Figure 1 - M3833 processor.
Complete multimodal imaging for applications in chemiluminescence, fluorescence, and brightfield imaging
is achieved with the LAS-3000 image analysis
system (Fujifilm Life Science USA, Stamford, CT).
At the core of the system is Super CCD technology1
(Fujifilm). The octagonal-shaped pixels in the M3833
processor (Fujifilm) (Figure 1) are situated on a 45°
angle, creating a pattern similar to the layout of the
optical sensing neuron of the human eye. This results
in increased sensitivity, improved signal-to-noise, and
a much wider dynamic range. The 3.2-M pixel charge-coupled
device (CCD) (1536 × 2048) is laid out on a
1-in. chip, resulting in well sizes capable of producing
true 16-bit depth image data. This standard resolution
can be increased to 6.3 M pixels (3072 × 2048) using
proprietary interpolation algorithms.
The LAS-3000 uses a two-stage Peltier system
with an additional fan to cool the Super CCD
to –30 °C, and provides ultrahigh binning states
to maximize sensitivity. The CCD coupled to a
F0.85 Fujinon lens (Fujifilm) makes the image
analysis system one of the fastest for chemiluminescence
Figure 2 - Standard architecture of a) conventional CCD, b) Super CCD, and
c) interpolated pixel resolution.
While a conventional CCD has rectangular pixels
arranged in columns and rows (Figure 2a), the
Super CCD has octagonal pixels approximately
10.75 μm × 10.75 μm in a honeycomb configuration
(Figure 2b). By rotating the pixels 45° to form
this interwoven layout, the CCD’s pixel pitch in
the horizontal and vertical directions is narrower
than in the diagonal direction. This gives a larger
relative area of the pixels per total size of the CCD
than possible with the conventional CCD structure,
and contributes to the high sensitivity and
high resolution attainable with the Super CCD.
In the high-resolution mode, virtual pixels are
created within the spatially interleaved real pixels
(Figure 2c) to effectively double the output and
provide high resolving capability.
Under normal CCD operating conditions, noise
arises as a result of both thermal and electronic
factors. The thermal noise is reduced
by cooling the CCD to –30 °C. The
residual thermal contribution, along
with the contribution of electronic
noise (which results from moving
charges around on the CCD), become
a function of the exposure time. In
the LAS-3000 system, this residual
noise inherent in the Super CCD is
corrected for by taking a series of dark
frame shots with the camera shutter
closed. Short exposures (1/100, 1/60,
1/30, 1/15, 1/8, 1/4, 1/2, 1, 2, 3, 4, 5,
6, 7, and 8 sec) are applied directly,
while the average of 16 × 9 sec is used
for exposure from 9 sec to 2 hr. A single
2-hr dark frame is used for exposure
times over 2 hr. Dark frames are
subtracted to perform the correction.
Although only the highest-quality scientific-grade Super CCDs are used to
build LAS-3000 systems, some pixels
will exhibit hot (always on) and cold
(always off) behavior. These pixels are
identified and their data modified using
nearest neighbor algorithms to correct
for this. Finally, additional lens and
flat frame corrections are performed
within the system to address radial
variability generated by the lens and
the nonuniformity of the illumination fields used
in certain applications.
Binning and image processing
Binning is a method of combining several pixels
into one large pixel when reading out the electrons
accumulated on the CCD after exposure.
The light receiving area of a combined pixel
increases to enhance sensitivity. The LAS-3000
offers standard binning (1 × 2 pixels), high binning
(2 × 4 pixels), super binning (4 × 8 pixels),
and ultra binning (8 × 16 pixels) modes. In the
ultra mode, the exposure time is nearly 60 times
faster than in the standard mode.
Figure 3 - Resolution for individual binning states with and without smoothing
While binning can dramatically reduce exposure
times and increase sensitivity, the downside is that
is does so at the expense of resolution (see Figure 3).
Thus, images become increasingly more pixilated
as the binning is increased. The LAS-3000 is still
able to produce visually palatable images, even in
the ultra binning mode, using a smoothing feature
that converts high, super, and ultra binning images
to the same pixel size and number as the standard
image. This result can be visualized in either the
binned or smooth formats, and the smoothing function
does not impact the analytical accuracy of the
Figure 4 - Quantum efficiency curve for the M3833 processor.
All CCDs display variable efficiency in transducing
photons of different wavelengths. A plot of this spectral response is known as a quantum
efficiency curve. A representative curve for the
M3833 processor is shown in Figure 4. In general,
the highest efficiency is realized in the
visible light range between 400 nm and 700 nm,
while poor response is observed with photons in
the UV or IR ranges. Peak response for the processor
is observed in the green range, optimizing
the LAS-3000 for chemiluminescent applications
using either alkaline phosphatase (i.e.,
CDP-Star, PerkinElmer, Boston, MA) or any of
the newer green shifted substrates for horseradish
peroxidase (i.e., SuperSignal, Pierce, Rockford,
IL). This property of the CCD also makes
the image analysis system well suited for fluorescence
applications using signals from the blue
into the near-IR.
Finally, one of the biggest advantages of the Super CCD
over film and other video systems is the large dynamic
range. Consistent measurement of system dynamic
range has been problematic due to a lack of appropriate
standards. Glowells™ (LUX Biotechnology, Edinburgh,
U.K.) is a product that makes evaluation and
ongoing system validation simple and accurate. Glowells
are certified reference standards for calibration of
optical equipment such as luminometers, microplate
readers, and CCD-based bioimaging systems. The light
output of the standard is generated using 70 milli-becquerel
(MBq) of gaseous tritium per unit and is calibrated
and traceable to National Physical Laboratory (NPL, Middlesex, U.K.) standards. With a half-life of 12.3 years, the standards remain virtually unchanged
over the instrument lifetime. The Glowell low light
imaging standard (LLIS) delivers light peaks in the blue
(450 nm), green (525 nm), and red (650 nm), ranging
over five orders of magnitude. Yellow (555 nm) and
custom light outputs are also available upon request.
Figure 5 - Glowell standard data for LAS-3000 standard, high, super, and ultra binning states.
As shown in Figure 5, the M3833 processor in the
LAS-3000 system is capable of delivering over
four orders of magnitude of linear dynamic range
in all four of the binning modes available. For this,
analysis exposure times were adjusted to ensure
that no regions reached saturation (i.e., intensity
beyond 65,000 AUs). Even in the standard (slowest)
binning mode, the exposure time to achieve
four of the five light units did not exceed 5 sec.
This performance almost doubles the typical 2.5
orders of linear dynamic range attainable with
most film systems. The expanded dynamic range
along with true 16-bit well depth greatly improves
analytical accuracy, particularly when comparing
intensities that are closely matched or are at the
extremes of detection. In conclusion, the architecture
of Super CCD, coupled with the optimized
design of the LAS-3000 system, offer significant
benefits to the researcher.
Yamada, T.; Ikeda, K.; Yong-Gwan, K.; Wakoh, H.;
Toma, T.; Sakamoto, T.; Ogawa, K.; Okamoto, E.;
Masukane, K.; Oda, K.; Masafumi, I. A progressive
scan CCD image sensor for DSC applications. IEEE J.
Solid State Cir. 2000, 35, 2044–54.
Dr. Pizzonia is Director of Applications, Life Sciences Group,
Fujifilm Life Science USA, 419 West Ave., Stamford, CT
06902, U.S.A.; tel.: 800-446-5450, ext. 6259; fax: 203-363-3879; e-mail: email@example.com.