Collinear Optical Coherence and Confocal Fluorescence Microscopies
Introduction
Materials, measurements, and methods that can speed tissue
engineered medical products (TEMPs) to market are highly desirable.
One means to this end is to use non-destructive imaging techniques
to optimize the relationships between scaffold structure, environment,
and cell response. The interaction of these variables influences
the success of the TEMPs. Yet, the precise nature of these interactions
has yet to be worked out in most instances. A significant difficulty
in furthering the understanding of the interaction between these
factors and cell behavior is the lack of high-resolution, non-destructive
imaging techniques that can penetrate deeply into the TEMP.
Optical coherence microscopy (OCM) has been chosen as the technique
to image the scaffold, cell, and tissue structures because of
its unique combination of high resolution (~1 µm) and
high sensitivity (>100dB). We have coupled OCM with confocal
fluorescence microscopy (CFM) in a collinear microscope that
is capable of non-invasively monitoring both structure and cell
function in a TEMP.
Experimental Approach
In the OCM component of the microscope, 1.3 µm light
is passed into a Michelson interferometer which splits the beam
into reference and sample arm paths. The length of the reference
arm is varied, and when the optical pathlength of the light
reflected from the scaffold equals the pathlength of the oscillating
reference arm, constructive interference occurs. The effect
is to filter out light that is scattered from out-of-focus light
much more effectively than in conventional confocal microscopy.
The CFM component of the microscope consists of a visible wavelength
beam (usually 488 nm) that is used to simultaneously examine
the fluorescence of stained cells. Confocal gating is achieved
through the use of a pinhole placed just before the detector.
3D images are obtained by moving the scaffold within the combined
beams with high precision motorized stages.
Left - A 3D rendering of OCM/CFM data from a cultured poly(e-caprolactone)
(PCL) scaffold is shown. The PCL scaffold is displayed as
transparent blue with pores again in black. The cells detected
by the CFM channel are in red.
Right – An example of data reduction: Chord length distribution
graph of pore diameters calculated from an image obtained
on the OCM.
Future Directions
We are building a self-sustaining bioreactor for the OCM/CFM
to perform in vitro (4-D) imaging of cell/scaffold interactions.
Cell morphology, population, and matrix protein expression will
be monitored in real time. The goal is to visualize how the
cellular interface with a 3-D scaffold changes over time. This
will provide insight into designing and developing TEMPs.
Publications
Landis, F.A., Cicerone, M.T., Cooper, J.A., Washburn, N.R.,
and Dunkers, J.P. “Developing Metrology for Tissue Engineering:
Collinear Optical Coherence and Confocal Fluorescence Microscopies”,
IEEE Bioimaging Conference Proceedings, 4/2004.
Dunkers, J.P., Cicerone, M.T., and Washburn, N.R. “Collinear
Optical Coherence and Confocal Fluorescence Microscopies for
Tissue Engineering”, Optics Express, 11(23), p. 3074, 2003.
Dunkers, J.P. and Cicerone, M.T. “Scaffold Structure and
Cell Function Through Multimodal Imaging and Quantitative Visualization”,
Biomaterials Forum, 25(3), p. 8, 2003.
Contributors:
Forrest A. Landis
James A. Cooper
Judith E. Devaney (ITL)
Martin Chiang
Joy P. Dunkers
Jeannie Stephens
John G. Hagedorn (ITL)
Xianfeng Wang
Marcus T. Cicerone
Newell R. Washburn
Steven G. Satterfield (ITL)
Biomaterials Group
Polymers Division
Materials Science and Engineering Laboratory
