The same is with the Nikon A1R, slower scan speeds result in
more intense images. Which makes sense to me.
I have worked with a Zeiss LSM510 for nearly 8 years and I have noticed
that there is probably a compensation factor applied to the images as an
attempt to keep the mean intensity constant. The result are weird effects on
intensity upon changes in scanning speed.
I have never been happy with that. I rather prefer the more
intuitive behavior of the Olympus and Nikon systems.
Leoncio
From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Andreas
Bruckbauer
Sent: Monday, May 10, 2010 3:46 PM
To: [hidden email]
Subject: Re: Comparison Zeiss 710 vs Nikon A1 ... full comparison
requires matching resolution and scan speed
On the Olympus FV1000 the signal varies (intentionally) with the
scan speed, longer dwell time = more photons. Zeiss seems to give out something
like counting rates which are then the same for different dwell times. It would
be good if the manufacturers give more details.
best wishes
Andreas
-----Original Message-----
From: George McNamara <[hidden email]>
To: [hidden email]
Sent: Sun, 9 May 2010 14:58
Subject: Re: Comparison Zeiss 710 vs Nikon A1 ... full comparison requires
matching resolution and scan speed
Hi listserv,
Repeating what I posted in a message yesterday:
I've noticed on both the
Leica SP5, Zeiss LSM510 and Zeiss LSM710 that "faster is brighter"
for many fluorophores. That is, a 0.4 us dwell time results in a brighter image
than a 4 us which is brighter than a 40 us (then repeated 0.4 us, which
resulted in the same brightness as the first 0.4 us dwell time - so not
photobleaching -- not all fluorophores in the specimen [Desmid slide from
Carolina] changed brightness). This implies that <0.4 us might be even
brighter - i.e. resonant scanner mode. A couple of possible explanations (not
mutually exclusive):
a) photophysics (possibly
caused by fluorophore-O2 photochemistry) - re TRex (PubMed 19337661), papers by
Sanden/Spielmann/Widergren et al (PubMed 20375039, 20196585, 19007245,
17385841).
b) calibration method(s) by
Zeiss and/or Leica that try to (but do not always) match output at all
settings.
c) other???
(suggestions/comments welcome!).
See also
high speed scanning has the potential to increase fluorescence
yield and to reduce photobleaching. Borlinghaus RT. Microsc Res Tech. 2006
Sep;69(9):689-92.PMID: 16878313
Since I can obtain brighter or dimmer fluorescence on the same
field of view on the same microscope system by simply changing scan speed, I
submit that between microscope comparisons are just anecdotes unless every
variable is controlled and reported. Even then there may be variables under the
hood that the confocal manufacturer has not mentioned, such as the possibility
that the system is "calibrated" to all scan speeds output more or
less the same intensity.
Comments with respect to the post below...
At 03:43 PM 4/11/2010, you wrote:
Hi Pedro,
Seems you have short listed A1 and 710. Both the systems are released almost
the same time. We have little experience in handling the 710 system but
sufficiently exposed with A1. I have mentioned my opinion and observation about
these two systems earlier that is given below.
A1 gives 4X4K image size, whereas 710 gives 6X6K. But do we really need 6KX6K?
When we tried taking 6K images, we have experienced photo bleaching in some
attempts.
GM: With a 100x1.4+ NA objective lens at the LSM710's 0.6x zoom (and perfect
specimen, i.e. refractive index matching and/or imaging right at the coverglass
... and coverglass 170.0 um)
XY resolution = 0.6 * 405 nm (the laser line) / 1.4 = 173 nm (assuming pinhole
1.0 Airy unit, infinitely bright specimen could put sqrt(2) in the
denominator).
Nyquist sampling for a 2D image; 173 nm / 3 = 57 nm.
(I'm not at the LSM710 so don't have the actual image field of view, but this
should be close):
For 100x lens and 0.6x zoom, field of view should be about 160 um, so
160 um / 6000 pixels = 0.026 um = 26 nm.
So, 6kx6k is oversampling by about a factor of two. For a very stable specimen
(photostability and vibration isolation), perfect imaging conditions, lots of
time (since probably will need Z-series for best deconvolution) and optimized
confocal deconvolution algorithm, the 6kx6k setting might be useful. How much
more useful than 4kx4k ... please do the experiment, publish and send me the
reference.
GM Most confocal microscope software have "optimize" XY resolution
buttons - when in doubt, click on it. Incidentally, Paul Goodwin, Applied
Precision, told me Biotium CF405 fluorophore is very good for immunofluorescence
(context: OMX nanoscope). Might be a good starting point for an experiment such
as above. DAPI or Hoechst 33342 or 33358 in a perfect mounting medium could
also be a good choice for the above.
A1 has continuously variable zoom up to 1000X but LSM 710 is
limited to 50X. However, we were not given proper training about how to use
variable zoom up to 1000X. In my view, the 1000X variable zoom is not that
important factor.
Fortunately, in 710 there is a master pinhole unlike the earlier 510 that takes
time for alignment. However, 710 uses conventional rectangular pinhole but A1
has unique hexagonal pinhole resulting better images. We have checked the same
sample in both the systems.
GM: Managing both a 4-pinhole LSM510 and a LSM710, I teach users the same thing
on both for multi-color fluorescence (ex. "colocalization"): for the
longest wavelength emission channel, select 1 Airy button, then match the
pinhole for the other channels. This way the (nominal) optical section
thickness is matched. In multitrack mode the pinhole setting can be changed
between tracks.
The spectral detector of LSM 710 has 34 channels with
simultaneously acquisition unlike the previous model 510 that was with 4x8=32.
However, the spectral step size is 3nm (3x34=102nm wavelength resolution) and
it is not flexible like A1 that has 2.5nm, 6nm and 32nm (resulting 2.5x32=80nm,
6x32=192nm and 10x32= 320nm wavelength resolution). A1 has surprisingly
effective unmixing efficiency even in the close range.
GM: LSM710/ZEN's 32-channel QUASAR detector default is ~10 nm, but is
adjustable (5 and 2.5 nm if I recall). When using the flanking PMTs the
spectral window for these can be adjusted in 1 nm steps - this is done with two
prism/slits - see the LSM710 internals schematic on the Zeiss web site. Going
to 1 nm might be useful to play around with the peaks vs off-peaks for Europium
or Terbium (which have long fluorescence lifetimes so scan speed would need to
be very slow, might be interesting if you have the time to do a spectral scan
overnight).
In LSM710 high wavelength range up to 1100 nm is possible for
optimized transmission. I am not sure if the A1 has the same capability. In our
(little) experience, 710 gives wonderful sensitivity. Their software Zen 2009
is compatible with Vista/Windows 7. Even anisotropy imaging is possible with
LSM 710 (do not know about A1). But you need to purchase this module (extra
cost).
Nikon software is robust has almost all of the regular modules. However, for
Zen we need to pay extra for the add on modules. Some of the regular functions
are "optional" with Zen.
For simultaneous photo activation and imaging, one need to incorporate Duo
system (two heads) into LSM 710 that adds up the cost. However, A1R scan head
serves this purpose without any cumbersome modifications and the speed and
performance is relatively incomparable. Though there are some annoying
"noise" is generated while we use the resonant scanner the speed and
performance is still impressive.
If you are regularly going for live cell imaging and Ca++ imaging, I feel A1
has so many features and it is not bleaching the dyes or induce unexpected
phototoxicity. Though, we found 710 has slightly better sensitivity, we often
face bleaching and laser induced toxicity problems for the same set of experiments.
GM: See top of message - scan speed, resolution, laser power at specimen, need
to be matched to make valid comparison.
Eager to know the inputs of other users.
No commercial interest.
Roshma.
On Sun, Apr 11, 2010 at 4:34 PM, Pedro J Camello <[hidden email]> wrote:
Hi all,
has anybody in the list
compared Nikon A1 vs Zeiss 710. We´re purchasing a
spectral micro with a TIRF
module and motorization. Any input will be
really wellcome (off list if you prefer)
A second question, what is
the most close to "live cell" sample to make
real tests in confocal? I´m
going to travel to test a couple of micros,
and to carry or prepapre real
living cells is rather complicated for us.
We´re especially interested in ion (Ca2+) experiments.
Thanks
--
Dr Pedro J Camello
Dpt Physiology
Faculty of Veterinary
Sciences
University of Extremadura
10071 Caceres
Spain
Ph: 927257000 Extension
51321/51290
Fax:927257110
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF
(Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip
(the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara
| Free forum by Nabble | Edit this page |