Biomedical Engineering Reference
In-Depth Information
lasers. Without simultaneous acquisition, dynamic changes can take place in a biological
sample between frames, which introduces registration errors, and image throughput is
lowered, an important parameter when imaging cell dynamics.
In order to simultaneously capture both fluorescence and quantitative phase images, we
used a Bayer-mosaic-pattern color CCD to separate fluorescence and phase information by
color channel. While there is some crosstalk between color channels (
B
5%, see earlier), the
illumination wavelengths and fluorophores can be carefully chosen to minimize these
effects. The system as shown in
Figure 14.8
consists of a transmission-geometry QPM
illuminated by a 532 nm laser source. Two additional laser sources at 632.8 and 376 nm are
incorporated at the unused port of the recombining beamsplitter and illuminate the entire
field of view on the sample after passing through the condenser lens and sample arm MO.
Fluorescence emission is then imaged using the same optical pathway as the transmitted
QPM light onto the CCD by the MO and tube lens. Excitation light from the 376 and
632.8 nm sources is suppressed by a band-pass filter that transmits all other visible light
(including the QPM illumination).
In our experiments, we examined rat ventricular cardiomyocytes and recorded their
contractions at 5 fps. The sample chamber was filled with growth media to keep the cells
alive and topped by a coverslip to reduce phase noise from media surface fluctuations. The
cell nuclei were labeled with Hoescht 34580 dye (Invitrogen, Carlsbad, CA, USA), which
was excited by the 376 nm laser and had an emission peak at 440 nm, which is closely
BS
λ
= 532 nm
Sample
MO
BPF
MO
CCD
BS
Tube lens
Condenser lens
λ
= 376 nm
DF1
λ
= 632.8 nm
Figure 14.8
Quantitative phase and fluorescence microscope using a color camera to multiplex the imaging
modalities. Source: Adapted from Ref.
[24]
.
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