Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence

Helen E. Parker; James M. Stone; Adam D. L. Marshall; Tushar R. Choudhary; Robert R. Thomson; Kevin Dhaliwal; Michael G. Tanner

doi:10.1364/BOE.10.001856

Biomedical Optics Express
Vol. 10,
Issue 4,
pp. 1856-1869
(2019)
•https://doi.org/10.1364/BOE.10.001856

Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence

Helen E. Parker, James M. Stone, Adam D. L. Marshall, Tushar R. Choudhary, Robert R. Thomson, Kevin Dhaliwal, and Michael G. Tanner

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Helen E. Parker, James M. Stone, Adam D. L. Marshall, Tushar R. Choudhary, Robert R. Thomson, Kevin Dhaliwal, and Michael G. Tanner, "Fibre-based spectral ratio endomicroscopy for contrast enhancement of bacterial imaging and pulmonary autofluorescence," Biomed. Opt. Express 10, 1856-1869 (2019)

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- Endoscopy and Fiber Optics
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About this Article
History
- Original Manuscript: December 12, 2018
- Revised Manuscript: February 21, 2019
- Manuscript Accepted: February 25, 2019
- Published: March 15, 2019

Abstract

Fibre-based optical endomicroscopy (OEM) permits high resolution fluorescence microscopy in endoscopically accessible tissues. Fibred OEM has the potential to visualise pathologies targeted with fluorescent imaging probes and provide an in vivo in situ molecular pathology platform to augment disease understanding, diagnosis and stratification. Here we present an inexpensive widefield ratiometric fibred OEM system capable of enhancing the contrast between similar spectra of pathologically relevant fluorescent signals without the burden of complex spectral unmixing. As an exemplar, we demonstrate the potential of the platform to detect fluorescently labelled Gram-negative bacteria in the challenging environment of highly autofluorescent lung tissue in whole ex vivo human lungs.

Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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References

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Year
Author
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T. H. Craven, N. Avlonitis, N. McDonald, T. Walton, E. Scholefield, A. R. Akram, T. S. Walsh, C. Haslett, M. Bradley, and K. Dhaliwal, “Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils,” Sci. Rep. 8(1), 13490 (2018).
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A. Megia-Fernandez, B. Mills, C. Michels, S. V. Chankeshwara, K. Dhaliwal, and M. Bradley, “Highly selective and rapidly activatable fluorogenic Thrombin sensors and application in human lung tissue,” Org. Biomol. Chem. 15(20), 4344–4350 (2017).
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[Crossref] [PubMed]
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[Crossref] [PubMed]
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[Crossref] [PubMed]
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[Crossref] [PubMed]
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2018 (6)

N. Krstajić, B. Mills, I. Murray, A. Marshall, D. Norberg, T. H. Craven, P. Emanuel, T. R. Choudhary, G. O. S. Williams, E. Scholefield, A. R. Akram, A. Davie, N. Hirani, A. Bruce, A. Moore, M. Bradley, and K. Dhaliwal, “Low-cost high sensitivity pulsed endomicroscopy to visualize tricolor optical signatures,” J. Biomed. Opt. 23(7), 1–12 (2018).
[Crossref] [PubMed]

T. H. Craven, N. Avlonitis, N. McDonald, T. Walton, E. Scholefield, A. R. Akram, T. S. Walsh, C. Haslett, M. Bradley, and K. Dhaliwal, “Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils,” Sci. Rep. 8(1), 13490 (2018).
[Crossref] [PubMed]

A. Megia-Fernandez, B. Mills, C. Michels, S. V. Chankeshwara, N. Krstajić, C. Haslett, K. Dhaliwal, and M. Bradley, “Bimodal fluorogenic sensing of matrix proteolytic signatures in lung cancer,” Org. Biomol. Chem. 16(43), 8056–8063 (2018).
[Crossref] [PubMed]

A. R. Akram, S. V. Chankeshwara, E. Scholefield, T. Aslam, N. McDonald, A. Megia-Fernandez, A. Marshall, B. Mills, N. Avlonitis, T. H. Craven, A. M. Smyth, D. S. Collie, C. Gray, N. Hirani, A. T. Hill, J. R. Govan, T. Walsh, C. Haslett, M. Bradley, and K. Dhaliwal, “In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A,” Sci. Transl. Med. 10(464), eaal0033 (2018).
[Crossref] [PubMed]

S. Seth, A. R. Akram, K. Dhaliwal, and C. K. I. Williams, “Estimating Bacterial and Cellular Load in FCFM Imaging,” J. Imaging 4(1), 11 (2018).
[Crossref]

E. Pedretti, M. G. Tanner, T. R. Choudhary, N. Krstajić, A. Megia-Fernandez, R. K. Henderson, M. Bradley, R. R. Thomson, J. M. Girkin, K. Dhaliwal, and P. A. Dalgarno, “High-speed dual color fluorescence lifetime endomicroscopy for highly-multiplexed pulmonary diagnostic applications and detection of labeled bacteria,” Biomed. Opt. Express 10(1), 181–195 (2018).
[Crossref] [PubMed]

2017 (5)

A. Perperidis, H. E. Parker, A. Karam-Eldaly, Y. Altmann, K. Dhaliwal, R. R. Thomson, M. G. Tanner, and S. McLaughlin, “Characterization and modelling of inter-core coupling in coherent fiber bundles,” Opt. Express 25(10), 11932–11953 (2017).
[Crossref] [PubMed]

J. M. Stone, H. A. C. Wood, K. Harrington, and T. A. Birks, “Low index contrast imaging fibers,” Opt. Lett. 42(8), 1484–1487 (2017).
[Crossref] [PubMed]

A. S. Luthman, S. Dumitru, I. Quiros-Gonzalez, J. Joseph, and S. E. Bohndiek, “Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array,” J. Biophotonics 10(6-7), 840–853 (2017).
[Crossref] [PubMed]

M. A. van der Putten, L. E. MacKenzie, A. L. Davies, J. Fernandez-Ramos, R. A. Desai, K. J. Smith, and A. R. Harvey, “A multispectral microscope for in vivo oximetry of rat dorsal spinal cord vasculature,” Physiol. Meas. 38(2), 205–218 (2017).
[Crossref] [PubMed]

A. Megia-Fernandez, B. Mills, C. Michels, S. V. Chankeshwara, K. Dhaliwal, and M. Bradley, “Highly selective and rapidly activatable fluorogenic Thrombin sensors and application in human lung tissue,” Org. Biomol. Chem. 15(20), 4344–4350 (2017).
[Crossref] [PubMed]

2016 (2)

N. Krstajic, A. R. Akram, T. R. Choudhary, N. McDonald, M. G. Tanner, E. Pedretti, P. A. Dalgarno, E. Scholefield, J. M. Girkin, A. Moore, M. Bradley, and K. Dhaliwal, “Two-color widefield fluorescence microendoscopy enables multiplexed molecular imaging in the alveolar space of human lung tissue,” J. Biomed. Opt. 21(4), 46009 (2016).
[Crossref] [PubMed]

A. Lopez, D. V. Zlatev, K. E. Mach, D. Bui, J. J. Liu, R. V. Rouse, T. Harris, J. T. Leppert, and J. C. Liao, “Intraoperative Optical Biopsy during Robotic Assisted Radical Prostatectomy Using Confocal Endomicroscopy,” J. Urol. 195(4 Pt 1), 1110–1117 (2016).
[Crossref] [PubMed]

2015 (2)

A. R. Akram, N. Avlonitis, A. Lilienkampf, A. M. Perez-Lopez, N. McDonald, S. V. Chankeshwara, E. Scholefield, C. Haslett, M. Bradley, and K. Dhaliwal, “A labelled-ubiquicidin antimicrobial peptide for immediate in situ optical detection of live bacteria in human alveolar lung tissue,” Chem. Sci. (Camb.) 6(12), 6971–6979 (2015).
[Crossref] [PubMed]

T. Aslam, A. Miele, S. V. Chankeshwara, A. Megia-Fernandez, C. Michels, A. R. Akram, N. McDonald, N. Hirani, C. Haslett, M. Bradley, and K. Dhaliwal, “Optical molecular imaging of lysyl oxidase activity - detection of active fibrogenesis in human lung tissue,” Chem. Sci. (Camb.) 6(8), 4946–4953 (2015).
[Crossref] [PubMed]

2014 (3)

T. Cheng, Q. Liu, R. Zhang, Y. Zhang, J. Chen, R. Yu, and G. Ge, “Lysyl oxidase promotes bleomycin-induced lung fibrosis through modulating inflammation,” J. Mol. Cell Biol. 6(6), 506–515 (2014).
[Crossref] [PubMed]

G. Lu and B. Fei, “Medical hyperspectral imaging: a review,” J. Biomed. Opt. 19(1), 10901 (2014).
[Crossref] [PubMed]

S. van der Walt, J. L. Schönberger, J. Nunez-Iglesias, F. Boulogne, J. D. Warner, N. Yager, E. Gouillart, T. Yu, and scikit-image contributors, “scikit-image: image processing in Python,” PeerJ 2, e453 (2014).
[Crossref] [PubMed]

2013 (2)

M. Hughes, T. P. Chang, and G.-Z. Yang, “Fiber bundle endocytoscopy,” Biomed. Opt. Express 4(12), 2781–2794 (2013).
[Crossref] [PubMed]

Y. Pan, J. P. Volkmer, K. E. Mach, J. J. Liu, R. V. Rouse, D. Sahoo, T. C. Chang, M. Van De Rijn, E. Skinner, S. S. Gambhir, I. Weissman, and J. C. Liao, “Endoscopic molecular imaging of human bladder cancer using a CD47 antibody,” Mol. Imaging Biol. 15(S1), 2–1590 (2013).
[Crossref]

2012 (2)

A. Churg, S. Zhou, and J. L. Wright, “Series “matrix metalloproteinases in lung health and disease”: Matrix metalloproteinases in COPD,” Eur. Respir. J. 39(1), 197–209 (2012).
[Crossref] [PubMed]

A. D. Elliott, L. Gao, A. Ustione, N. Bedard, R. Kester, D. W. Piston, and T. S. Tkaczyk, “Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer,” J. Cell Sci. 125(Pt 20), 4833–4840 (2012).
[Crossref] [PubMed]

2011 (1)

A. Davey, D. F. McAuley, and C. M. O’Kane, “Matrix metalloproteinases in acute lung injury: mediators of injury and drivers of repair,” Eur. Respir. J. 38(4), 959–970 (2011).
[Crossref] [PubMed]

2009 (1)

M. B. Wallace and P. Fockens, “Probe-based confocal laser endomicroscopy,” Gastroenterology 136(5), 1509–1513 (2009).
[Crossref] [PubMed]

2007 (2)

L. Thiberville, S. Moreno-Swirc, T. Vercauteren, E. Peltier, C. Cavé, and G. Bourg Heckly, “In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy,” Am. J. Respir. Crit. Care Med. 175(1), 22–31 (2007).
[Crossref] [PubMed]

J. D. Hunter, “Matplotlib: A 2D graphics environment,” Comput. Sci. Eng. 9(3), 90–95 (2007).
[Crossref]

2006 (1)

F. Chua and G. J. Laurent, “Neutrophil elastase: mediator of extracellular matrix destruction and accumulation,” Proc. Am. Thorac. Soc. 3(5), 424–427 (2006).
[Crossref] [PubMed]

2005 (2)

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[Crossref] [PubMed]

2002 (2)

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol. 3(12), 906–918 (2002).
[Crossref] [PubMed]

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Akram, A. R.

S. Seth, A. R. Akram, K. Dhaliwal, and C. K. I. Williams, “Estimating Bacterial and Cellular Load in FCFM Imaging,” J. Imaging 4(1), 11 (2018).
[Crossref]

Altmann, Y.

Aslam, T.

Avlonitis, N.

Bedard, N.

Birks, T. A.

J. M. Stone, H. A. C. Wood, K. Harrington, and T. A. Birks, “Low index contrast imaging fibers,” Opt. Lett. 42(8), 1484–1487 (2017).
[Crossref] [PubMed]

Bohndiek, S. E.

Boulogne, F.

Bourg Heckly, G.

Bradley, M.

Bruce, A.

Bui, D.

Campbell, R. E.

J. Zhang, R. E. Campbell, A. Y. Ting, and R. Y. Tsien, “Creating new fluorescent probes for cell biology,” Nat. Rev. Mol. Cell Biol. 3(12), 906–918 (2002).
[Crossref] [PubMed]

Cavé, C.

Chang, T. C.

Chang, T. P.

M. Hughes, T. P. Chang, and G.-Z. Yang, “Fiber bundle endocytoscopy,” Biomed. Opt. Express 4(12), 2781–2794 (2013).
[Crossref] [PubMed]

Chankeshwara, S. V.

Chen, J.

Cheng, T.

Cheung, E. L. M.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[Crossref] [PubMed]

Choudhary, T. R.

Chua, F.

F. Chua and G. J. Laurent, “Neutrophil elastase: mediator of extracellular matrix destruction and accumulation,” Proc. Am. Thorac. Soc. 3(5), 424–427 (2006).
[Crossref] [PubMed]

Churg, A.

Cocker, E. D.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[Crossref] [PubMed]

Collie, D. S.

Conchello, J.-A.

J. W. Lichtman and J.-A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[Crossref] [PubMed]

Craven, T. H.

Dalgarno, P. A.

Davey, A.

Davie, A.

Davies, A. L.

Desai, R. A.

Dhaliwal, K.

S. Seth, A. R. Akram, K. Dhaliwal, and C. K. I. Williams, “Estimating Bacterial and Cellular Load in FCFM Imaging,” J. Imaging 4(1), 11 (2018).
[Crossref]

Dumitru, S.

Elliott, A. D.

Emanuel, P.

Fei, B.

G. Lu and B. Fei, “Medical hyperspectral imaging: a review,” J. Biomed. Opt. 19(1), 10901 (2014).
[Crossref] [PubMed]

Fernandez-Ramos, J.

Flusberg, B. A.

B. A. Flusberg, E. D. Cocker, W. Piyawattanametha, J. C. Jung, E. L. M. Cheung, and M. J. Schnitzer, “Fiber-optic fluorescence imaging,” Nat. Methods 2(12), 941–950 (2005).
[Crossref] [PubMed]

Fockens, P.

M. B. Wallace and P. Fockens, “Probe-based confocal laser endomicroscopy,” Gastroenterology 136(5), 1509–1513 (2009).
[Crossref] [PubMed]

Gambhir, S. S.

Gao, L.

Ge, G.

Girkin, J. M.

Gouillart, E.

Govan, J. R.

Gray, C.

Haraguchi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[Crossref] [PubMed]

Harrington, K.

J. M. Stone, H. A. C. Wood, K. Harrington, and T. A. Birks, “Low index contrast imaging fibers,” Opt. Lett. 42(8), 1484–1487 (2017).
[Crossref] [PubMed]

Harris, T.

Harvey, A. R.

Haslett, C.

Henderson, R. K.

Hill, A. T.

Hirani, N.

Hiraoka, Y.

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Supplementary Material (2)

Name	Description
Visualization 1	Ratiometric fibre-based fluorescent imaging of lung tissue
Visualization 2	Ratiometric fibre-based imaging of lung tissue and fluorescently labelled bacteria

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Fig. 1 Ratiometric imaging system. Panoptes imaging fibre (a) consisting of a tessellated array of 8100 cores (b). A blue LED (c) is coupled via a dichroic mirror (d) into the fibre bundle. The fluorescence emission is separated by a second dichroic mirror (e) about 605 nm. The long wavelength path is interrupted by an optical chopper (f) and recombined with another dichroic mirror (g) onto a monochromatic camera (h). A PC (j) is used to control a triggering unit (i) with outputs to the camera and the chopper.

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Fig. 2 (left) Example fluorescence emission of SmartProbe labelled P. aeruginosa (black) and human lung (red) at excitation of 470 nm at 5.5 µW acquired using a spectrometer (USB2000 + VIS-NIR-ES, Ocean Optics) coupled into a spectroscopy setup analogous to our imaging arrangement. SmartProbe labelled bacteria have greater short wavelength contributions than typical lung tissue autofluorescence. Profile of dichroic mirrors used to separate the short and long channels determines the short channel wavelength range (green fill) and the long channel wavelength range (red fill). (right) Relative visibility of SmartProbe labelled bacteria defined as the relative spectral ratio value of SmartProbe labelled bacteria to lung tissue for a range of potential cut-off wavelengths.

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Fig. 3 Initial, semi-supervised processing of data set that sets out parameters for core data extraction.

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Fig. 4 Schematic of processing methodology used to analyse data and produce final images. (a) How wavelength dependent propagation of light through different cores is accounted for through normalisation of the data with a brightfield image. (b) How spectral ratio values are calculated following a core matching protocol and interpolation.

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Fig. 5 (a) Images from negative USAF target with (left to right) 100% 1 mM NBD, 25% 1 mM NBD 75% 1 mM fluorescein, and 100% 1 mM fluorescein with spectral ratio values clipped to 0.75 - 4. (b) Spectra of the three solutions taken at an illumination wavelength of 470 nm at 5.5 µW, using a commercial spectrometer (USB2000 + VIS-NIR-ES, Ocean Optics). (c) Histograms of the spectral ratio values contained within USAF images summed over the whole image for ten sequential frames. The histograms have been weighted by intensity and normalised by area for ease of comparison. Broadening of the 100% fluorescein histogram is due to a low signal to noise ratio above the cut-off wavelength at 605 nm. Note that a shift of the spectra towards shorter wavelengths corresponds to an increase in the spectral ratio calculated.

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Fig. 6 (top three rows) Typical examples of imaging lung tissue in an ex vivo lung perfusion (EVLP) model prior to instillation of labelled bacteria. (bottom three rows) Typical examples of EVLP lung imaging after in situ delivery of SmartProbe labelled P. aeruginosa. All images are the same FOV presented in four modes. (from left to right) Intensity image as would appear on a widefield fluorescence fibred OEM system; spectral ratio image (auto-scaled); combined image with auto-scaling; combined image with scaling clipped from a spectral ratio of 0.3 to 0.4 to enhance SmartProbe labelled P. aeruginosa visibility (see Visualization 2); histograms of the spectral ratio contained with the image. Selected images are shown enlarged in Fig. 7.

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Fig. 7 (Top) Typical ex vivo imaging of lung tissue from EVLP model in alveolar space before and after in situ instillation of pre-labelled bacteria. Greyscale images show how imaging appears in the widefield imaging system without spectral ratio analysis enabled. Coloured images display spectral ratio and intensity with rescaling of spectral ratio to 0.3 - 0.4 to enhance SmartProbe labelled P. aeruginosa visibility. (Bottom) Typical histogram of spectral ratio across 300 frame videos of lung before and after delivery of labelled bacteria. A tabulated series of images can be seen in Fig. 6.

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Fig. 8 Typical examples of skin tissue. From left to right, intensity only image; spectral ratio only image with auto-contrast; combined image; histogram of the spectral ratio within the image weighted by intensity.

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Equations (1)

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R = \frac{I_{s c}}{I_{l c}} = \frac{[\sum_{i = 1}^{n} p_{i} (m_{s c t})] {[\sum_{i = 1}^{n} p_{i} (m_{s c b})]}^{- 1}}{[\sum_{i = 1}^{n} p_{i} (m_{l c t})] {[\sum_{i = 1}^{n} p_{i} (m_{l c b})]}^{- 1}}