Re: A1

[GeneMaverick]

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Dear Heiko Düssmann, Michael Mancini, Simon Watkins, Stephen Cody and other
confocal aficionados,

In additions to the comments given by Mancini, Simon and Steve, I would like
to express some of my inputs related to the newly introduced A1 Confocal
system. Though I am working for Nikon, I am expressing my views as a fellow
researcher.

I presume that Nikon has designed this system to cater the upcoming needs of
live cell imaging and molecular interaction analysis. As the molecular
biological processes are happening in vivo at nano/micro second levels, we
require higher speed without compromising the resolution and generating
unwanted artifacts and stray noise interference. Due to high optical
efficiency and 16 million pixels resolution, high quality confocal images
can be achieved that will bring inter/intra cellular nuance into the
limelight.  

Nikon claims that the most rapid biological events can be seen in ultra high
resolution with the new system that is the evolved version of the present
real time spectral confocal system C1si that has so many unique features
like diffraction efficiency enhancement system based multiple gratings
(DEES), weak signal sensitivity through dual integration signal processing
(DISP), pre-calibrated synchronized 32 channel multi-anode PMTs,
high-efficiency fluorescence transmission technology to achieve high optical
transmission and most importantly the faster spectral unmixing algorithm
that enables high speed spectral images without any molecular crosstalk in
real time.

The A1 system has standard paired galvonometers that gives high resolution
images up to 4K x 4K pixels, whereas A1R incorporates two independent galvo
systems – high speed resonant and high resolution non-resonant hybrid
scanner system, offering the speed of 30 frames per second at 512 x 512
pixels. The resonant scanner is mounted along with the non-resonant scanner
gives industry’s fastest 230 fps at 512 x 64 pixels and facilitates
ultra-high-speed imaging with out compromising image quality.

Scanning can be done by three modes through using the resonant scanner alone
for high speed imaging up to 230 fps, using the non-resonant scanner along
for high resolution imaging up to 4K x 4K pixels and by combining both
resonant scanner and non-resonant scanner for simultaneous photo-stimulation
imaging. This mechanism enables simultaneous photo-activation and imaging
with improved sensitivity and reduced photo-toxicity that is vital for most
of the sensitive functional cell dynamics applications.

The hybrid scanning system also enables high speed imaging up to 420fps
(2.4ms/frame) at 512 x 32 pixels. This supports advanced live cell imaging
works more efficiently. The system comes with the analysis software for FRAP
and FRET as standard.    

The newly fully automated standard fluorescence detector with 4 PMTs enables
to acquire 4 color images simultaneously. This detector unit has changeable
filters, enabling simple onsite installation of emission filter and mirror
sets. In combination with four lasers, simultaneous observation of four
different fluorescence labels is possible.

When compare to C1si confocal system, the spectral detection performance
with A1 is enhanced further along with V-filtering function that expands the
range of use of spectral images. Through V-filtering function, up to four
preferred spectral ranges can be chosen from 32 channels and the intensity
of each range can be adjusted independently and this allows selection of
desired spectral range and flexibility to handle new fluorescence probes.

Together with the high speed AD conversion circuit, the new signal
processing technology allows simultaneous 32 channel spectral image
acquisition at 512 x 512 pixels in 0.5 seconds. At 512 x 64 pixels, images
can be acquired with the speed of 16 frames per second.

The industry’s first low incidence 12 degree angle dichroic mirror enhances
30% more fluorescence efficiency and 99% transmission in combination with
high performance sputtering as the reflection-transmission characteristics
have lower polarization dependence, when compare to conventional incidence
angle (45 degree) method, where reflection-transmission characteristics have
high polarization dependence.
 
Ideally speaking, the pinhole shape should be circular. With the A1 system,
the continuously variable hexagonal pinhole, which replaces the standard
four-sided aperture, considerably sharpen the image quality and it allows
30% more light resulting brighter images with the same sectioning
performance as the area of hexagon inscribing a circle is bigger than the
area of square inscribing to the same circle. With tthis hexagonal pinhole
design, maximum confocality is maintained while achieving higher brightness
equivalent to that of an ideal circular pinhole.

Virtual Adaptable Aperture System (VAAS) pinhole unit provides better images
with less flare as the light that a confocal pinhole rejects is collected by
another detector. It allows simulation of different sectionings and slice
thicknesses after image acquisition and has a better control over the
attainment of experimental data. This unique detector system allows virtual
adjustment of the confocality and sensitivity by collecting more photons
during the initial image acquisition to generate a high resolution image.
VAAS detection is an upgrade option that is expected in October 2008.

Two laser input ports are incorporated in the scan head to use various laser
lines.  4 laser unit is used as a standard and 3 laser board can be added as
an option. Hence, 7 lasers with 9 lines are available in maximum.  In
addition, it also has IR laser port as an option.

Along with the newly introduced Ti-E inverted microscope, A1/A1R confocal
system set a new standard for advanced time-lapse studies of rapid cellular
interactions to bring biological imaging to life. However, to understand
this system and the over all performance we need to wait further as the
optional spectral detector is yet to be launched in April 2008. In addition,
the VAAS system, which is again an optional up grade, will be available only
in October 2008.

Gene Maverick.