Limits of Leica tile scan / navigator modules

[daj1u06]

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Hi all,

I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.

Thanks in advance,
Dave Johnston,
Biomedical Imaging Unit,
University of Southampton.

[Rhonda Powell]

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Hi Dave,

This may not be exactly what you are asking, but the total file size for export of one stitched image is ~2 gb.

I've been able to stitch together an entire slide of ovine placentome in brightfield (one Z plane) in navigator or in stage experiments- these are sometimes 2000 pictures or more, and push ~20 gb. It will image it with no issue, but the only way to export it is individual .TIF (not as the gigantic stitched image).

There is also a checkbox feature on one of our Leica systems for something called "data streaming"- it allows you to save data directly to the hard drive. I haven't checked to see if that is an option on all of our systems, but that might be a solution.

Hope that helps!
Rhonda

Rhonda Reigers Powell
Research Lab Manager
CLEMSON LIGHT IMAGING FACILITY
Clemson University, Division of Research
190 Collings Street
Life Sciences Facility, Room G024/G030
Clemson SC 29634
P: 864-656-1264
clemson.edu/light-imaging

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
Sent: Thursday, November 7, 2019 5:24 AM
To: [hidden email]
Subject: Limits of Leica tile scan / navigator modules

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Hi all,

I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.

Thanks in advance,
Dave Johnston,
Biomedical Imaging Unit,
University of Southampton.

[Cromey, Douglas W - (dcromey)]

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You can get around the 2GB file size limit for TIF export in the Leica LAS software by opening LIF files in FIJI. I took me about a day and a half to sort this out, but here are the instructions I give my local users: http://microscopy.arizona.edu/sites/default/files/sites/default/files/upload/exporting_large_LIF_data_files.pdf

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
Life Sciences North, Room 463
1333 N. Martin Ave, Tucson, AZ  85721 USA

office:  LSN 463        email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
Home of: "Microscopy and Imaging Resources on the WWW"

UA Microscopy Alliance - http://microscopy.arizona.edu/

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Rhonda Reigers Powell
Sent: Thursday, November 7, 2019 6:30 AM
To: [hidden email]
Subject: Re: Limits of Leica tile scan / navigator modules

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Hi Dave,

This may not be exactly what you are asking, but the total file size for export of one stitched image is ~2 gb.

I've been able to stitch together an entire slide of ovine placentome in brightfield (one Z plane) in navigator or in stage experiments- these are sometimes 2000 pictures or more, and push ~20 gb. It will image it with no issue, but the only way to export it is individual .TIF (not as the gigantic stitched image).

There is also a checkbox feature on one of our Leica systems for something called "data streaming"- it allows you to save data directly to the hard drive. I haven't checked to see if that is an option on all of our systems, but that might be a solution.

Hope that helps!
Rhonda

Rhonda Reigers Powell
Research Lab Manager
CLEMSON LIGHT IMAGING FACILITY
Clemson University, Division of Research
190 Collings Street
Life Sciences Facility, Room G024/G030
Clemson SC 29634
P: 864-656-1264
clemson.edu/light-imaging

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
Sent: Thursday, November 7, 2019 5:24 AM
To: [hidden email]
Subject: Limits of Leica tile scan / navigator modules

*****
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Hi all,

I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.

Thanks in advance,
Dave Johnston,
Biomedical Imaging Unit,
University of Southampton.

[0000001ed7f52e4a-dmarc-request]

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As I understand the Leica systems save the individual files to a temp folder on the hard disk during imaging, so you can generate files which are much larger than the computer RAM, I have done 50GB without problem (without stitching). But the tile scan is not yet saved to the *.lif file at the end of the scan, this takes another 20 min on our system. Programs like Imaris, Arivis or Aivia should be able to handle these files and load the required data dynamically. I did the stitching using BigStitcher in Fiji, but this required a lot of RAM. 
best wishes
Andreas

-----Original Message-----
From: Rhonda Reigers Powell <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 7 Nov 2019 13:30
Subject: Re: Limits of Leica tile scan / navigator modules

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Hi Dave,

This may not be exactly what you are asking, but the total file size for export of one stitched image is ~2 gb.

I've been able to stitch together an entire slide of ovine placentome in brightfield (one Z plane) in navigator or in stage experiments- these are sometimes 2000 pictures or more, and push ~20 gb. It will image it with no issue, but the only way to export it is individual .TIF (not as the gigantic stitched image).

There is also a checkbox feature on one of our Leica systems for something called "data streaming"- it allows you to save data directly to the hard drive. I haven't checked to see if that is an option on all of our systems, but that might be a solution.

Hope that helps!
Rhonda

Rhonda Reigers Powell
Research Lab Manager
CLEMSON LIGHT IMAGING FACILITY
Clemson University, Division of Research
190 Collings Street
Life Sciences Facility, Room G024/G030
Clemson SC 29634
P: 864-656-1264
clemson.edu/light-imaging

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
Sent: Thursday, November 7, 2019 5:24 AM
To: [hidden email]
Subject: Limits of Leica tile scan / navigator modules

*****
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*****

Hi all,

I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.

Thanks in advance,
Dave Johnston,
Biomedical Imaging Unit,
University of Southampton.

[Glyn Nelson]

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Hi Doug,

At risk of hijacking this thread and heading off at a tangent, I looked at your recommendations to your users.  I would suggest an automated conversion of lif files in IMageJ/Fiji.  This link:
https://forum.image.sc/t/produce-max-projections-from-lif-files-in-imagej/93/8

has a macro that will open all images in lif files and save them as tifs, including flattening stacks to MIPs and saving those too.  It would be easy to add/ convert  save to include the line ' run("RGB Color"); ' to make your RGB image first if desired.  The macro can also deal with tilescans or multi-position lif files (these are a bit more complicated to extract the images from automatically due to the lif file structure)- it also captures the lif filename as well as the imagename, making data auditing/ tracking easier.

To come back to the original question, I agree with others that the system will be able to capture the data as long as there is enough hard drive space where it makes the temp file (in Preferences), and the reason I reply here is that the stitching itself may be easier to manage using ImageJ offline, as the acquisition PC will a: struggle, and b:block usage for image acquistion whilst spending hours trying to stitch that image!

Hope it helps,

Glyn.

[Steffen Dietzel]

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Hi Dave and others,

some of our users are doing time laps stitched movies who may get well
above 50 Gbyte on an SP8 MP system with a HP Z640 with 32 Gbyte RAM. So
far we did not run into a hard limit. The soft limit was computer speed
in dealing with the file *after* acquisition. It helped a lot when I put
two additional SSDs in the machine. A 1 TByte SSD holds the temporary
files that Andreas was mentioning, a 2 TByte SSD is for the saved lif
file. This made saving after acquisition much faster since it now goes
from one SSD to another. A fast network connection to a server certainly
also helps.

We usually do not export files in LASX, except for a quick screenshot
for the lab book or so. I actually discourage this. Given that files
should be kept in the original format to keep the meta data, this would
mean to double the required disk space. We import the lif file in Fiji
or Imaris. So far we did not run into a problem this way.

So, to come back to the original question, I believe acquisition itself
will not be a problem. And everything thereafter (stitching, saving)
will probably work but possibly very sloooooow. If this runs over night
or week-end you might not care much if the stitching will need several
hours, when it is automatically started. But you also could try to do
the stitching on a more powerful computer. If you don't have an offline
license, you could try to use the USB-Dongles from the microscope
computer for this. It might block the microscope computer for less time
than doing it at the microscope.

It certainly is worth trying to go for stitching. Personally, I love
these giant images where you can go exploring visually in 3D for hours
without getting bored (assuming the computer can fit the whole data set
in RAM and the graphics board is up to the task). We had a cleared
sample that was recording for 57 hours (only 28 Gbyte though), I am
still amazed by the overview and detail this data set has.

Steffen


Am 07.11.2019 um 22:15 schrieb Andreas Bruckbauer:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> As I understand the Leica systems save the individual files to a temp folder on the hard disk during imaging, so you can generate files which are much larger than the computer RAM, I have done 50GB without problem (without stitching). But the tile scan is not yet saved to the *.lif file at the end of the scan, this takes another 20 min on our system. Programs like Imaris, Arivis or Aivia should be able to handle these files and load the required data dynamically. I did the stitching using BigStitcher in Fiji, but this required a lot of RAM.
> best wishes
> Andreas
>
> -----Original Message-----
> From: Rhonda Reigers Powell <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Thu, 7 Nov 2019 13:30
> Subject: Re: Limits of Leica tile scan / navigator modules
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Dave,
>
> This may not be exactly what you are asking, but the total file size for export of one stitched image is ~2 gb.
>
> I've been able to stitch together an entire slide of ovine placentome in brightfield (one Z plane) in navigator or in stage experiments- these are sometimes 2000 pictures or more, and push ~20 gb. It will image it with no issue, but the only way to export it is individual .TIF (not as the gigantic stitched image).
>
> There is also a checkbox feature on one of our Leica systems for something called "data streaming"- it allows you to save data directly to the hard drive. I haven't checked to see if that is an option on all of our systems, but that might be a solution.
>
> Hope that helps!
> Rhonda
>
> Rhonda Reigers Powell
> Research Lab Manager
> CLEMSON LIGHT IMAGING FACILITY
> Clemson University, Division of Research
> 190 Collings Street
> Life Sciences Facility, Room G024/G030
> Clemson SC 29634
> P: 864-656-1264
> clemson.edu/light-imaging
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Dave Johnston
> Sent: Thursday, November 7, 2019 5:24 AM
> To: [hidden email]
> Subject: Limits of Leica tile scan / navigator modules
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
>
> I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.
>
> Thanks in advance,
> Dave Johnston,
> Biomedical Imaging Unit,
> University of Southampton.
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[George McNamara]

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Our Leica SP8 LAS X temp folder is now Hyper M2 PCIe card with Silicon
Power NVMe SSDs. Installing in SP8's HP Z440 PC described below.

==> my thanks to those in a previous thread who corrected me about NVMe
M.2 vs SATA-6 SSDs formats and performance. For here, 1 NVMe is
typically ~5x faster than one SATA-6 SSD.

8 TB can be achieved with (prices are current amazon.com):

$244 each ...Silicon Power 2TB NVMe M.2 PCIe Gen3x4 2280 TLC R/W up to
3,400/3,000MB/s SSD (SU002TBP34A80M28AB)

$57 ...ASUS Hyper M.2 X16 PCIe 3.0 X4 Expansion Card V2 Supports 4 NVMe
M.2 (2242/2260/2280/22110) Up to 128 Gbps for Intel VROC and AMD Ryzen
Threadripper NVMe RAID

So: about $1050 for 8 TB fast drive (a lot lower price than the HP
equivalent). Useful for both Leica "temp" folder and short term saving
of Leica LIF files ... 1 Gb file saves a lot quicker on the Hyper M.2
than on any of the original HP Z640 drives. I am guessing in not too
distant future the "PCIe version 3" will be supplanted by "version 4,
whose x16 slots are 2x faster than version 3 (requires "version 4"
motherboard and Threadripper CPU with enough threads ==> new PC).
Similarly, new NVidia RTX TITAN ($2000 educational pricing, 16 Teraflop,
24 Gb ram) is PCIe version 3 ... should see performance improvement when
converted to "version 4" and put into new motherboard etc PC.

further accessorize ... and improve data access ... these two items are
what I have in our image core (10 Gbe switch for FV3000RS, my office PC,
40 GB file server):

$97 ... 10Gbe Ethernet card ...ASUS XG-C100C 10G Network Adapter Pci-E
X4 Card with Single RJ-45 Port

$600 ... 8-port 10Gbe Ethernet switch ...NETGEAR 8-Port 10G
Multi-Gigabit Ethernet Unmanaged Switch (XS508M) - with 1 x 10G SFP+,
Desktop/Rackmount, and ProSAFE Limited Lifetime Protection

$ ... CAT7 cables ... future proof local network (don't tell my I.T. ...
including to our floor's fiber optics closet ... also will need an
adapter for RJ-45 --> "fiber port").

Future plans ... 100Gbe Ethernet cards (now ~$800 each) ... 100 Gbe
Ethernet switch(es) ... ~$800 per port at ~$12K for 16-port switch ...
compared to cost of a roomful of confocal and multiphoton microscopes,
and I'd be happy for someone to donate an Olympus VS200 or similar
fluorescence slide scanner(s) to our image core, 100 Gbe Ethernet is
trivial, but 10x faster than 10 Gbe (ignoring PC, server, etc, performance).

I now have Hyper M.2 cards in several PCs (and plan to add more). Some
tips, especially for the Leica SP8 Z440:

* document the "Leica dual Ethernet card" static IP and full DNS address
details!!! the PCIe slot this was in was needed to fit the Hyper M.2 ...
moving the card to another slot causes "windows amnesia. That slot was
freed up by removing some un-needed card (Firewire for Leica, multi
thingy RS232 for my FV3000RS's HP Z640).

!!!  ==> My thanks to whoever at Leica printed a label with the IP
address on the outside of the card. I have (just) enough experience to
figure out the rest (with some help from some internet sites). Simpler
is to go into network details and save screenshots of all the details.

* Hyper M.2 card uses an PCIe x16 slot, but BIOS needs to be configured
as "x4x4x4x" bifurcation. Both Leica SP8 HP Z640 and Olympus FV3000RS
Z440 have this in BIOS.

enjoy,

George

p.s. our new FISHscope has nice HD 4K monitor:

$373 ... LG 32UD59-B 32" 3840x2160 Ultra HD 4k LED Monitor with FreeSync

will upgrade various other image core PC's with same (or someday better)
monitor or dual monitors.

FISHscope (single molecule RNA FISH is main application, also capable of
immunofluorescence ... no incubator for now, so could acquire AausFP1
and other FPs, but not intended for timelapse imaging):

http://confocal.jhu.edu/current-equipment/fishscope

web page slightly out of date ... went live on iLab last week and
Lumencor SPECTRA III-360* is installed and "5plex" in Olympus cellSens.
I have details at office on connecting the "360" to run with cellSens
(NI6501 card benefits from additional accessories).

With respect to immunofluorescence (or getting Brilliants connected to
oligos) Future plans for III-360*, which has 368 nm LED in place of
standard "Teal", and has 390 nm LED is to be able to acquire Brilliant
Ultraviolets (BUV395 and ites tandems) AND Brilliant Violets (BV421 and
its tandems), and Brilliant Blues, BYG (future: more than one BYG), by
adding "Long pass" filter cubes to the IX83 microscope (currently just
have Semrock PPenta cube purchased through AVR Optics), Brilliants at

https://www.bdbiosciences.com/en-eu/instruments/research-instruments/research-cell-analyzers/facsymphony#tab-2

Reagents tab, table shows 21plex possible now. Sure, would be more fun
with simultaneous acquisition, Babcock 2018 shows compact and
inexpensive 4 simultaneous cameras for fluorescence,

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789017/figure/Fig1/

and text mentions plans to go to eight cameras (if microscope
manufacturers would put dichroic inside left <---> right camera port,
could simply stick two 4cam on each side ... there is also potential for
simultaneous out a bottom "Keller port" and out the back). I think eight
ORCA-Fusions would be nice, and computer(s) would benefit from fast
drives (i.e. Hyper M.2's) and 100 Gbe Ethernet.



On 11/8/2019 3:59 AM, Glyn Nelson wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Doug,
>
> At risk of hijacking this thread and heading off at a tangent, I looked at your recommendations to your users.  I would suggest an automated conversion of lif files in IMageJ/Fiji.  This link:
> https://forum.image.sc/t/produce-max-projections-from-lif-files-in-imagej/93/8
>
> has a macro that will open all images in lif files and save them as tifs, including flattening stacks to MIPs and saving those too.  It would be easy to add/ convert  save to include the line ' run("RGB Color"); ' to make your RGB image first if desired.  The macro can also deal with tilescans or multi-position lif files (these are a bit more complicated to extract the images from automatically due to the lif file structure)- it also captures the lif filename as well as the imagename, making data auditing/ tracking easier.
>
> To come back to the original question, I agree with others that the system will be able to capture the data as long as there is enough hard drive space where it makes the temp file (in Preferences), and the reason I reply here is that the stitching itself may be easier to manage using ImageJ offline, as the acquisition PC will a: struggle, and b:block usage for image acquistion whilst spending hours trying to stitch that image!
>
> Hope it helps,
>
> Glyn.

[Cammer, Michael]

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We've had similar problems with our Zeiss widefield system.  After a few Zen upgrades we can now capture and stitch large tiled regions, but they cannot be exported.  The files can be stitched and saved as CZI, but neither will Zen export as TIF nor will BioFormats inport the CZI.   This was as of a year ago.  Our users stopped trying or moved their slide scanning to the histopathology core which has a system dedicated for this type of work.  We have one user who breaks the tiling up into chunks to get around this problem.
Cheers-

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY  10016
Office: 646-501-0567 Cell: 914-309-3270  [hidden email]  
http://nyulmc.org/micros  http://microscopynotes.com/





-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel
Sent: Friday, November 8, 2019 5:18 AM
To: [hidden email]
Subject: Re: Limits of Leica tile scan / navigator modules


Hi Dave and others,

some of our users are doing time laps stitched movies who may get well above 50 Gbyte on an SP8 MP system with a HP Z640 with 32 Gbyte RAM. So far we did not run into a hard limit. The soft limit was computer speed in dealing with the file *after* acquisition. It helped a lot when I put two additional SSDs in the machine. A 1 TByte SSD holds the temporary files that Andreas was mentioning, a 2 TByte SSD is for the saved lif file. This made saving after acquisition much faster since it now goes from one SSD to another. A fast network connection to a server certainly also helps.

We usually do not export files in LASX, except for a quick screenshot for the lab book or so. I actually discourage this. Given that files should be kept in the original format to keep the meta data, this would mean to double the required disk space. We import the lif file in Fiji or Imaris. So far we did not run into a problem this way.

So, to come back to the original question, I believe acquisition itself will not be a problem. And everything thereafter (stitching, saving) will probably work but possibly very sloooooow. If this runs over night or week-end you might not care much if the stitching will need several hours, when it is automatically started. But you also could try to do the stitching on a more powerful computer. If you don't have an offline license, you could try to use the USB-Dongles from the microscope computer for this. It might block the microscope computer for less time than doing it at the microscope.

It certainly is worth trying to go for stitching. Personally, I love these giant images where you can go exploring visually in 3D for hours without getting bored (assuming the computer can fit the whole data set in RAM and the graphics board is up to the task). We had a cleared sample that was recording for 57 hours (only 28 Gbyte though), I am still amazed by the overview and detail this data set has.

Steffen


Am 07.11.2019 um 22:15 schrieb Andreas Bruckbauer:

> *****
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> *****
>
> As I understand the Leica systems save the individual files to a temp folder on the hard disk during imaging, so you can generate files which are much larger than the computer RAM, I have done 50GB without problem (without stitching). But the tile scan is not yet saved to the *.lif file at the end of the scan, this takes another 20 min on our system. Programs like Imaris, Arivis or Aivia should be able to handle these files and load the required data dynamically. I did the stitching using BigStitcher in Fiji, but this required a lot of RAM.
> best wishes
> Andreas
>
> -----Original Message-----
> From: Rhonda Reigers Powell <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Thu, 7 Nov 2019 13:30
> Subject: Re: Limits of Leica tile scan / navigator modules
>
> *****
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> *****
>
> Hi Dave,
>
> This may not be exactly what you are asking, but the total file size for export of one stitched image is ~2 gb.
>
> I've been able to stitch together an entire slide of ovine placentome in brightfield (one Z plane) in navigator or in stage experiments- these are sometimes 2000 pictures or more, and push ~20 gb. It will image it with no issue, but the only way to export it is individual .TIF (not as the gigantic stitched image).
>
> There is also a checkbox feature on one of our Leica systems for something called "data streaming"- it allows you to save data directly to the hard drive. I haven't checked to see if that is an option on all of our systems, but that might be a solution.
>
> Hope that helps!
> Rhonda
>
> Rhonda Reigers Powell
> Research Lab Manager
> CLEMSON LIGHT IMAGING FACILITY
> Clemson University, Division of Research
> 190 Collings Street
> Life Sciences Facility, Room G024/G030 Clemson SC 29634
> P: 864-656-1264
> clemson.edu/light-imaging
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Dave Johnston
> Sent: Thursday, November 7, 2019 5:24 AM
> To: [hidden email]
> Subject: Limits of Leica tile scan / navigator modules
>
> *****
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> NalOXMU-7wvnVXyWFCOqI9sIOC5z6tghLPJ1NjRec&s=Iw9qDHiBEdZIy_JDqih6qvSJTO
> eOHP-2ZEFfIjjRsNo&e= Post images on
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> *****
>
> Hi all,
>
> I would be grateful to learn of any user experience of how far you can push the Leica SP8's tile scan or navigator modes with respect to number of tiles / slices / overall dataset size before either the aquisition or assembly software or the Leica issued PC (only 8GB ram) falls over. I have a user wanting to potentially do multichannel sequential imaging of 1cm sq of 30um deep tissue at system optimised Z at x20. That's 255 tiles (after 10% overlap) x probably 45 slices (as sections never flat) x maybe 3 sequential imaging sets and 6 channels. At 1024x1024 per tile that is a 60gb-ish data set taking about 60 hours to acquire!!!!! The software (navigator module) seems happy to set that up and to start acquiring it (hence the software-supplied time estimate) but whether it can finish it or assemble it is another matter entirely.
>
> Thanks in advance,
> Dave Johnston,
> Biomedical Imaging Unit,
> University of Southampton.
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

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[Michael Giacomelli-2]

*****
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*****

If I could offer some more advice, we have been using SFP+ ethernet
modules rather than 10Gbaset for 10gbit on our microscopes.  Reason
for that is that a 4 port SFP+ switch costs ~$140 (or 240 for 8 port)
on Amazon:

https://www.amazon.com/MikroTik-CRS305-1G-4S-Gigabit-Ethernet-RouterOS/dp/B07LFKGP1L/ref=sr_1_3

While you can get SFP+ PCIe ethernet cards for $30-40 if you shop
around.  For short connections (~20 ft) you can use an SFP+ direct
attach cable (DAC) for a few dollars.  For longer runs,  you can buy
fiber SFP+ modules (~$20 per port).  We were able to network a
workstation, a NAS and a microscope over 10gbit for ~220 dollars total
at 10gbit/s.

You can also buy 10GbaseT SFP+ modules for existing ethernet
equipment, but 10GbaseT in general is just really expensive even
compared to fiber, I think due to it being close to the limits of what
can be done over copper.  If you want faster, 40gbit QSFP+ isn't that
much more expensive than 10GbaseT, so that might be worth a look,
although in my experience, Windows 7/10 can require some work to get
them to transmit data that quickly.

Mike

On Fri, Nov 8, 2019 at 10:30 AM George McNamara
<[hidden email]> wrote:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=PkZfas0YqnpiNFtBQ8czReczlyTtpPFzdCEbaw7t-zo&e=  and include the link in your posting.
> *****
>
> Our Leica SP8 LAS X temp folder is now Hyper M2 PCIe card with Silicon
> Power NVMe SSDs. Installing in SP8's HP Z440 PC described below.
>
> ==> my thanks to those in a previous thread who corrected me about NVMe
> M.2 vs SATA-6 SSDs formats and performance. For here, 1 NVMe is
> typically ~5x faster than one SATA-6 SSD.
>
> 8 TB can be achieved with (prices are current amazon.com):
>
> $244 each ...Silicon Power 2TB NVMe M.2 PCIe Gen3x4 2280 TLC R/W up to
> 3,400/3,000MB/s SSD (SU002TBP34A80M28AB)
>
> $57 ...ASUS Hyper M.2 X16 PCIe 3.0 X4 Expansion Card V2 Supports 4 NVMe
> M.2 (2242/2260/2280/22110) Up to 128 Gbps for Intel VROC and AMD Ryzen
> Threadripper NVMe RAID
>
> So: about $1050 for 8 TB fast drive (a lot lower price than the HP
> equivalent). Useful for both Leica "temp" folder and short term saving
> of Leica LIF files ... 1 Gb file saves a lot quicker on the Hyper M.2
> than on any of the original HP Z640 drives. I am guessing in not too
> distant future the "PCIe version 3" will be supplanted by "version 4,
> whose x16 slots are 2x faster than version 3 (requires "version 4"
> motherboard and Threadripper CPU with enough threads ==> new PC).
> Similarly, new NVidia RTX TITAN ($2000 educational pricing, 16 Teraflop,
> 24 Gb ram) is PCIe version 3 ... should see performance improvement when
> converted to "version 4" and put into new motherboard etc PC.
>
> further accessorize ... and improve data access ... these two items are
> what I have in our image core (10 Gbe switch for FV3000RS, my office PC,
> 40 GB file server):
>
> $97 ... 10Gbe Ethernet card ...ASUS XG-C100C 10G Network Adapter Pci-E
> X4 Card with Single RJ-45 Port
>
> $600 ... 8-port 10Gbe Ethernet switch ...NETGEAR 8-Port 10G
> Multi-Gigabit Ethernet Unmanaged Switch (XS508M) - with 1 x 10G SFP+,
> Desktop/Rackmount, and ProSAFE Limited Lifetime Protection
>
> $ ... CAT7 cables ... future proof local network (don't tell my I.T. ...
> including to our floor's fiber optics closet ... also will need an
> adapter for RJ-45 --> "fiber port").
>
> Future plans ... 100Gbe Ethernet cards (now ~$800 each) ... 100 Gbe
> Ethernet switch(es) ... ~$800 per port at ~$12K for 16-port switch ...
> compared to cost of a roomful of confocal and multiphoton microscopes,
> and I'd be happy for someone to donate an Olympus VS200 or similar
> fluorescence slide scanner(s) to our image core, 100 Gbe Ethernet is
> trivial, but 10x faster than 10 Gbe (ignoring PC, server, etc, performance).
>
> I now have Hyper M.2 cards in several PCs (and plan to add more). Some
> tips, especially for the Leica SP8 Z440:
>
> * document the "Leica dual Ethernet card" static IP and full DNS address
> details!!! the PCIe slot this was in was needed to fit the Hyper M.2 ...
> moving the card to another slot causes "windows amnesia. That slot was
> freed up by removing some un-needed card (Firewire for Leica, multi
> thingy RS232 for my FV3000RS's HP Z640).
>
> !!!  ==> My thanks to whoever at Leica printed a label with the IP
> address on the outside of the card. I have (just) enough experience to
> figure out the rest (with some help from some internet sites). Simpler
> is to go into network details and save screenshots of all the details.
>
> * Hyper M.2 card uses an PCIe x16 slot, but BIOS needs to be configured
> as "x4x4x4x" bifurcation. Both Leica SP8 HP Z640 and Olympus FV3000RS
> Z440 have this in BIOS.
>
> enjoy,
>
> George
>
> p.s. our new FISHscope has nice HD 4K monitor:
>
> $373 ... LG 32UD59-B 32" 3840x2160 Ultra HD 4k LED Monitor with FreeSync
>
> will upgrade various other image core PC's with same (or someday better)
> monitor or dual monitors.
>
> FISHscope (single molecule RNA FISH is main application, also capable of
> immunofluorescence ... no incubator for now, so could acquire AausFP1
> and other FPs, but not intended for timelapse imaging):
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__confocal.jhu.edu_current-2Dequipment_fishscope&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=1DLRNO8QUi4RzrpX4X69oxNGYlgFWu7LRsQMTZdz6EE&e=
>
> web page slightly out of date ... went live on iLab last week and
> Lumencor SPECTRA III-360* is installed and "5plex" in Olympus cellSens.
> I have details at office on connecting the "360" to run with cellSens
> (NI6501 card benefits from additional accessories).
>
> With respect to immunofluorescence (or getting Brilliants connected to
> oligos) Future plans for III-360*, which has 368 nm LED in place of
> standard "Teal", and has 390 nm LED is to be able to acquire Brilliant
> Ultraviolets (BUV395 and ites tandems) AND Brilliant Violets (BV421 and
> its tandems), and Brilliant Blues, BYG (future: more than one BYG), by
> adding "Long pass" filter cubes to the IX83 microscope (currently just
> have Semrock PPenta cube purchased through AVR Optics), Brilliants at
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.bdbiosciences.com_en-2Deu_instruments_research-2Dinstruments_research-2Dcell-2Danalyzers_facsymphony-23tab-2D2&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=F17TIibcVD0E-AqGBoqwQbZVGBTHJy8TKsYrU2l4ZJg&e=
>
> Reagents tab, table shows 21plex possible now. Sure, would be more fun
> with simultaneous acquisition, Babcock 2018 shows compact and
> inexpensive 4 simultaneous cameras for fluorescence,
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih.gov_pmc_articles_PMC5789017_figure_Fig1_&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=N4lYqYbGmaBQOE6wG2TmIrgUlawqGjS8wSUO3RyHTxk&e=
>
> and text mentions plans to go to eight cameras (if microscope
> manufacturers would put dichroic inside left <---> right camera port,
> could simply stick two 4cam on each side ... there is also potential for
> simultaneous out a bottom "Keller port" and out the back). I think eight
> ORCA-Fusions would be nice, and computer(s) would benefit from fast
> drives (i.e. Hyper M.2's) and 100 Gbe Ethernet.
>
>
>
> On 11/8/2019 3:59 AM, Glyn Nelson wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=ZHWSFhDusNUr5W32UlAwK6fgwmROq0g7K8gyLY7hM34&e=
> > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=PkZfas0YqnpiNFtBQ8czReczlyTtpPFzdCEbaw7t-zo&e=  and include the link in your posting.
> > *****
> >
> > Hi Doug,
> >
> > At risk of hijacking this thread and heading off at a tangent, I looked at your recommendations to your users.  I would suggest an automated conversion of lif files in IMageJ/Fiji.  This link:
> > https://urldefense.proofpoint.com/v2/url?u=https-3A__forum.image.sc_t_produce-2Dmax-2Dprojections-2Dfrom-2Dlif-2Dfiles-2Din-2Dimagej_93_8&d=DwIDaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=EKlTYxfkaqX6co6NvgDhJVr58hU4rdPz_or_8WhQCbs&s=SpjyDtV703GYWkwLe5Y04_t-2WEg_p-3Q7GgWM38GGY&e=
> >
> > has a macro that will open all images in lif files and save them as tifs, including flattening stacks to MIPs and saving those too.  It would be easy to add/ convert  save to include the line ' run("RGB Color"); ' to make your RGB image first if desired.  The macro can also deal with tilescans or multi-position lif files (these are a bit more complicated to extract the images from automatically due to the lif file structure)- it also captures the lif filename as well as the imagename, making data auditing/ tracking easier.
> >
> > To come back to the original question, I agree with others that the system will be able to capture the data as long as there is enough hard drive space where it makes the temp file (in Preferences), and the reason I reply here is that the stitching itself may be easier to manage using ImageJ offline, as the acquisition PC will a: struggle, and b:block usage for image acquistion whilst spending hours trying to stitch that image!
> >
> > Hope it helps,
> >
> > Glyn.

--
Michael Giacomelli, Ph.D.
Assistant Professor
Department of Biomedical Engineering
University of Rochester