Our lab has used an optical coherence microscope (OCM) to assess both the structure of tissue-engineered corneal constructs and their transparency. Currently, we are not able to resolve cells versus collagen matrix material in the images produced. We would like to distinguish cells in order to determine if they are viable while growing in culture and also if they are significantly contributing to the light scattering in the tissue. In order to do this, we are currently investigating the use of immunogold labeling. Gold nanoparticles are high scatterers and can create contrast in images. We have conjugated gold nanoparticles to antibodies specific to the α5 β1 integrins expressed in some corneal cells. This choice of target should allow assessment of the phenotypic behavior of the cells in the tissue, as different integrins are expressed in different phenotypes. This study applies the immunogold technique to study cultured corneal cells using the OCM with the ultimate goal of monitoring cellular behavior in engineered tissue and corroborating results from standard histological methods

Haskell, Richard C.

Orwin, Elizabeth J.

Raub, Chris B.

English

Keck Graduate Institute

Claremont Colleges
Scholarship @ Claremont
All HMC Faculty Publications and Research HMC Faculty Scholarship
9-1-2004
Immunogold Labeling to Enhance Contrast in
Optical Coherence Microscopy of Tissue
Engineered Corneal Constructs
Chris B. Raub
Elizabeth J. Orwin
Harvey Mudd College
Richard C. Haskell
Harvey Mudd College
This Conference Proceeding is brought to you for free and open access by the HMC Faculty Scholarship at Scholarship @ Claremont. It has been
accepted for inclusion in All HMC Faculty Publications and Research by an authorized administrator of Scholarship @ Claremont. For more
information, please contact scholarship@cuc.claremont.edu.
Recommended Citation
Raub, C.B., E.J. Orwin, and R.C. Haskell. "Immunogold labeling to enhance contrast in optical coherence microscopy of tissue
engineered corneal constructs." Proceedings of the 26th Annual International Conference of the IEEE, Engineering In Medicine and
Biology Society, Vol. 1 (2004): 1210-1213.
Abstract— Our lab has used an optical coherence
microscope (OCM) to assess both the structure of tissue-
engineered corneal constructs and their transparency.
Currently, we are not able to resolve cells versus collagen
matrix material in the images produced.  We would like to
distinguish cells in order to determine if they are viable while
growing in culture and also if they are significantly
contributing to the light scattering in the tissue.  In order to do
this, we are currently investigating the use of immunogold
labeling.  Gold nanoparticles are high scatterers and can create
contrast in images.  We have conjugated gold nanoparticles to
antibodies specific to the α5β1 integrins expressed in some
corneal cells.  This choice of target should allow assessment of
the phenotypic behavior of the cells in the tissue, as different
integrins are expressed in different phenotypes.  This study
applies the immunogold technique to study cultured corneal
cells using the OCM with the ultimate goal of monitoring
cellular behavior in engineered tissue and corroborating results
from standard histological methods.
Keywords—Cornea, tissue engineering, optical coherence
microscopy
I.  INTRODUCTION
Recently, it has been discovered that there is a cellular
contribution to corneal transparency, making observation of
our tissues on the molecular level essential.  In response, the
characterization of the phenotype of corneal keratocytes in
normal and wounded corneas has been the subject of much
investigation. Stromal wounding in response to damage or
surgical procedures initiates a distinct response in corneal
keratocytes [1].  Keratocytes in the wounded region are
activated to become repair fibroblasts, which proliferate and
synthesize both extracellular matrix (ECM) components and
enzymes to degrade ECM.  The repair fibroblasts then
differentiate further into either myofibroblasts or scar
keratocytes.  Myofibroblasts, which express α-smooth
muscle actin, act to contract the wound [2].  The transition
from repair fibroblast to myofibroblast is reversible upon
completion of the healing process. In contrast, if a repair
fibroblast differentiates into a scar keratocyte, the process is
irreversible and results in the formation of a corneal scar.
        The three phenotypically distinct cell types are
characterized by certain features.  30% by mass of the water
soluble protein in quiescent rabbit keratocytes is comprised
of the “corneal crystallins” transketolase (TKT) and
aldehyde dehydrogenase 1 (ALDH1) [3].  In addition, it has
been discovered that there is at least a 50% reduction in
expression levels of these proteins when corneal haze
following injury is present.  At this time, cells have been
activated to the repair or myofibroblast phenotype.  It is
hypothesized from these results that these two proteins act
similarly to crystallin proteins in the lens of the eye – that is,
they aggregate in the cell cytoplasm and contribute to index
matching of the cytosol with the surrounding extracellular
environment.  As further evidence for this theory, Jester and
colleagues have shown that light scatter from normal
keratocytes in a transparent cornea comes entirely from the
nucleus of the cell – the cell body does not scatter light [3].
Corneal stromal cells differentially express integrin
receptors. Specifically, myofibroblasts express the α5β1
integrin at higher levels than do repair fibroblasts, whereas
quiescent keratocytes do not express the α5β1 integrin [4,5].
Therefore, the α 5β1  integrin is a good marker for
nontransparent corneal stromal cell phenotypes.
Immunogold labeling methods in electron microscopy
are well established [6,7].  Immunogold labeling has
recently been used in conjunction with reflectance confocal
imaging to enhance contrast of cancer cells both in cervical
tissue sections and in epithelial tissue constructs [8].  These
studies suggest that gold labeling in general, and
immunogold labeling in particular, might have application
in studies of engineered tissue using optical coherence
microscopy.
II.  METHODOLOGY
A. Gold Nanoparticle Formation
Gold nanoparticles were made using the citrate
reduction of Au (III) [9].  The amount of citrate was varied
to produce 16 nm, 71.5 nm, and 147 nm diameter gold
nanoparticles.  Upon complete formation of the particles, the
16 nm and 71.5 nm gold colloids were orange-red and deep
red, respectively, whereas the 147 nm particles appeared as
a purple precipitate.  Further boiling of the particle solutions
did not produce additional color change.
A test of particle monodispersity is to add PBS to a
solution of gold particles.  In the presence of >7 mM
electrolytes, gold particles form aggregates, which appear as
a purple precipitate [6,7].  2 ml each of PBS and the 16 nm
and 71.5 nm gold colloids were combined, producing a
purple precipitate.  Gold nanoparticles in the presence of 10
mM HEPES buffer pH 7.4 did not form precipitates when
exposed to PBS.
B. Gold Particle Imaging
Naked colloidal gold particles were imaged with phase
contrast microscopy and OCM.  Phase contrast microscopy,
Immunogold Labeling to Enhance Contrast in Optical Coherence Microscopy of
Tissue Engineered Corneal Constructs
C. B. Raub1, E. J. Orwin2, R. Haskell3
1Department of Biology, Harvey Mudd College, Claremont, CA, USA
2Department of Engineering, Harvey Mudd College, Claremont, CA, USA
3Department of Physics, Mudd College, Claremont, CA, USA
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0-7803-8439-3/04/$20.00©2004 IEEE 1210
Proceedings of the 26th Annual International Conference of the IEEE EMBS 
San Francisco, CA, USA • September 1-5, 2004 
with a 100x objective, revealed aggregates 1-10 µm in
diameter in the 147 nm diameter gold particle solution, and
much smaller, more abundant particles in the 71.5 nm gold
particle solution.  These particles were monodisperse or in
small clusters.  No particles were detected in the 16 nm
diameter gold particle solution using phase contrast
microscopy.
OCM images were taken with a gain of 1000 using an
850 nm SLD light source.  The resolution of the instrument
is 5 µm laterally and 10 µm vertically.
C. Immunogold Conjugation and Labeling of Cells
16 nm and 71.5 nm gold particles were conjugated to
antibodies, and human corneal myofibroblasts were
immunogold labeled using a procedure modified from
Sokolov et. al. [8].  71.5 nm gold was incubated with goat
anti-mouse IgG (F(ab’)2 antibody (Sigma)  for 24 hours at 4
°C, cells were trypsinized instead of being treated with
collagenase, and cells were incubated in PBS/10%FBS
containing 0.02 µg/µl mouse anti-α 5β1 non-blocking
antibody (Sigma), pelleted, and washed before incubation
with immunogold conjugates for 6 hours at 4 °C.
Spectrophotometry was used to confirm conjugation of gold
particles to antibody.  Indirect immunofluorescence using
the same antibodies as above (but with a FITC-conjugated
secondary antibody fragment) confirmed the presence of
α5β1 on corneal myofibroblasts and confirmed antibody
specificity.
Corneal myofibroblasts, grown on glass coverslips and
fixed in methanol for 20 minutes at room temperature, were
labeled with naked 16 nm and 71.5 nm gold by incubating
the cells with the gold colloids diluted once in 20 mM
HEPES buffer, pH 7.4.
D. Immunogold Labeling of Tissue-engineered Collagen
Sponges
Freeze-dried bovine type I collagen sponges were
prepared according to previous methods [10]. Sponges were
labeled either with naked 71.5 nm gold or were seeded with
human corneal myofibroblasts at 400,000 cells/cm3 and then
allowed to incubate in Dulbecco’s Modified Eagle’s
Medium at 37 °C for 24 hours.  They were then washed in
PBS and incubated with the anti-α5β1 primary and 71.5 nm
gold-conjugated secondary antibodies previously mentioned.
Antibody incubation occurred as previously mentioned,
except that tissues were incubated with primary antibody for
12 hours at 4 °C and with 71.5 nm gold-conjugated
secondary antibodies for 9 hours at 4 °C.
III. RESULTS
A. OCM Images of Gold Particles in Solution
Differences were observed in the amount of
backscattering coming from dilute aqueous solutions of the
gold particles (Fig. 1).  Larger particles scattered more
intensely.  Sedimentation of particle aggregates was detected
in the 147 nm particle solution.
Fig. 1.  OCM Images of gold particles in aqueous solution.  Blue is
relatively low backscattering; red is relatively high backscattering.  16 nm
diameter gold nanoparticles (left panel) scatter less intensely than 71.5 nm
diameter gold nanoparticles (central panel), which scatter less intensely
than 1-10 µm diameter gold aggregates of 147 nm gold particles (right
panel).  Images are 500 µm x 500 µm x 500 µm.
B. Labeling of Collagen Sponge with Naked Gold Particles
Naked 71.5 nm diameter gold nanoparticles adhered to a
tissue-engineered collagen matrix (Fig. 2).  Images of the
same matrix location over the course of 3 hours show 71.5
nm diameter gold nanoparticles settling on the upper half of
the matrix (Fig. 2, central panels).  Extensive washing
removed only a fraction of the nanoparticles (Fig. 2, bottom
panel).  After washing, backscattered intensity of the matrix
was greater at all depths than that of the pre-incubation
sponge.
Fig. 2.  OCM images of a collagen sponge (side view) incubated with 71.5
nm diameter gold particles.  The images are of a tissue-engineered collagen
sponge before (top panel) and 10, 30, 40, and 180 minutes after addition of
71.5 nm gold particles (central panels, from left to right).  The bottom panel
is the same imaging volume after extensive rinsing of the sponge with
deionized water.  Images are 500 µm wide.
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1211
C. Labeling of Cells with Naked Gold Nanoparticles
Naked 16 nm and 71.5 nm gold nanoparticles adhered to
methanol-fixed corneal myofibroblasts, staining the cells
orange-red and red, respectively (Fig. 3).  After 48 hours
incubation, 16 nm nanoparticles appeared, in general, to
stain the cells more intensely than 71.5 nm gold
nanoparticles and to stain the cytoplasm more than the cell
nuclei, although there was variability in staining intensity
and location.  From this data, we were led to wonder
whether immunogold labeling of stromal cells could be
assessed colorimetrically using brightfield microscopy.
D. Immunogold Labeling of Human Corneal Stromal Cells
Brightfield microscopy of cells in suspension labeled
with 71.5 nm gold-antibody conjugates revealed intense red
staining on part or all of some cell surfaces, whereas control
cells displayed no red staining (Fig. 4).  The variability of
the gold staining may indicate differential antigen
expression on different cell phenotypes, inefficient gold
labeling, or both.  The fraction of red-stained cells was low,
and the stained cells were typically morphologically less
spherical than stained cells.
Fig. 3.  Unfiltered brightfield images of methanol-fixed corneal
myofibroblasts incubated with 16 nm (top panel) and 71.5 nm (bottom
panels) gold.  Total magnification is 400x.  In general, nuclei stained less
intensely than other cellular material, but there were exceptions (top panel,
upper left corner).  71.5 nm gold-stained cells generally displayed less
intense coloration (bottom left panel).
Fig. 4.  Unfiltered brightfield images of living corneal stromal cells
(top panel) and of cells incubated with primary antibody followed by 71.5
nm gold-conjugated secondary antibody fragments (central and bottom
panels).  For the incubations, cell suspensions were in PBS/10% FBS and
were placed on coverslips for imaging.
E. Incubation of cells on collagen sponges with gold
conjugates
Corneal fibroblasts were seeded at high density on
collagen sponges and subsequently incubated with the
previously mentioned primary antibody and 71.5 nm gold-
conjugated secondary antibody fragment.  After each
incubation, the sponge was washed in PBS and imaged with
the OCM (Fig. 5).  Cell-seeded sponge images after
incubation with the conjugate appeared to have brighter
regions than images of the sponge before incubation with
the conjugates, although the difference is not large.  The
post-incubation sponge images had a significantly higher
average scattering intensity per sponge voxel (one-tailed
two-sample t-test, n1=4, n2=10, p=0.0007).
Fig. 5.  OCM images of a corneal fibroblast-seeded collagen sponge before
incubation with gold conjugate (left panel) and after (right panel).  These
images were taken after incubating the sponge in primary antibody (left
panel) for 12 hours followed by gold conjugate (right panel) for 9 hours.
Incubations occurred at 4 °C with gentle agitation.  Images are 500 µm x
500 µm wide and 700 µm deep.
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1212
IV.  DISCUSSION
Our OCM data shows that larger gold particles in
solution scatter more intensely than smaller particles.  As
gold particles get larger, their conjugation to antibodies
becomes more difficult and they increase attenuation of the
OCM sample beam.  Both factors contribute to an upper size
limit for practical use of gold particles as OCM contrast
agents.
We have shown that through the use of monodisperse,
uniform gold nanoparticles, we can achieve improved
contrast of OCM images of tissue-engineered corneal
construct.  Naked gold particles most likely bind to collagen
through noncovalent charge interactions and Van der Waals
forces [6,7].
We can also monitor gold nanoparticle binding to
corneal stromal cells with a simple brightfield inverted
microscope.  This method can help correlate OCM data of
changes in cellular backscattering properties to immunogold
labeling.  It should be noted that brightfield microscopy
cannot distinguish between antibody-specific and
nonspecific gold binding to cells.  Since all cells were
incubated with gold conjugates in PBS, however, it seems
likely that unbound gold would aggregate and thus would
not contribute to cellular labeling.  In fact, brightfield
images revealed several micron-sized aggregates of gold
particles in the gold conjugate solution.
Although more data needs to be taken with OCM,
brightfield microscopy, and electron microscopy to confirm
whether immunogold-labeled cells are contributing to the
OCM signal increase of cell-seeded collagen sponges, the
results of the current study show that our OCM is sensitive
to the addition of gold colloid (both bare and conjugated to
antibodies) to a complex tissue sample.  Ideally, biological
contrast agents should produce clearly visible and striking
changes to OCM images of labeled biological tissue.
Further experimentation with immunogold labeling is
needed to optimize cellular contrast enhancement.
 
V.  CONCLUSION
Our data suggest that gold nanoparticles are promising
contrast-enhancing agents for engineered tissue and cells
imaged with OCM.  OCM can detect gold nanoparticles and
gold conjugates, both in solution and associated with
collagen and cell-seeded engineered tissue.  Brightfield
microscopy can detect binding of gold particles to cells and
cell material.  Immunogold labeling of human corneal
stromal cells can potentially be used as a method for
assessing cell phenotype of living cells in engineered tissue.
This would allow monitoring of the way cells respond to
physical forces on the tissue, the addition of soluble factors
to the media, and other changes in cellular environment.
Other cell surface markers can be targeted to label different
cell phenotypes.  This study provides an initial step towards
using OCM to monitor living cells and cellular phenotype in
engineered tissue.
ACKNOWLEDGMENT
We would like to thank Dr. Barbara Hoeling and Dr.
Daniel Petersen for their contributions to the OCM, and
Sung-Eun Lee and Tonya Icenogle for their contributions to
engineered tissue and bioreactor design.
REFERENCES
[1] M. E. Fini. “Keratocyte and Fibroblast Phenotypes in the
Repairing Cornea,” Progress in Retinal and Eye Research,vol.
18, pp. 529-551, 1999.
[2] J. V. Jester, P. A. Barry, G. J. Lind, W. M. Petroll, R. Garana,
and H. D. Cavanaugh, “Corneal keratocytes: in situ and in vitro
organization of cytoskeletal contractile proteins”, Invest.
Ophthalmol. Vis. Sci., vol. 35(2), pp. 730-43, 1994.
[3] J. V. Jester, T. Moller-Pedersen, J. Huang, C. M. Sax, W. T.
Kays, H. D. Cavanaugh, W. M. Petroll, and J. Piatigorsky, “The
Cellular Basis for Corneal Transparency: Evidence for ‘Corneal
Crystallins’,” Journal of Cell Science, vol. 112, pp. 613-622,
1999.
[4] S. K. Masur, J. K. Cheung, and S. Antohi.  “Identification of
integrins in cultured corneal fibroblasts and in isolated
keratocytes,” Investigative Ophthalmology and Visual Science,
vol. 34, pp. 2690-8, 1993.
[5] S. K. Masur, R. J.  Conors, Jr, J. K. Cheung, and S. Antohi.
1999.  “Matrix adhesion characteristics of corneal
myofibroblasts,” Investigative Ophthalmology and Visual
Science, vol. 40, pp. 904-910, 1999.
[6] J. L. M. Leunissen, and J. R. De Mey, Immuno-Gold Labeling in
Cell Biology (Verklij, AJ, and Leunissen, JLM, Eds.), Boca
Raton, FL: CRC Press, 1989.
[7] J. Roth, Techniques in Immunochemistry (Bullock, GR, and
Petrusz, P, Eds.), Vol. 2, New York, NY: Academic Press, 1983.
[8] K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R.
Lotan, and R. Richards-Kortum, “Real-Time Optical Imaging of
Precancer Using Anti-Epidermal Growth Factor Receptor
Antibodies Conjugated to Gold Nanoparticles,” Cancer
Research, vol. 63, pp. 1999-2004, 2003.
[9] F. Frens, “Controlled nucleation for the regulation of the particle
size in monodisperse gold suspensions,” Nature Physical
Science, vol. 241, pp. 20-22, 1973.
[10] E. J. Orwin, and A. Hubel, “In vitro culture characteristics of
corneal epithelial, endothelial, and keratocytes in a native
collagen matrix,” Tissue Engineering, vol. 6(4), pp. 307-319,
2000.
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1213


Immunogold Labeling to Enhance Contrast in Optical Coherence Microscopy of Tissue Engineered Corneal Constructs

Our lab has used an optical coherence microscope (OCM) to assess both the structure of tissue-engineered corneal constructs and their transparency. Currently, we are not able to resolve cells versus collagen matrix material in the images produced. We would like to distinguish cells in order to determine if they are viable while growing in culture and also if they are significantly contributing to the light scattering in the tissue. In order to do this, we are currently investigating the use of immunogold labeling. Gold nanoparticles are high scatterers and can create contrast in images. We have conjugated gold nanoparticles to antibodies specific to the alpha/sub 5/beta/sub 1/ integrins expressed in some corneal cells. This choice of target should allow assessment of the phenotypic behavior of the cells in the tissue, as different integrins are expressed in different phenotypes. This study applies the immunogold technique to study cultured corneal cells using the OCM with the ultimate goal of monitoring cellular behavior in engineered tissue and corroborating results from standard histological methods

Raub, Christopher B., \u2704

Haskell, Richard C

Scholarship@Claremont

Claremont CollegesScholarship @ ClaremontAll HMC Faculty Publications and Research HMC Faculty Scholarship9-1-2004Immunogold Labeling to Enhance Contrast inOptical Coherence Microscopy of TissueEngineered Corneal ConstructsChris B. RaubElizabeth J. OrwinHarvey Mudd CollegeRichard C. HaskellHarvey Mudd CollegeThis Conference Proceeding is brought to you for free and open access by the HMC Faculty Scholarship at Scholarship @ Claremont. It has beenaccepted for inclusion in All HMC Faculty Publications and Research by an authorized administrator of Scholarship @ Claremont. For moreinformation, please contact scholarship@cuc.claremont.edu.Recommended CitationRaub, C.B., E.J. Orwin, and R.C. Haskell. "Immunogold labeling to enhance contrast in optical coherence microscopy of tissueengineered corneal constructs." Proceedings of the 26th Annual International Conference of the IEEE, Engineering In Medicine andBiology Society, Vol. 1 (2004): 1210-1213.Abstract— Our lab has used an optical coherencemicroscope (OCM) to assess both the structure of tissue-engineered corneal constructs and their transparency.Currently, we are not able to resolve cells versus collagenmatrix material in the images produced.  We would like todistinguish cells in order to determine if they are viable whilegrowing in culture and also if they are significantlycontributing to the light scattering in the tissue.  In order to dothis, we are currently investigating the use of immunogoldlabeling.  Gold nanoparticles are high scatterers and can createcontrast in images.  We have conjugated gold nanoparticles toantibodies specific to the α5β1 integrins expressed in somecorneal cells.  This choice of target should allow assessment ofthe phenotypic behavior of the cells in the tissue, as differentintegrins are expressed in different phenotypes.  This studyapplies the immunogold technique to study cultured cornealcells using the OCM with the ultimate goal of monitoringcellular behavior in engineered tissue and corroborating resultsfrom standard histological methods.Keywords—Cornea, tissue engineering, optical coherencemicroscopyI.  INTRODUCTIONRecently, it has been discovered that there is a cellularcontribution to corneal transparency, making observation ofour tissues on the molecular level essential.  In response, thecharacterization of the phenotype of corneal keratocytes innormal and wounded corneas has been the subject of muchinvestigation. Stromal wounding in response to damage orsurgical procedures initiates a distinct response in cornealkeratocytes [1].  Keratocytes in the wounded region areactivated to become repair fibroblasts, which proliferate andsynthesize both extracellular matrix (ECM) components andenzymes to degrade ECM.  The repair fibroblasts thendifferentiate further into either myofibroblasts or scarkeratocytes.  Myofibroblasts, which express α-smoothmuscle actin, act to contract the wound [2].  The transitionfrom repair fibroblast to myofibroblast is reversible uponcompletion of the healing process. In contrast, if a repairfibroblast differentiates into a scar keratocyte, the process isirreversible and results in the formation of a corneal scar.        The three phenotypically distinct cell types arecharacterized by certain features.  30% by mass of the watersoluble protein in quiescent rabbit keratocytes is comprisedof the “corneal crystallins” transketolase (TKT) andaldehyde dehydrogenase 1 (ALDH1) [3].  In addition, it hasbeen discovered that there is at least a 50% reduction inexpression levels of these proteins when corneal hazefollowing injury is present.  At this time, cells have beenactivated to the repair or myofibroblast phenotype.  It ishypothesized from these results that these two proteins actsimilarly to crystallin proteins in the lens of the eye – that is,they aggregate in the cell cytoplasm and contribute to indexmatching of the cytosol with the surrounding extracellularenvironment.  As further evidence for this theory, Jester andcolleagues have shown that light scatter from normalkeratocytes in a transparent cornea comes entirely from thenucleus of the cell – the cell body does not scatter light [3].Corneal stromal cells differentially express integrinreceptors. Specifically, myofibroblasts express the α5β1integrin at higher levels than do repair fibroblasts, whereasquiescent keratocytes do not express the α5β1 integrin [4,5].Therefore, the α 5β1  integrin is a good marker fornontransparent corneal stromal cell phenotypes.Immunogold labeling methods in electron microscopyare well established [6,7].  Immunogold labeling hasrecently been used in conjunction with reflectance confocalimaging to enhance contrast of cancer cells both in cervicaltissue sections and in epithelial tissue constructs [8].  Thesestudies suggest that gold labeling in general, andimmunogold labeling in particular, might have applicationin studies of engineered tissue using optical coherencemicroscopy.II.  METHODOLOGYA. Gold Nanoparticle FormationGold nanoparticles were made using the citratereduction of Au (III) [9].  The amount of citrate was variedto produce 16 nm, 71.5 nm, and 147 nm diameter goldnanoparticles.  Upon complete formation of the particles, the16 nm and 71.5 nm gold colloids were orange-red and deepred, respectively, whereas the 147 nm particles appeared asa purple precipitate.  Further boiling of the particle solutionsdid not produce additional color change.A test of particle monodispersity is to add PBS to asolution of gold particles.  In the presence of >7 mMelectrolytes, gold particles form aggregates, which appear asa purple precipitate [6,7].  2 ml each of PBS and the 16 nmand 71.5 nm gold colloids were combined, producing apurple precipitate.  Gold nanoparticles in the presence of 10mM HEPES buffer pH 7.4 did not form precipitates whenexposed to PBS.B. Gold Particle ImagingNaked colloidal gold particles were imaged with phasecontrast microscopy and OCM.  Phase contrast microscopy,Immunogold Labeling to Enhance Contrast in Optical Coherence Microscopy ofTissue Engineered Corneal ConstructsC. B. Raub1, E. J. Orwin2, R. Haskell31Department of Biology, Harvey Mudd College, Claremont, CA, USA2Department of Engineering, Harvey Mudd College, Claremont, CA, USA3Department of Physics, Mudd College, Claremont, CA, USAAuthorized licensed use limited to: to the Claremont Colleges!. Downloaded on June 16, 2009 at 16:47 from IEEE Xplore.  Restrictions apply.0-7803-8439-3/04/$20.00©2004 IEEE 1210Proceedings of the 26th Annual International Conference of the IEEE EMBS San Francisco, CA, USA • September 1-5, 2004 with a 100x objective, revealed aggregates 1-10 µm indiameter in the 147 nm diameter gold particle solution, andmuch smaller, more abundant particles in the 71.5 nm goldparticle solution.  These particles were monodisperse or insmall clusters.  No particles were detected in the 16 nmdiameter gold particle solution using phase contrastmicroscopy.OCM images were taken with a gain of 1000 using an850 nm SLD light source.  The resolution of the instrumentis 5 µm laterally and 10 µm vertically.C. Immunogold Conjugation and Labeling of Cells16 nm and 71.5 nm gold particles were conjugated toantibodies, and human corneal myofibroblasts wereimmunogold labeled using a procedure modified fromSokolov et. al. [8].  71.5 nm gold was incubated with goatanti-mouse IgG (F(ab’)2 antibody (Sigma)  for 24 hours at 4°C, cells were trypsinized instead of being treated withcollagenase, and cells were incubated in PBS/10%FBScontaining 0.02 µg/µl mouse anti-α 5β1 non-blockingantibody (Sigma), pelleted, and washed before incubationwith immunogold conjugates for 6 hours at 4 °C.Spectrophotometry was used to confirm conjugation of goldparticles to antibody.  Indirect immunofluorescence usingthe same antibodies as above (but with a FITC-conjugatedsecondary antibody fragment) confirmed the presence ofα5β1 on corneal myofibroblasts and confirmed antibodyspecificity.Corneal myofibroblasts, grown on glass coverslips andfixed in methanol for 20 minutes at room temperature, werelabeled with naked 16 nm and 71.5 nm gold by incubatingthe cells with the gold colloids diluted once in 20 mMHEPES buffer, pH 7.4.D. Immunogold Labeling of Tissue-engineered CollagenSpongesFreeze-dried bovine type I collagen sponges wereprepared according to previous methods [10]. Sponges werelabeled either with naked 71.5 nm gold or were seeded withhuman corneal myofibroblasts at 400,000 cells/cm3 and thenallowed to incubate in Dulbecco’s Modified Eagle’sMedium at 37 °C for 24 hours.  They were then washed inPBS and incubated with the anti-α5β1 primary and 71.5 nmgold-conjugated secondary antibodies previously mentioned.Antibody incubation occurred as previously mentioned,except that tissues were incubated with primary antibody for12 hours at 4 °C and with 71.5 nm gold-conjugatedsecondary antibodies for 9 hours at 4 °C.III. RESULTSA. OCM Images of Gold Particles in SolutionDifferences were observed in the amount ofbackscattering coming from dilute aqueous solutions of thegold particles (Fig. 1).  Larger particles scattered moreintensely.  Sedimentation of particle aggregates was detectedin the 147 nm particle solution.Fig. 1.  OCM Images of gold particles in aqueous solution.  Blue isrelatively low backscattering; red is relatively high backscattering.  16 nmdiameter gold nanoparticles (left panel) scatter less intensely than 71.5 nmdiameter gold nanoparticles (central panel), which scatter less intenselythan 1-10 µm diameter gold aggregates of 147 nm gold particles (rightpanel).  Images are 500 µm x 500 µm x 500 µm.B. Labeling of Collagen Sponge with Naked Gold ParticlesNaked 71.5 nm diameter gold nanoparticles adhered to atissue-engineered collagen matrix (Fig. 2).  Images of thesame matrix location over the course of 3 hours show 71.5nm diameter gold nanoparticles settling on the upper half ofthe matrix (Fig. 2, central panels).  Extensive washingremoved only a fraction of the nanoparticles (Fig. 2, bottompanel).  After washing, backscattered intensity of the matrixwas greater at all depths than that of the pre-incubationsponge.Fig. 2.  OCM images of a collagen sponge (side view) incubated with 71.5nm diameter gold particles.  The images are of a tissue-engineered collagensponge before (top panel) and 10, 30, 40, and 180 minutes after addition of71.5 nm gold particles (central panels, from left to right).  The bottom panelis the same imaging volume after extensive rinsing of the sponge withdeionized water.  Images are 500 µm wide.Authorized licensed use limited to: to the Claremont Colleges!. Downloaded on June 16, 2009 at 16:47 from IEEE Xplore.  Restrictions apply.1211C. Labeling of Cells with Naked Gold NanoparticlesNaked 16 nm and 71.5 nm gold nanoparticles adhered tomethanol-fixed corneal myofibroblasts, staining the cellsorange-red and red, respectively (Fig. 3).  After 48 hoursincubation, 16 nm nanoparticles appeared, in general, tostain the cells more intensely than 71.5 nm goldnanoparticles and to stain the cytoplasm more than the cellnuclei, although there was variability in staining intensityand location.  From this data, we were led to wonderwhether immunogold labeling of stromal cells could beassessed colorimetrically using brightfield microscopy.D. Immunogold Labeling of Human Corneal Stromal CellsBrightfield microscopy of cells in suspension labeledwith 71.5 nm gold-antibody conjugates revealed intense redstaining on part or all of some cell surfaces, whereas controlcells displayed no red staining (Fig. 4).  The variability ofthe gold staining may indicate differential antigenexpression on different cell phenotypes, inefficient goldlabeling, or both.  The fraction of red-stained cells was low,and the stained cells were typically morphologically lessspherical than stained cells.Fig. 3.  Unfiltered brightfield images of methanol-fixed cornealmyofibroblasts incubated with 16 nm (top panel) and 71.5 nm (bottompanels) gold.  Total magnification is 400x.  In general, nuclei stained lessintensely than other cellular material, but there were exceptions (top panel,upper left corner).  71.5 nm gold-stained cells generally displayed lessintense coloration (bottom left panel).Fig. 4.  Unfiltered brightfield images of living corneal stromal cells(top panel) and of cells incubated with primary antibody followed by 71.5nm gold-conjugated secondary antibody fragments (central and bottompanels).  For the incubations, cell suspensions were in PBS/10% FBS andwere placed on coverslips for imaging.E. Incubation of cells on collagen sponges with goldconjugatesCorneal fibroblasts were seeded at high density oncollagen sponges and subsequently incubated with thepreviously mentioned primary antibody and 71.5 nm gold-conjugated secondary antibody fragment.  After eachincubation, the sponge was washed in PBS and imaged withthe OCM (Fig. 5).  Cell-seeded sponge images afterincubation with the conjugate appeared to have brighterregions than images of the sponge before incubation withthe conjugates, although the difference is not large.  Thepost-incubation sponge images had a significantly higheraverage scattering intensity per sponge voxel (one-tailedtwo-sample t-test, n1=4, n2=10, p=0.0007).Fig. 5.  OCM images of a corneal fibroblast-seeded collagen sponge beforeincubation with gold conjugate (left panel) and after (right panel).  Theseimages were taken after incubating the sponge in primary antibody (leftpanel) for 12 hours followed by gold conjugate (right panel) for 9 hours.Incubations occurred at 4 °C with gentle agitation.  Images are 500 µm x500 µm wide and 700 µm deep.Authorized licensed use limited to: to the Claremont Colleges!. Downloaded on June 16, 2009 at 16:47 from IEEE Xplore.  Restrictions apply.1212IV.  DISCUSSIONOur OCM data shows that larger gold particles insolution scatter more intensely than smaller particles.  Asgold particles get larger, their conjugation to antibodiesbecomes more difficult and they increase attenuation of theOCM sample beam.  Both factors contribute to an upper sizelimit for practical use of gold particles as OCM contrastagents.We have shown that through the use of monodisperse,uniform gold nanoparticles, we can achieve improvedcontrast of OCM images of tissue-engineered cornealconstruct.  Naked gold particles most likely bind to collagenthrough noncovalent charge interactions and Van der Waalsforces [6,7].We can also monitor gold nanoparticle binding tocorneal stromal cells with a simple brightfield invertedmicroscope.  This method can help correlate OCM data ofchanges in cellular backscattering properties to immunogoldlabeling.  It should be noted that brightfield microscopycannot distinguish between antibody-specific andnonspecific gold binding to cells.  Since all cells wereincubated with gold conjugates in PBS, however, it seemslikely that unbound gold would aggregate and thus wouldnot contribute to cellular labeling.  In fact, brightfieldimages revealed several micron-sized aggregates of goldparticles in the gold conjugate solution.Although more data needs to be taken with OCM,brightfield microscopy, and electron microscopy to confirmwhether immunogold-labeled cells are contributing to theOCM signal increase of cell-seeded collagen sponges, theresults of the current study show that our OCM is sensitiveto the addition of gold colloid (both bare and conjugated toantibodies) to a complex tissue sample.  Ideally, biologicalcontrast agents should produce clearly visible and strikingchanges to OCM images of labeled biological tissue.Further experimentation with immunogold labeling isneeded to optimize cellular contrast enhancement. V.  CONCLUSIONOur data suggest that gold nanoparticles are promisingcontrast-enhancing agents for engineered tissue and cellsimaged with OCM.  OCM can detect gold nanoparticles andgold conjugates, both in solution and associated withcollagen and cell-seeded engineered tissue.  Brightfieldmicroscopy can detect binding of gold particles to cells andcell material.  Immunogold labeling of human cornealstromal cells can potentially be used as a method forassessing cell phenotype of living cells in engineered tissue.This would allow monitoring of the way cells respond tophysical forces on the tissue, the addition of soluble factorsto the media, and other changes in cellular environment.Other cell surface markers can be targeted to label differentcell phenotypes.  This study provides an initial step towardsusing OCM to monitor living cells and cellular phenotype inengineered tissue.ACKNOWLEDGMENTWe would like to thank Dr. Barbara Hoeling and Dr.Daniel Petersen for their contributions to the OCM, andSung-Eun Lee and Tonya Icenogle for their contributions toengineered tissue and bioreactor design.REFERENCES[1] M. E. Fini. “Keratocyte and Fibroblast Phenotypes in theRepairing Cornea,” Progress in Retinal and Eye Research,vol.18, pp. 529-551, 1999.[2] J. V. Jester, P. A. Barry, G. J. Lind, W. M. Petroll, R. Garana,and H. D. Cavanaugh, “Corneal keratocytes: in situ and in vitroorganization of cytoskeletal contractile proteins”, Invest.Ophthalmol. Vis. Sci., vol. 35(2), pp. 730-43, 1994.[3] J. V. Jester, T. Moller-Pedersen, J. Huang, C. M. Sax, W. T.Kays, H. D. Cavanaugh, W. M. Petroll, and J. Piatigorsky, “TheCellular Basis for Corneal Transparency: Evidence for ‘CornealCrystallins’,” Journal of Cell Science, vol. 112, pp. 613-622,1999.[4] S. K. Masur, J. K. Cheung, and S. Antohi.  “Identification ofintegrins in cultured corneal fibroblasts and in isolatedkeratocytes,” Investigative Ophthalmology and Visual Science,vol. 34, pp. 2690-8, 1993.[5] S. K. Masur, R. J.  Conors, Jr, J. K. Cheung, and S. Antohi.1999.  “Matrix adhesion characteristics of cornealmyofibroblasts,” Investigative Ophthalmology and VisualScience, vol. 40, pp. 904-910, 1999.[6] J. L. M. Leunissen, and J. R. De Mey, Immuno-Gold Labeling inCell Biology (Verklij, AJ, and Leunissen, JLM, Eds.), BocaRaton, FL: CRC Press, 1989.[7] J. Roth, Techniques in Immunochemistry (Bullock, GR, andPetrusz, P, Eds.), Vol. 2, New York, NY: Academic Press, 1983.[8] K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R.Lotan, and R. Richards-Kortum, “Real-Time Optical Imaging ofPrecancer Using Anti-Epidermal Growth Factor ReceptorAntibodies Conjugated to Gold Nanoparticles,” CancerResearch, vol. 63, pp. 1999-2004, 2003.[9] F. Frens, “Controlled nucleation for the regulation of the particlesize in monodisperse gold suspensions,” Nature PhysicalScience, vol. 241, pp. 20-22, 1973.[10] E. J. Orwin, and A. Hubel, “In vitro culture characteristics ofcorneal epithelial, endothelial, and keratocytes in a nativecollagen matrix,” Tissue Engineering, vol. 6(4), pp. 307-319,2000.Authorized licensed use limited to: to the Claremont Colleges!. Downloaded on June 16, 2009 at 16:47 from IEEE Xplore.  Restrictions apply.1213

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Immunogold Labeling to Enhance Contrast in Optical Coherence Microscopy of Tissue Engineered Corneal Constructs

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