WP 4: Imaging of cell seeded scaffolds and cell coated surfaces

State of the art in the field

Microscopic as well as macroscopic imaging technologies based on optical spectroscopy or fluorescence detection are increasingly evolving as important tools for investigation of the morphology as well as function and viability of living cells, cell cultures and tissues [1, 2, 3]. Employing (quasi-) confocal or two-photon excitation microscopy 3D-images of cells, cell networks and tissues can be recorded with high (sub-µm) spatial and high (ps) temporal resolution. By near infrared spectroscopy tissue constituents and hence physiological parameters like blood supply and tissue oxygenation have been determined [4] in vivo. Furthermore, functional information has been revealed applying optical methods to detect fluorescence-labelled molecular probes like monoclonal antibodies, DNA/RNA fragments or peptides. In particular, optical “molecular” imaging is discussed in the literature as very promising for functional imaging due to it´s high sensitivity and specificity [5]. Beside high sensitivity/specificity the vision connected with molecular imaging is to correlate function or viability of cells to the number of molecular targets. Investigations in this area are metrologically challenging at the present, since quantitative approaches to localise and determine fluorophore concentrations in cells and tissues are not available. To this end, knowledge about the (non-specific) autofluorescence background, the localisation of specific fluorescence labels in 3D as well as quantitative evaluation of the quantum efficiency of fluorophore-ligand conjugates under native conditions, the number of fluorophores per molecular probe and the binding probability of the labelled probes to an target must be known.

Optical Coherence Tomography (OCT) is a non-invasive optical imaging modality capable of producing 3D images through highly scattering media [6]. The technique has been widely demonstrated for imaging the morphology of human tissue, from oesophagus to retina [7] and some research has been carried out on engineered tissue [8]. Ultra-high resolution implementations of OCT have also been demonstrated with sub-cellular resolution [9] but further development of the technology is required for diagnostic or therapeutic use. Whilst tissue and cellular morphology are important indicators of the normality or abnormality, changes are only a symptom of an underlying chemical process. Such molecular processes cause the optical properties of a material to change. Therefore, over the past three years, our research has concentrated on exploiting the advantages of OCT imaging (resolution, sensitivity [10], penetration depth and speed) as a platform technology for measuring both the morphology and spatio-temporal variations in optical properties within complex materials [11, 12] such as cells [13] and tissue [14]. This area of functional optical imaging holds tremendous promise for non-invasively and rapidly mapping tissue and cellular health, an important advance in the field of regenerative medicine.

Movement beyond the current state of art

The lack of quantitative methods for fluorescence-based molecular imaging is an acknowledged and still not solved problem. In the project proposed here the capability of confocal or quasi-confocal one-photon excitation as well as two-photon excitation fluorescence microscopy for this purpose will be explored. The 3D information of the morphology of cells, cell networks and cells in scaffolds together with the localisation of specific fluorophores will be determined by these techniques. Furthermore, the methodologies applied will be developed to reduce - or to correct for at least – autofluorescence background and to gain quantitative knowledge about the local (specific) fluorophore concentration in the tissue.

At present there are no commercial instruments available that are capable of mapping the 3D distribution of optical properties within tissue engineered products. In fact, the current state of the art is only just demonstrating the potential for such measurements. Therefore, in this work-package a novel angularly scanned form of high resolution OCT will be developed. This will be used to map in three dimensions the refractive index, dispersion and optical absorption of cells located at the surface and primary sub-surface of a tissue scaffold. The proposed technique offers a major advance in non-invasive diagnostic tools for tissue engineering, with the potential to be scaled-up for online quality testing of commercially engineered tissue products. Complimentary to this, a secondary novel broadband optical transmission tomography technique will be developed that will be used to reconstruct a 3D spectroscopic map of absorption as well as refractive index and morphology within the engineered tissue. This latter technique will have approximately 100 micrometer spatial resolution and will provide functional information regarding oxygenation the spatial concentration of waste products and other analytes of interest that have spectroscopic features within the light source spectrum. Our second approach has the advantage over the first that it relies on forward scattered light and therefore has much greater penetration depth so that it can image bulk samples. However, it requires access to top and bottom of the tissue construct. Both of these new metrological techniques will have a significant impact on metrology for regenerative medicine, since there are no other techniques available with similar potential sensitivity or specificity. Applications for these techniques are wider than just tissue engineering, with possibilities in bio-materials, tissue optics, clinical medicine and the optical characterisation of complex structures in other fields such as optical waveguides and polymer composites.

Scientific tasks

Exploration and comparison of one-photon and two photon excitation confocal laser-scanning vs. one-photon quasi-confocal microscopy (i.e. wide-field of view camera-based microscopy plus image deconvolution techniques) for non-destructive 3D “tomographic” imaging of cells on surfaces and cell seeded scaffolds. Development of methods to reduce or to correct for autofluorescence background and to quantify local concentrations of specific fluorophores in cells and tissues.

Parallel development of angularly scanned optical reflection and transmission tomographic techniques based upon optical coherence tomography. These will use a limited angle tomographic reconstruction approach based upon Fourier slice theorem. Data processing techniques will then be investigated for robustly extracting refractive index, dispersion and spectral absorption information inherently present in the interferometric signal and generating three-dimensional spatial maps of these quantitative properties. The relationship between the measured optical properties and the engineered tissue and cell health will be explored by comparing our data with that obtained by other partners using complimentary techniques such as fluorescence and CARS.

Technical risks

It is an open question at the present if the proposed microscopic techniques can be applied with viable cells and reveal 3D information on a µm scale with statistical precision sufficient for quality control in tissue engineering. Furthermore, it is not clear if viability or function of grown cells can be determined and “quantified” with the help of autofluorescent or flurescence-labelled molecular probes employing quantitative fluorescence microscopic approaches. The proposed project will explore this question. At present it is unclear whether limited angle Fourier reconstruction methods can yield sufficient accuracy within strongly refracting material. However, there is a recent precedent with similar optical work being carried out on single cells [8]. The main difficulty with the reflection technique is that the same point within the material must be identified in scans at multiple angles in order for the tomographic reconstruction method to work. This may be non-trivial in a complex material, however we do have some similar experience in feature identification from our previous OCT work [6, 7, 9].

Expected outputs

  • patent(s) where novel methodologies are developed
  • publication of results in scientific journals
  • laboratory set-up for quality control of cells on surfaces and cells in scaffolds

    • Selected references

    [1] Park, YK, Popescu, G, Badizadegan, K, Dasari, RR, and Feld, M.S. Optics Express 14 (18), 8263, (2006)
    [2] http://www.cyto.purdue.edu/cdroms/microscopy/vol1/index3.htm
    [3] http://www.mih.unibas.ch/Booklet/Booklet96/Booklet96.html
    [4] D. Grosenick et al.; Phys.Med.Biol. 49, 1165-1182 (2004)
    [5] R. Weissleder, U. Mahmood; Molecular Imaging; Radiology 219, 316-333 (2001); and references herein
    [6] PH Tomlins and RK Wang, “Theory, developments and applications of optical coherence tomography”, J. Phys. D: Appl. Phys., Vol. 38 pp. 2519-2535 (2005)
    [7] RA Leitgeb, W Drexler, A Unterhuber, B Hermann, T Bajraszewski, T Le, A Stingl and AF Fercher, “Ultrahigh resolution Fourier domain optical coherence tomography”, Opt. Express, Vol. 12, pp. 2156-2165 (2004)
    [8] C Mason, JF Markusen, MA Town, P Dunnill and RK Wang, “The potential of optical coherence tomography in the engineering of living tissue”, Phys. Med. Biol., Vol 49 pp. 1097-1115 (2004)
    [9] W Drexler, U Morgner, FX Kartner, C Pitris, SA Boppart, XD Li, EP Ippen and JG Fujimoto, “In vivo ultrahigh-resolution optical coherence tomography”, Opt. Lett., Vol. 24 pp. 1221-1223 (1999)
    [10] Peter H Tomlins and Ruikang K Wang, "Digital phase stabilisation to improve detection sensitivity for optical coherence tomography", Meas. Sci. Tech. (In Press 2007)
    [11] Peter H Tomlins, Will M. Palin, Adrian C. Shortall and Ruikang K Wang, "Time-resolved simultaneous measurement of group index and phyiscal thickness during photopolymerisation of resin-based dental composite", J. Biomed. Opt., Vol. 12, 014020 (2007)
    [12] Peter H Tomlins and Ruikang K Wang, "Matrix approach to quantitative refractive index analysis by Fourier domain optical coherence tomography", J. Opt. Soc. Am. A, Vol. 23, pp. 1897-1907 (2006)
    [13] W Choi, C Fang-Yen, K Badizadegan, S Oh, N Lue, RR Dasari and MS Feld, “Tomographic Phase Microscopy”, Nature Methods, DOI:10.1038/NMETH1078 (2007)
    [14] Peter H Tomlins, Matthew Tedaldi, Robert A Ferguson and Ruikang K Wang, "Stereoscopic Optical Coherence Tomography in the Frequency Domain for Refractive Index Sensitive Imaging", Proc. SPIE, Vol. 6627 (2007)

    For more information: Dr. Paul Tomlins