Biomedical Engineering Reference
In-Depth Information
labeling antibodies with extrinsic fluorescent agents alter the strength and specificity of the
protein-ligand interactions [36], but techniques such as flow cytometry can also negatively
impact cell viability and alter the differentiation potential of the sorted stem cells [37]. As
such, significant effort has been invested in developing microscopy-based methods with
which to achieve the real-time and label-free screening of stem cell self-renewal and
differentiation.
To this end, Lo and co-workers have demonstrated that oblique-incidence reflectivity
difference (OI-RD) microscopy is an effective method for the label-free, real-time detection
of cell-surface markers and applied this technique to analyze the presence of stage-specific
embryonic antigen 1 (SSEA1), a well-established stem-cell marker, on mouse ESCs and
mouse induced pluripotent stem cells (iPSCs) [38]. Specifically, Lo
et al
. immobilized
mouse stem cells and non-stem cell controls (A19 fibroblasts and HEK293T cells) onto a
glass surface as a type of “cell microarray.” Afterwards, the cell microarray was reacted
with unlabeled SSEA1 antibodies and the status of SSEA1 antibody binding on the cell
surface was monitored with OI-RD microscopy (Figure 19.7). Oblique-incidence reflectiv-
ity difference microscopy utilizes a p-polarized He-Ne laser beam at λ = 633 nm that passes
through a photoelastic modular so that the output beam is polarization modulated at
Ω = 50 kHz [39]. This polarized laser beam, with adjustable phase, is reflected off the glass
slide surface containing cells, and when the surface is exposed to a probe solution, the
binding event that occurs between the antigen and its substrate results in a magnitude and
phase shift of the electromagnetic field that is associated with the reflected light [40]. Using
this method, binding curves were obtained for individual pixels and used to determine the
K
d
. By obtaining binding curves for undifferentiated mESCs and miPSCs as well as differ-
entiated mESCs and mIPSCs (Figure 19.8), it was determined that the
K
d
is lower for dif-
ferentiated cells when compared to undifferentiated cells. As higher
K
d
would correspond
with lower binding affinities, and considering the fact that the number of SSEA1 surface
markers should be higher on undifferentiated cells than differentiated cells, the observation
that SSEA1 antibody shows higher binding affinity with differentiated cells is surprising.
However, the authors explain that this can be caused by differences in the presentation of
the SSEA1 surface marker at different stages of differentiation. In particular, given that the
Ol-RD
illumination
Ol-RD
detection
Laser (
λ
)
PEM
PD
PS
A
OBJ
FTL
Figure 19.7
Sketch of an oblique-incidence reflectivity difference (OI-RD) scanning optical
microscope consisting of illumination and detection optics and a sample cartridge that holds a
functionalized glass slide and a fluidic inlet/outlet assembly for each of eight chambers [38]. By
scanning a focused optical beam along the
y
axis and moving the sample-holding stage along the
x
axis, the scanner detects real-time changes on the microarray as a result of reaction or other processing
by measuring the amplitude and phase changes of the reflected beam. PEM, photoelatic modulator;
PS, phase shifter; FTL,
f
-theta lens; OBJ, objective; A, analyzer; PD, photodiode. Reproduced from
Lo
et al
., 2011 with permission from BioTechniques.
