91ÌÒÉ«

To date our laser scanning confocal microscopy systems shave been developed around Leica TCS SP2 and SP5 microscope frames. For tunable confocal microscopy, we implemented an ultrafast laser-pumped supercontinuum excitation source with electronic spectral selection for spectral versatility including automated excitation spectroscopy [1]. For multiphoton microscopy, we introduced femtosecond pulses from a mode-locked Ti:Sapphire laser via the IR port. For studies of the immunological synapse (IS) between two interacting live cells, we incorporated an optical tweezer set-up to manipulate the IS plane of interest to be horizontal, so that it could be imaged at higher resolution and higher frame rate [2]

For cell-based studies we also developed multibeam multiphoton microscopy utilising a LaVision Biotec TriMScope, which we have applied to FLIM, hyperspectral imaging and polarisation-resolved fluorescence microscopy. Currently this is being adapted for application to automated multiwell plate imaging for high content analysis.

For clinical imaging we worked with JenLab GmbH to implement multispectral multiphoton FLIM microscopy on their clinical DermaInspect platform and we applied this to ex vivo [3] and in vivo studies of skin cancer, including in clinical studies at the Department of Dermatology in the University of Modena and Reggio Emilia [4].

These clinical multiphoton microscopy studies highlighted the constraints of the large multiphoton microscope frame and the resulting limitations on the areas of a patient’s body to which it could be conveniently applied. We also noted the relatively long in vivo measurement times (~tens of seconds per field of view) could result in multiphoton FLIM data being compromised by motion artefacts. Accordingly, we developed a compact handheld multiphoton microscope [5] that incorporated a low-coherence spectral domain interferometer to measure the distance from the objective lens to the patient’s skin and maintained this at a constant value using a piezo-electric actuator to adjust the position of the objective lens. A key feature of this instrument was the use of a negative curvature microstructured optical fibre developed at the University of Bath to provide flexible delivery of the ultrashort excitation pulses [6] from a femtosecond Ti:Sapphire laser.

Laser scanning confocal and multiphoton microscopy references

1. 
   C. Dunsby, P. M. P. Lanigan, J. McGinty, D. S. Elson, J. Requejo-Isidro, I. Munro, N. Galletly, F. McCann, B. Treanor, B. Önfelt, D. M. Davis, M. A. A. Neil, and P. M. W. French, J Phys D: Appl. Phys. 37 (2004) 3296-3303
2. 
   S. Oddos, C. Dunsby, M. A Purbhoo, A. Chauveau, D. M Owen, M. A A Neil, D. M Davis & P. M W French
   Biophysical Journal: Biophysical Letters 95 (2008) L66-L68
3. ,
   R. Patalay, C. Talbot, Y. Alexandrov, M. O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. A. Neil, K. König, P. M. W. French, A. Chu, G. W. H. Stamp and C. Dunsby,
   PLoS ONE 7 (2012) e43460
4. ,
   S. Seidenari, F. Arginelli, C. Dunsby, P. M. W. French, K. König, C. Magnoni, M. Manfredini, C. Talbot and G. Ponti,
   Experimental Dermatology 21 (11), pp. 831–836, 2012
5. 
   B. Sherlock, S. Warren, J. Stone, M. Neil, C. Paterson, J. Knight, P. French and C. Dunsby
   Biomed. Opt. Exp. 6 (2015) 1876-1884
6. Ben Sherlock, Fei Yu, Jim Stone, Sean Warren, Carl Paterson, Mark A. A. Neil, Paul M. W. French, Jonathan Knight and Chris Dunsby
   J. Biophoton, 9 (2016) 715–720

Our work on FLIM began in 1996 with the combination of gated optical image intensifiers (GOI) from Kentech Instruments Ltd that operated up to kHz repetition rates, which we combined with in-house developed ultrafast Cr:LiSAF laser amplifier based systems, initially amplifying Ti:Sapphire lasers [1] and later utilising all-solid-state diode pumped Cr:LiSAF laser oscillator and amplifier systems [2]. We showed that such time-gated FLIM systems could discriminate lifetime differences as small as ~10 ps [3] and subsequently demonstrated FLIM with higher repetition rate mode-locked laser oscillators and gain-switched laser diodes [4].

We continue to work with Kentech Instruments Ltd to improve the performance and capabilities of wide-field time-gated FLIM, characterising GOI performance [5] to optimise FLIM data acquisition. We demonstrated “single-shot” FLIM using a segmented intensifier [6] and an improved GOI with enhanced spatial and resolution and signal-to-noise ratio [7]. This time-gated FLIM hardware is complemented by in-house FLIM software that is incorporated in , our open source FLIM data analysis software tool [8] that includes global analysis capabilities for analysing time sequences and multiwell plate FLIM data.

We subsequently extended wide-field time-gated FLIM to a wide range of instruments including wide-field optically sectioned FLIM using structured illumination [9] or using Nipkow “spinning disc” microscopy [10] – that latter approach providing suitable for live cell FLIM FRET [11] and high content analysis. Wide-field FLIM was also applied to FLIM endoscopy, FLIM optical tomography, to line-scanning “push-broom” hyperspectral FLIM [12] and 6D excitation-emission-lifetime-resolved fluorescence microscopy [13] and to polarisation-resolved FLIM for time-resolved anisotropy studies [14].

To realise versatile laser scanning confocal or multiphoton FLIM, we implemented time-correlated single photon counting (TCSPC) detection, utilising the X1 port of the SP5 microscope frame to mount external photon counting photomultipliers. For many applications, we implemented a beamsplitter with two hybrid photon counting photomultipliers at the X1 port. This could enable 2-channel multispectral FLIM or polarisation-resolved FLIM as discussed below.

We have implemented multibeam multiphoton FLIM microscopy by integrating our wide-field GOI-based approach to realise rapid 3D FLIM [15], which also combined with multibeam multiphoton imaging of time-resolved fluorescence anisotropy. We have also implemented multibeam multiphoton FLIM with TCSPC using a 16 channel multi-anode photomultiplier [16].

When exciting biological samples with ultrashort pulses for FLIM, it is important to minimise the laser intensity to reduce photobleaching and phototoxicity. For multiphoton excitation, the high intensity is required, but for single photon excitation, we found it advantageous to stretch the excitation pulse duration to >10 ps by passing femtosecond excitation pulses through a glass block. This is not necessary for pulses from fibre-laser-pumped supercontinuum sources.

FLIM references

1. 
   K. Dowling, S.C.W. Hyde, J.C. Dainty, P.M.W. French and J.D. Hares,
   Opt. Commun. 135 (1997) 27
2. ,
   R. Jones K. Dowling, M. J. Cole, D. Parsons-Karavassilis, M.J. Lever, P.M.W. French, J.D. Hares and A.K.L. Dymoke-Bradshaw,
   Electron Lett. 35, (1999) 256-258
3. 
   K. Dowling, M. J. Dayel, M. J. Lever, P.M.W. French, J. D. Hares and A. K. L Dymoke-Bradshaw,
   Opt Lett, 23 (1998) 810-812
4. 
   D. S. Elson, J. Siegel, S. E. D. Webb, S. Lévêque-Fort, M. J. Lever, P. M. W. French, K. Lauritsen, M. Wahl, R. Erdmann
   Opt Lett, 27 (2002) 1409-1411
5. 
   J. McGinty, J. Requejo-Isidro, I. Munro, C.B. Talbot, C. Dunsby, P. Kellett, J. D. Hares, M.A.A. Neil and P.M.W. French
   J. Phys. D: Appl. Phys. 42 (2009) 135103
6. ,
   D. S. Elson, I. Munro, J. Requejo-Isidro, J. McGinty, C. W. Dunsby, N. Galletly, G. W. Stamp M. A. A. Neil, P. A. Kellett, A. Dymoke-Bradshaw, Jonathon Hares and P. M. W. French
   New Journal of Physics 6 (2004), art. no.-180
7. 
   H. Sparks*, F. Görlitz*, D.J. Kelly, S.C. Warren, P.A. Kellett, A.K.L. Dymoke-Bradshaw, J.D. Hares, M. A.A. Neil, C. Dunsby, P.M.W. French
   Rev Sci Instrum. 88 (2017) 013707.
8. ,
   S.C. Warren, A. Margineanu, D. Alibhai, D.J. Kelly, C. Talbot, Y. Alexandrov, I. Munro, M. Katan, C. Dunsby and P.M.W. French, PLoS ONE 8 (2013) e70687
9. 
   M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, P. M. W. French, M. J. Lever, L. O. Sucharov, M. A. A. Neil,
   R. Juškaitis and T. Wilson
   Opt Lett, 25 (2000) 1361
10. 
    D. M. Grant, D. S. Elson, D. Schimpf, C. W. Dunsby, J. Requejo-Isidro, I. Munro, G. Stamp, M. A. A. Neil, P. M. W. French and P. Courtney
    Opt Lett. 30 (2005) 3353
11. 
    D. Grant, J. McGinty, E.J. McGhee, T.D. Bunney, D.M. Owen, C.B. Talbot, W. Zhang, S. Kumar, I. Munro, P. Lanigan, G. Kennedy, C. Dunsby, A.I. Magee, P. Courtney, M. Katan, M.A.A. Neil & P.M.W. French
    Opt. Expr.15 (2007) 15656 – 15673
12. 
    P. De Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. J. Lever, P. Anand, M. A. A. Neil and P. M. W. French
    Microscopy 91ÌÒÉ« and Technique 70 (2007) 481-484
13. 
    D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A.A. de Beule, M. A. A. Neil and P. M. W. French
    Opt. Lett. 32 (2007) 3408-3410
14. 
    J. Siegel, K. Suhling, S. Lévêque-Fort, S.E.D. Webb, D.M. Davis, D. Phillips, P.M.W. French and Y. Sabharwal
    Rev Sci Instrum, 74, (2003) 182-192
15. 
    R. K.P. Benninger,O. Hofmann, J. McGinty, J. Requejo-Isidro,I. Munro, M. A. A. Neil,
    A. J. deMello and P. M.W. French,
    Optics Express, 13 (2005) 6275
16. 
    S. Kumar, C. Dunsby, P. A. A. De Beule, D. Owen, U. Anand, A. I. Magee, D. M. Davis, M. A. A. Neil, P. Anand, C. Benham, A. Naylor and P. M. W. French
    Opt. Expr.15 (2007) 12548-12561

Polarisation-resolved measurements of anistropy in fluorescence excitation or emission can provide information about fluorophore molecular orientation and/or size via the fluorophore’s rotational decorrelation time. By adding a dual-channel image splitter incorporating a polarising beam splitter (PBS) to the LaVision TriMScope, we developed an instrument for rapid depth-resolved imaging of fluorescence anisotropy (emission coefficient) and linear dichroism (i.e. fluorescence excitation coefficient) [1]. This was applied to imaging membrane dyes reporting the distribution of orientations of lipids in cell membranes and in nanotubes connecting interacting (immune and target) cells.

We have also worked extensively on polarisation-resolved FLIM, also described as time-resolved fluorescence anisotropy imaging (TR-FAIM). This was first implemented using a dual-channel polarisation-resolved imager (PRI) with time-gated wide-field FLIM on a single-photon excited epifluorescence microscope [2] and later implemented on the LaVision TriMScope [3]. The latter system was used to study molecular interactions in microfluidic reactors, e.g. [4], noting that the binding of a fluorescent ligand to a larger molecule can significantly increase the rotational decorrelation time and thereby the steady-state anisotropy coefficient.

We also implemented laser scanning TR-FAIM in our Leica TCS SP5 microscope by using a polarising beamsplitter with two hybrid photomultipliers at the X1 port. Thus we could realise laser-scanning fluorescence anistropy imaging (with steady-state detection) or time-resolved fluorescence anistropy imaging with TCSPC detection in each polarisation-resolved channel [5], which we applied to homoFRET readouts of fluorescent protein-based biosensors, noting that FRET between similar fluorophores reduces the fluorescence anisotropy.

Polarisation-resolved fluorescence imaging references

1. ,
   Richard K. P. Benninger; Björn Önfelt; Daniel M. Davis; Mark Neil and Paul French
   Biophysical Journal, 88 (2005) 609
2. 
   J. Siegel, K. Suhling, S. Lévêque-Fort, S.E.D. Webb, D.M. Davis, D. Phillips, P.M.W. French and Y. Sabharwal
   Rev Sci Instrum, 74, (2003) 182-192
3. 
   R. K.P. Benninger,O. Hofmann, J. McGinty, J. Requejo-Isidro,I. Munro, M. A. A. Neil,
   A. J. deMello and P. M.W. French,
   Optics Express, 13 (2005) 6275
4. 
   Richard K. P. Benninger, Bjorn Önfelt, Oliver Hofmann, Daniel M. Davis, Mark A. A. Neil, Paul M. W. French and Andrew J. deMello
   Angewandte Chemie International Edition, 46 (2007) 2228-2231
5. .
   Warren SC, Margineanu A, Katan M, Dunsby C, French PM
   Int J Mol Sci, 16 (2015):14695-14716

We have implemented “push-broom” hyperspectral imaging, using cylindrical optics to generate a linear (x) excitation beam and stage-scanning (y) the sample through this line of excitation to acquire a 2D x-y map of the emission. At each stage position, the (x) line of fluorescence emission is imaged to the entrance slit of an imaging spectrograph and the resulting (x-λ) image is captured by an EMCCD that supports binning in each direction, enabling the number of spectral channels to be selected. The line illumination combined with the entrance slit of the spectrograph results in semi-confocal microscopy and optical sectioning, thereby also enabling z-resolution.

To further enhance this multidimensional fluorescence imaging, we can use ultrashort pulsed excitation and install a GOI after the imaging spectrograph to realise hyperspectral time-gated FLIM, resulting in x-y-z-λ_em-τ resolved imaging [1]. We applied this instrument to characterise autofluorescence of biological tissue, indicating the opportunity to use autofluorescence lifetime as a label-free diagnostic of atherosclerosis.

We have further extended this approach by also implementing excitation hyperspectral imaging with an electronically tunable excitation source – providing 6D (x-y-z-λ_em-τ-λ_ex) resolved imaging [2]. Recently, we extended this instrument to also resolve polarisation and have applied it to characterise luminescence from optical defects in diamond. This includes hyperspectral imaging, FLIM and steady-state exaction resolved imaging for photoluminescence studies [3].

Hyperspectral imaging references

1. 
   P. De Beule, D. M. Owen, H. B. Manning, C. B. Talbot, J. Requejo-Isidro, C. Dunsby, J. McGinty, R. K. P. Benninger, D. S. Elson, I. Munro, M. J. Lever, P. Anand, M. A. A. Neil and P. M. W. French
   Microscopy 91ÌÒÉ« and Technique 70 (2007) 481-484
2. 
   D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A.A. de Beule, M. A. A. Neil and P. M. W. French
   Opt. Lett. 32 (2007) 3408-3410
3. 
   Jones, Daniel; Kumar, Sunil; Lanigan, Peter; McGuinness, Colin; Dale, Matthew; Twitchen, Daniel; Fisher, David; Martineau, Philip; Neil, Mark; Dunsby, Christopher; French,
   Methods Appl. Fluoresc. 8 (2020) 014004

Oblique plane microscopy (OPM) is a type of light-sheet fluorescence microscopy that utilises a single microscope objective lens for both fluorescence excitation and imaging. It can be implemented on a standard microscope frame, making it compatible with most existing sample formats including samples arrayed in multiwell plates. OPM utilises remote refocussing to image a tilted plane in the specimen and this also facilitates convenient rapid scanning of the light sheet to provide fast (up to 25 vol/s) volumetric imaging. This can be applied to time-resolved 3D studies of dynamic processes, e.g. of calcium sparks in cardiomyocytes and high-throughput 3D imaging of 3D cell cultures in multiwell plate arrays for time-lapse 3D high content imaging.