We have developed a wide-range of fluorescence instruments including microscopes, macroscopes, endoscopes and tomographic imaging systems. Our fluorescence microscopes have largely been developed around commercial microscope frames (including the IX71, IX73, IX81 and IX83 frames from Olympus, the TCS SP2 and TCS SP5 confocal microscope platforms from Leica Microsystems and the Axiovert 200 frame from Carl Zeiss).
openScopes: As we typically write our own instrument control and data acquisition software and design our own instrument configurations based on repurposing legacy instruments, we believe that it should be easy for other laboratories to replicate our instruments. To this end we are working to share all our instrument control and data acquisition and analysis software – using open source platforms where possible.
We have also designed a low-cost, modular microscope frame that can be used for conventional (wide-field) light microscopy and extended to a range of advanced modalities. This “openFrame” system should enable advanced microscopy modalities such as super-resolved microscopy, fluorescence lifetime imaging and/or high content analysis to be implemented at relatively low cost, particularly where it is possible to utilise cost-effective components such as multimode laser diodes or LEDs for excitation and CMOS cameras for detection. Complete (upgradeable) microscope systems can be assembled for £10,000-£20,000, depending on the functionality.
Fluorescence Microscopy
- Laser scanning confocal and multiphoton microscopy
- Fluorescence lifetime imaging (FLIM)
- Polarisation-resolved fluorescence imaging
- Hyperspectral imaging
- Oblique plane microscopy
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
- An electronically tunable ultrafast laser source for fluorescence imaging including fluorescence lifetime imaging microscopy
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 - High-speed, high-resolution imaging of intercellular immune synapses using optical tweezers
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 - Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas,
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 - Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: Morphologic Features for Non-Invasive Diagnostics,
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 - Fibre-coupled handheld multiphoton microscope with active motion compensation
B. Sherlock, S. Warren, J. Stone, M. Neil, C. Paterson, J. Knight, P. French and C. Dunsby
Biomed. Opt. Exp. 6 (2015) 1876-1884 - Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre
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 FLIMfit, 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
- 2-D Fluorescence Lifetime Imaging using a Time-Gated Image Intensifier
K. Dowling, S.C.W. Hyde, J.C. Dainty, P.M.W. French and J.D. Hares,
Opt. Commun. 135 (1997) 27 - Fluorescence lifetime imaging using a diode-pumped all-solid-state laser system,
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 - Fluorescence Lifetime Imaging with picosecond resolution for biomedical applications
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 - Fluorescence lifetime system for microscopy and multi-well plate imaging using a blue picosecond diode laser
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 - Signal-to-Noise Characterisation of Time-Gated Intensifiers used for Wide-field Time-Domain FLIM
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 - Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier,
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 - Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging
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. - Rapid global fitting of large fluorescence lifetime imaging microscopy datasets,
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 - Whole-field optically sectioned fluorescence lifetime imaging
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 - Wide-Field Optically-Sectioned Fluorescence Lifetime Imaging using a Nipkow Disk Microscope and a Tunable Ultrafast Continuum Excitation Source
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 - High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events
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 - Rapid Hyperspectral Fluorescence Lifetime Imaging
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 Research and Technique 70 (2007) 481-484 - Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source
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 - Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) – Imaging the mobility of a fluorophore
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 - Time resolved fluorescence imaging of solvent interactions in microfluidic devices
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 - Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging
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
- Fluorescence Imaging of Two-Photon Linear Dichroism: Cholesterol Depletion Disrupts Molecular Orientation in Cell Membranes,
Richard K. P. Benninger; Björn Önfelt; Daniel M. Davis; Mark Neil and Paul French
Biophysical Journal, 88 (2005) 609 - Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) – Imaging the mobility of a fluorophore
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 - Time resolved fluorescence imaging of solvent interactions in microfluidic devices
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 - Fluorescence‐Lifetime Imaging of DNA–Dye Interactions within Continuous‐Flow Microfluidic Systems
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 - Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.
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-λem-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-λem-τ-λex) 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
- Rapid Hyperspectral Fluorescence Lifetime Imaging
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 Research and Technique 70 (2007) 481-484 - Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source
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 - Multidimensional luminescence microscope for imaging defect colour centres in diamond
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.