WP 6: Label free cell behaviour and function measurement
State of the art
As a relatively new field tissue engineering has experienced a number of metrology challenges that have hindered the potential impact of new products entering the market place. When tissue engineering was conceived around 30 years ago it was thought to be the trigger for a new revolution in personalized medicine with damaged or diseased organs being quickly replaced with off the shelf products, negating the continual shortfall of organs on the transplantation lists. However, since its conception only a handful of tissue such as skin, bone, cartilage, capillary and periodontal tissues have shown clinical promise. One of the biggest challenges to the tissue engineering field is measuring the behavior of cells within a scaffold support structure, this is particularly important when the clinical efficacy of a tissue engineered product is related to its active cellular component. Current methods for measuring cell behavior in tissue engineering are less than adequate, in many cases relying on destructive methods or by making inferences about cell behavior based on factors released by the cells. In both these cases a direct measurement of the cell in-situ is not possible and important product related information such as cell distribution, viability and differentiation state is not obtainable.
Over the last few years a number of imaging techniques have started to be developed which could offer huge benefits to fields such as tissue engineering. Many of these techniques require labeling of the cells or scaffolds in order to accurately make measurements within the 3D tissue engineered product. However, recently a number of label free technologies have been developed which offer the potential for rapid information rich imaging without compromising or altering the product composition or interfering with the behavior of the cells. In particular, label free imaging technologies such as coherent anti-Stokes Raman scattering (CARS) and imaging mass spectroscopy offer a great deal of potential.
Coherent anti-Stokes Raman scattering (CARS) microscopy has attracted a lot of interest as a new technique for vibrational imaging. CARS is a third-order nonlinear optical process that involves a pump beam at a frequency of wp, a Stokes beam at a frequency of ws, and a signal at the anti-Stokes frequency of 2 wp - ws generated in the phase matching direction. The vibrational contrast in CARS microscopy is created when the frequency difference (wp - ws) between the pump and the Stokes beams is tuned to be resonant with a Raman-active molecular vibration. CARS possesses a higher sensitivity than Raman microscopy because the coherent CARS radiations produce a large and directional signal. This leads to a lower average excitation power and consequently less photo damage to cells. Moreover, the nonlinear excitation intensity dependence of CARS provides inherent 3D sectioning capability, similar to multiphoton fluorescence microscopy. Duncan et al. constructed the first CARS microscope by use of two dye laser beams with a non collinear beam geometry. Zumbush and colleagues revived CARS microscopy by focusing a pair of collinearly overlapped near infrared femtosecond laser beams with an objective lens of high numerical aperture (NA). Under the tight focusing condition, the phase matching condition of CARS is satisfied in the collinear beam geometry which facilitates experimental implementation and provides superior image quality compared to the non-collinear beam geometry. The tight focusing by a high NA objective lens also improves the spatial resolution.
Mass spectroscopy (MS) imaging, specifically matrix assisted laser desorption ionisation (MALDI) and desorption electrospray surface ionisation (DESI) imaging, are recently developed soft ionization techniques which allow the analysis of biomolecules (proteins, peptides and sugars) and large organic molecules. Importantly these methods operate in ambient or near ambinet conditions. Until recently imaging MS has been restricted to the provision of elemental or very simple molecular information. Recent advances in the use of MALDI and DESI have resulted in the direct analysis of proteins, peptides and small molecules from surfaces. Both technologies are relatively new to the area of cell imaging and innovators have speculated on its utility in real time spatial analysis for biomarker monitoring in diseased tissues.. There is an urgent requirement to harness the mass spectrometry power of these techniques for cell surface imaging. To do this a major coordinated research effort is required focusing on key metrology issues including understanding the different ionisation processes, the projectile-surface interaction (DESI) the mass range over which desorption is effective, information depth and the molecular sensitivity. These key parameters allow the ultimate spatial resolution to be determined and the most effective routes to achieve that. The project partners bring together enormous experience in mass spectrometry and surface analysis covering chemistry, biology and physics that will make a major contribution to improving the sensitivity, spatial resolution, reproducibility and measurement uncertainty of these important techniques.
This work package will investigate the application of these new imaging technologies to the field of tissue engineering. In particular parameters such as sensitivity, depth resolution and reproducibility will be investigated.
Movement beyond the current state of art
Despite the promise of advanced imaging techniques such as CARS and MALDI/DESI imaging there are a number of technical drawbacks which hinder there wider uptake into emergent fields such as tissue engineering. If these issues can be addressed then advanced label free imaging will have a huge impact on the analysis and development of current and future cell based therapies. Raman based imaging such as CARS encounter problems in fields such as tissue engineering due to the existence of a non-resonant background that arises from the electronic contributions even in the absence of vibrational resonance. In addition, for aqueous solutions strong resonant background signals may be present due to the broad Raman band of water. This has started to be addressed recently through the development of techniques that have been demonstrated to enhance of the signal-to-background ratio in CARS microscopy. These include the use of near-infrared instead of visible excitation pulses to avoid two-photon electronic resonances and adaptation of the spectral widths of the laser pulses to the spectral line width of the Raman resonance under investigation. Further advancement has been achieved employing an epi-detection scheme for scatterers smaller than the wavelength and by time-resolved CARS (T-CARS) microscopy.
This project will go beyond the current state of the art by developing a scheme for scanning CARS based on the use of infrared femtosecond Er-doped fibre laser as pump laser and a supercontinuum in the infrared as very large band Stokes laser. The expected advantages of such a scheme are large band scanning and a spectral region background free for electronic transitions and transparent to the tissues, which will enhance the signal for 3D imaging.
MS based imaging, in particular DESI and MALDI imaging, have been shown to have potential in a range of applications from studying drug distribution and metabolism to examining protein expression in the brain. However, the application of this type of novel imaging has not been investigated with tissue-engineered products. There are a number of factors that could hinder its application such as resolution to the single cell level, cell identification based on protein mass and sensitivity issues due to possible matrix suppression effects. This project will address critical issues that could potentially limit the uptake of techniques such as MALDI and DESI imaging. These include the ionisation processes of the samples to be analysed as well as sensitivity, spatial resolution and depth profiles of the technology in relation to its application in tissue engineering. In addition the project will also gather information on the molecular size and range of compounds that can be detected for both techniques. Important factors such as reproducibility and quantification will be considered in order to enable analysts to use the techniques with confidence. Many of these fundamental issues have yet to be addressed but are essential for the wider use of the technology, especially if they are to be used routinely to measure tissue engineered products.
Scientific tasks
Development of a depth resolution model for evaluating CARS and imaging MS. Evaluate the DESI projectile-surface interaction including emitted material volume, efficiency, information depth and mass range of the desorption process. Design and manufacture of a bespoke imaging source for DESI. This will involve the design and testing of a DESI source for performing the required experiments into spatial resolution, depth profiling and ionisation mechanisms. Such fundamental information is essential for understanding how best to present samples for analysis and how to improve the resolution and utility of the technology. Comparison of DESI and MALDI imaging and assess the two molecular imaging sources for the provision of biological data. Correlation of results with CARS Develop a new scheme of scanning CARS for label free depth resolution imaging . An evaluation of the measurement uncertainty associated with quantitative biological imaging using CARS, DESI and MALDI. This will include repeatability, reproducibility and accuracy of the measurements.
Technical risks
The development of novel label free imaging systems is a difficult technical challenge. The institutes carrying out the tasks have considerable expertise in developing and evaluating instrumentation systems. Each has strong contacts with universities and high technology companies in the field where additional solutions can be explored.
Expected outputs
Assessment of the metrological application of new imaging CARS and MS technologies; High sensitivity imaging CARS, DESI and MALDI capabilities the provision of imaging standards Intercomparison of CARS, DESI and MALDI for quantifying biological parameters
Selected references
[1] Zumbusch, A., G. R. Holtom, and X. S. Xie. 1999. Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering. Phys.Rev. Lett. 82:4142–4145
[2] J.-X. Cheng, A. Volkmer, L.D. Book,X.S. Xie, J. Phys. Chem. B 105, 1277 (2001)
[3] H. Kano and H-o Hamaguchi ”Femtosecond coherent anti-Stokes Raman scattering spectroscopy using supercontinuum generated from a photonic crystal fiber” Applied Physics Letters, Vol. 85, No. 19, pp. 4298–4300
[4] Science 306 (2004) 471
For more information: Dr. Damien Marshall
