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COMMERCIAL DISCLAIMER: PerkinElmer is supplier of UltraVIEW LCI confocal
system based on Microlens Disk Scanning technology (Yokogawa)

Dear All,

Thanks everyone contributed to discussion on live cell imaging confocal based
on Microlens Disk Scanning technology.

As a person behind UltraVIEW in PerkinElmer, I feel invited to share my view
of the technology as unbiased as I could be.

The aim of UltraVIEW is to be an ultimate solution for range of applications,
not the ultimate optical solution. Applications, which we have targeted in
particular, are dynamic processes in 3D (live cells and tissues).

With number of users now counted in hundreds, I dare to say that Microlens
Disk Scanning technology is the first alternative technology that
successfully challenged classical single beam confocals. This technology has
effectively again opened a door for confocals based on spatial filters
(Nipkow, raster, grid, micro-mirrors, etc.) which now will inevitable make an
attempt to invade the confocal space.
The result is going to be more choice for users, which is certainly good. But
also the right decision will be likely more difficult to make, as
technologies will significantly differ in performance. In any case, few
general requirements for any confocal application can be identified and
alternative technical solutions scored in respect to them.

Looking into our targeted applications we concluded that if to capture
dynamic 3D processes in living sample one will need:
A) good X-Y resolution
B) good Z resolution/penetration
C) good speed in multi-D (z, wavelengths, time)
D) as little as possible damage to dyes or live samples.
Anybody with background in confocal microscopy will recognize this as the
worst-case scenario and any equipment is most likely to be tested to the
limits.

So, how technology in question (UltraVIEW) stands with respect to four
requirements? Paragraphs below are not to provide UltraVIEW "features and
benefits" but to touch some less visible information and misconceptions.

A) X-Y resolution
With lens of higher magnification, UltraVIEW X-Y resolution is as good as
with any other confocal. With 100x lens and 7 micron pixel size it
oversamples optical resolution above the Nyquest. With x20 lens you are not
able to resolve 0.2 micron objects, but we have not seen a strong demand for
that feature. Interestingly, many people when first time faced with the
technology ask why images looks better than with classical confocal? Some of
them even jumped into conclusion that UltraVIEW resolution is better that
classical confocals. This is unfortunately not true. UltraVIEW, as any other
confocal system, is ultimately limited by the wavelength-dependent optical
resolution. The advantage of UltraVIEW is in more effective control over the
S/N. This is due to 3 factors: a) CCD outperforms PMT in S/N, if later is not
used in photon counting mode. b) effective rapid averaging (up to
1000/second) c) more effective control over total dwell time per pixel. To
explain the last, the total dwell time in UltraVIEW is a sum of chosen number
of repetitive excitations (if you expose for 10 seconds, it will sum up to 10
000 of excitations each obviously having a dwell time related to the pixel
size).
All 3 factors contribute to the control over S/N and can improve contrast of
the images and resolving power of the system. For bright samples this is of
little importance, but for weak samples one can benefit significantly.
B) Z-resolution/penetration
Despite an attempt to question confocality of the Microlens Disk technology,
the truth is that for the range of samples you will have difficulty to notice
any the difference. However, one needs to accept that pinhole size is
optimized for higher magnification objectives and that at x10 magnification
it will not show the full confocal capacity.
I also want to leave no doubts for which type of samples Microlens Disk
technology will not be ideally suited. If you are to look into a big, solid,
bright, chunk of fluorescence (for example, several tenths of microns)
excitation peeks from adjacent pinholes will start shoving an effect and a
hallow effect will become apparent. Such a sample with a low magnification
lens has been used to question confocality of the technology, but somehow we
do not see very often such samples in real live cell applications.
Another misconception related to the Microlens Disk technology, is in
technology being limited in depth of penetration. Effectively for variety of
samples depth of penetration is very good (often better!). This is no direct
relation to the confocality, but to the fact that in deep sections, you are
more often fighting the battle of effective extraction/detection of photons
than ability to get sub-micron z-resolution. This is where UltraVIEW will
gain an advantage due to better control over S/N, and result can be truly
surprising!
In conclusion, for fine, sparse, structures in 3D, regardless of depth of
penetration, you should try to test UltraVIEW.
C) Speed
The result of Nipkow disk scanner is an effective array of parallel confocals
(in UltraVIEW about ~1000). Advantage of parallel information acquisition
over sequential is obvious.
Regardless of technical specification of fast cameras or scanning speed of
Nipkow disk, the real limits are in level of fluorescent signal you can
detect and how the detector, scanner and wavelength and Z control are tuned
to work together. If one is to compare UltraVIEW with a widefield camera
based system the reality is that after confocal discrimination the total
level of light will be less and the ultimate camera speed can seldom be
utilised. Currently, realistic speed (decent frame size/decent resolution,
depending on camera type) is video rate. With some improvements which will be
available shortly, double the video rate can be expected, but for more, you
probably need an intensifier, which, as it was pointed in discussion, will
compromise resolution, dynamic range and image quality. However, there are
advancements in camera technology (Jim mentioned one) to be expected shortly
and technology can immediately benefit from those.
D) Bleaching
I trust that by now, large part of the community has realized that Nipkow
disk bleaches less than single beam confocal, although nobody really knows
why.
It is clear that the effect is not related to the total power of excitation
which sample receives over time, but the way excitation is administrated to
the sample.
If one measure the total power of the excitation at the objective end, the
power will be in the similar range as for classical confocals, but what that
measurement really represents is a sum of ~1000 microbeams. So what is really
different?
Looking from the perspective of the molecule that is excited, in one
technology (Microlens Disk) molecule experience brief repetitive excitation
(up to 1000 times per second). Alternatively, in single beam scanner,
molecule is excited maybe few times per second with a full beam power.
Difference in bleaching of 1000 repetitive excitations, which in power equal
to one single excitation, suggests non-linear phenomenon in photobleaching.
I have discussed this on numerous occasion and response varied from "it is
nonsense, everyone knows that bleaching is linear in respect to the power of
excitation" to " everyone knows that, similar was shown for 2 photon
excitation as well". Any comments, suggestions are more than welcomed.

In summary, for applications orientated technical solutions we have found
that more critical is to successfully address the combination of requirements
than to perfect a single one. This is why mixing arguments for ultimate
optical solution and ultimate solution for range of applications can lead to
different conclusion.

I hope you have found my message interesting and informative.

Kind regards,

Fedja Bobanovic
Global Product Manager
PerkinElmer Life and Analytical Sciences
www.liveCELLimaging.com