Leica SP5 issues, HyD, TIRF

[George McNamara]

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Dear confocal listserv,
I wrote this fopr Vitaly but figured could be useful to others here.
enjoy,
George
****

Hi Vitaly,

I have two Leica SP5's - one has a Coherent Chameleon Ultra II MP laser
as well. I encourage checking our the most recent paper from our Leica
MP/SP5 - Abdulreda et al 2011 PNAS ... PubMed 21768391 ... go make some
popcorn and check out the movies ... if you make a really big batch,
check out Tomasz Zal's lab movies from PubMed 22327568 (Tomasz is at
MDACC, and has a nice setup with two MP lasers plus a very dedicated
first author to image the same spot twice a day for a month).

Current Leica confocal tips:

1. Confocal hardware:

    a. have each instrument on its own UPS.

    b. I leave the microscope and PC on 24/7.

    c. I leave the lasers on 24/5 ... or 24/7 if I have weekend users.

    d. I ask that users exit LAS AF at the end of their session, and
after waiting 30 seconds (because LAS AF does a few things after the
user interface closes,) then turn OFF the SP5 scanhead power. Only
exception is if next user is waiting.

    e. I require that every user switch the microscope to the 5x lens
(adjacent positions are 10x lens and empty) at the beginning and end of
their session - needed to protect the lenses, XY motorized stage and
galvo Z from the next user when the LAS AF software initializes the
motorized stage (memo to Leica: should not need to home the stage every
session).

2. Windows stuff:

     a. I leave the computer (windows XP) running 24/7, logged in as the
"generic user" (TCS User, no password). I find XP behaves better with
one login than each user having their own login (downside is that anyone
can sit down and mess with the PC - the building and floor has fair
security measures and if I found anyone in the room - and especially
touching the instrument or PC - who was not authorized, I would likely
call Security, have their campus ID confiscated, have them banned from
campus and termination initiated).

     b. Windows XP is pretty lame - after a year or so the file system
becomes slow. Simplest approach is to create a new (generic, no
password) account and after making sure the instrument is working
correctly, purge the old account (make sure all user data was saved to a
"data" drive, not lazily in the user path).

    c. All users are required to save to D:\Users\labname path, not to
C: ... I consider C: as being for the operating system only. Never let
C: get anywhere close to full.

    d. The PC noticeably slows down when the data drive (D:) gets close
to full. I backup old data (currently to USB2 portable drives since my
University has not been able to get me enough server disk space so far),
then purge all the old LIF, TIFF and JPEG files ... my policy is users
need to copy their data to a "shared" server drive and then move
immediately to their own space when they get back to their lab, so
deleting 1+ month old data from the confocal PC is not an issue
(frequently used settings can and should be saved through the
"sequential" scan dialog ... not that all settings are saved, i.e. high
res scanner should be run at 600 Hz not at the default 400 Hz when at 1x
zoom).

e. I have SmartDefrag on the confocal PC's
(http://download.cnet.com/Smart-Defrag/3000-2094_4-10759533.html )  and
defrag the hard drives weekly (automatically).

f. I have also installed an OCZ Agility SSD drive (SATA connector to the
motherboard) and an OCZ RevoDrive SSD card (PCI Express x4 slot) - about
twice as fast as the HP xw8200 PC hard drives (note: do not defrag
SSD's). I have the Windows Pagefile(s) on the SSD (not sure how much it
really helps). I do explain to users that they can "save to SSDs at
their own risk" - not that I can promise the LIF file will save
successfully to either the hard drive or SSDs, but the SSD's are an even
higher risk (they've been working fine for a year+ so far).

g. Leica now offers new (64-bit operating system) PC ... consider
investing in it.

3. Users need to monitor their LIF file size(s) and should not have more
than 1 Gigabyte in RAM in Leica LAS AF at any time (even better would be
to close the LIF file when it reaches 500 Mb). I had one user acquire a
3.5 Gb Z/Tiles can and then stitch the dataset, for another 3.5 Gb. This
took a long time to save and copy to network space (and eventually to
open on their PC). They would have been a lot better off paying
attention to my training about sensible acquisition settings.

Your issues:

LAS AF Acquisition software issues:

* Could be Windows XP issues - see above (clear off hard drive space,
defrag hard drives regularly, consider getting a solid state drive for
data and/or page file[s]). Do not let users save data on C: drive
(especially not in the "User" folder).

* If you have less than 4 Gb ram, max out the computer RAM that XP can
take advantage of (and make sure good quality RAM is used). I usually
buy RAM from www.crucial.com though might be safer to order the
"official" ram from HP. RAM is cheap - order identical RAM chips to get
to 4 Gb.

* If you are on an old version - talk to your Leica confocal service
engineer about the version they find best (several old versions are
available at ftp://ftp.llt.de/softlib/  but the service engineer should
handle the installation ... you might want to back up the PC before they
visit!).

HyD's:

* I had excellent experiences with the HyD's on the Leica STED/SP5 demo
machines last September at UM and in June 2011 at the Yale Microscopy
Workshop. While it is possible you have a "lemon" detector, more likely
is that your detectors are being mis-used: essential to keep the
excitation light level low. The HyD's are photon counting detectors -
more than one photon collected just causes problems.

* I noticed at the demo'd that the HyD detectors are "on" all the time -
even if they are not checked on (also seems to be true of my FLIM
detectors on the MP/SP5/FCS/FLIM system). This means it is essential
that every user make sure that the HyD detector emission bandpass
settings does not overlap any active laser line - I recommend being at
least 10 nm from any laser line.  Also good practice not to zap any PMT
with too much laser light (especially Ti:Sapphire!).

* More on HyD safety - the SP5 emission bandpass values assume pinhole
is set to 1.0 Airy unit. If the pinhole is open, more light gets through
- probably should require all users operate the SP5 only at pinhole 1 AU
(or use the standard PMT 3 only, and have the HyD detector bandpasses
far from laser lines).

* If your instrument is under service contract, you should be able to
arrange for Leica service to ship in a replacement HyD(s) and compare it
(them) to your current HyD detector(s). As to whether the new ones stay
is up to your service engineer - not you (if the replacement is clearly
superior, they would leave it on your instrument ... if equivalent, they
would probably switch back).  You should come up with test specimens
that you can use to carefully evaluate performance of your old vs the
new HyD. The service engineer might test using the official Leica test
specimen of Convallaria cross section, which would probably look ok even
with a lemon HyD (this is not just the Leica official test target -
Zeiss uses the same kind of slide).

* If you are not under service contract, getting back under service
contract should be a lot higher spending priority than a white light laser!

Leica White light laser:

* No personal experience.

* My (Florida based) Leica confocal salesperson, Charles Hemphill, says
the WLL is great - very versatile.

* I do not know what the annual service contract rate is on the WLL vs
standard lasers - be sure to crunch the numbers as to whether the
cost:benefit ratio makes sense.

* My SP5's have 405, 458/476/488/496/514 Argon, 561m 594 and 633 nm
laser lines (and AOBS). I can excite pretty much any typical fluorophore
with one of these lines (I essentially never both with 496 nm line -
weak compared to 488 and 514 nm). Most of my users are just using some
or all of 405, 488

Leica TIRF or alternatives:

* I do not have Leica TIRF.

* I saw the Leica TIRF at the Yale Microscopy Workshop June 2011 - part
of their GSD nanoscope setup. Their special objective lens holder for
GSD was very cool. Hopefully by now they have taken GSD to 3D (hint to
Leica: double helix poiint spread function license).

* If you are thinking of purchasing a Leica TIRF, consider doing so as
part of an "upgrade SP5 to SP8, get a TIRF or TIRF/GSD as part of
purchase price". Talk with your salesperson.

* Plan V: if Leica is not enthusiastic about giving you a nice price for
SP5 -> SP8 + TIRF/GSD, consider purchasing a Vutara SR-200 single
molecule localization nanoscope instead of Leica TIRF. Contact Vutara,
get names of their customers, and call the customers to find out how
well their Vutara instruments are working - Vutara had a nice demo setup
at the June 2011 Yale microscopy workshop. TIRF is limited to ~100 nm
axial resolution and only at the coverglass, the Vutara SML (and Nikon
N-STORM) are rated at ~50 nm axial precision localization and several
microns focus range (last time I checked, I believe both Leica GSD and
Zeiss ELYRA-PALm are TIRF only?).

* Plan T: check out http://www.tirftechnologies.com/  - Alex Asanov has
developed a TIRF illuminator / platform that can go on ANY microscope
and does not require buying a 1.45+ NA objective lens. Can sell you LED
and/or lasers for illumination for his system.

George

p.s. I'll probably post this message to the confocal listserv tonight.

George McNamara, Ph.D.

Image Core Manager

Analytical Imaging Core Facility (AICF), DRI 6025

*University of Miami*, Miller School of Medicine

Diabetes Research Institute, John P. Hussman* *Institute for Human
Genomics,

Interdisciplinary Stem Cell Institute, and UM/Sylvester Comprehensive
Cancer Center

*From:* Vitaly Boyko
*Sent:* Monday, August 13, 2012 10:29 AM
*To:* McNamara, George
*Subject:* Leica

Hi George,

  We are looking into SP5 upgrade with the white light laser.

I assume you have lots of stuff from Leica.

We have two SP5's, but having lots of issues with the instability of
acquisition software and HyDs.

Would you have any experience and/or detailed specs for the Leica white
light laser. Do you also have Leica's TIRFM?

With best regards,

Vitaly

www.mskcc.org <http://www.mskcc.org>