sergio Coda, Mikkel Brydegaard Sørensen, Gordon Kennedy, Rakesh Patalay; Ulrika Waitong-Brämming, Pieter A. A. De Beule, Mark A. A. Neil, Stefan Andersson-Engels, Niels Bendsøe, Paul M. W. French, Katarina Svanberg, and Chris Dunsby,
    J. Biophotonics 5 (2012) 240-254
  5. Detection of cartilage matrix degradation by autofluorescence lifetime
    H B Manning, M B Nickdel, K Yamamoto, J L Lagarto, D J. Kelly, C B Talbot, G Kennedy, J Dudhia, MJ Lever, C. Dunsby, PMW French and Y. Itoh
    Matrix Biology 32 (2013) 32–38
  6. Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fibre-optic probe
    S. Coda, A. J. Thompson, G. T. Kennedy, K. L. Roche, L. Ayaru, D. S. Bansi, G. W. Stamp, A. V. Thillainayagam, P. M. W. French and C. Dunsby,
    Biomedical Optics Express 5 (2014) 515-538
  7. Application of time-resolved autofluorescence to label-free in vivo optical mapping of changes in tissue matrix and metabolism associated with myocardial infarction and heart failure,
    J. Lagarto, B. T. Dyer, C. Talbot, M. B. Sikkel, N. S. Peters, P. M. W. French, A. R. Lyon and C. Dunsby,
    Biomed Opt Express 2015, 6 (2015) 324-346
  8. Characterization of NAD(P)H and FAD autofluorescence signatures in a Langendorff isolated-perfused rat heart model
    João L. Lagarto, Benjamin T. Dyer, Clifford B. Talbot, Nicholas S. Peters, Paul M. W. French, Alexander R. Lyon, and Chris Dunsby,
    Biomed. Opt. Expr, 9 (2018) 4961-4978
  9. A Fluorescence Lifetime Imaging Scanning Confocal Endomicroscope,
    G.T. Kennedy, H.B. Manning, D.S. Elson, M. A. A. Neil, G.W. Stamp, B. Viellerobe,F. Lacombe, C. Dunsbyand P.M.W. French
    Journal of Biophotonics, 3 (2010) 103-107
  10. In vivo fluorescence lifetime imaging confocal endomicroscopy reveals intra- and inter-tumor heterogeneity in chromatin binding by doxorubicin
    Hugh Sparks Hiroshi Kondo1, Steven Hooper, Ian Munro, Gordon Kennedy, Christopher Dunsby, Paul French, Erik Sahai
    Nature Communications, 9 (2018) 2662
  11. Adaptive phase compensation for ultracompact laser scanning endomicroscopy
    A. J. Thompson, C. Paterson, M. A. A. Neil, C. Dunsby and P. M. W. French
    Opt. Lett. 36 (2011) 1707-1709
  12. Adaptive multiphoton endomicroscope incorporating a polarization-maintaining multicore optical fibre
    Y. Kim, S. C. Warren, J. M. Stone, J. C. Knight, M. A. A. Neil1, C. Paterson, C. Dunsby and P. M. W. French,
    IEEE J Selected Topics in QE, 22 (2015) Article #: 6800708
  13. Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fibre using proximal detection
    Sean C. Warren*, Youngchan Kim*, James M. Stone, Claire Mitchell1, Jonathan C. Knight, Mark A. A. Neil, Carl Paterson, Paul M. W. French# and Chris Dunsby#
    Opt Exp. 24 (2016) 21474-21484
  14. Semi-random multicore fibre design for adaptive multiphoton endoscopy,
    Youngchan Kim, Sean Warren, Fernando Favero, James Stone, James Clegg, Mark Neil, Carl Paterson, Jonathan Knight, Paul French, and Chris Dunsby, Opt. Express 26, 3661-3673 (2018)