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