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Live cell imaging experiences, please

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Zoltan

Live cell imaging experiences, please

Dear All,

We are planning to run a few tests, where we follow cell development
in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
min time interval, for a total of 14 hours at room temp, using GFP and
DsRed settings. We had lots of success with the Leica SP5 system for
the past 2 years. We are wondering if this community has more input
about how other systems would perfom here? We are most interested in
Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
the setting up procedure, the stability of the system during the long
scans, and the handling of the large data files.
All inputs will be much appreciated! Happy New Year to all! With
best wishes,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
Univ. Cambridge, UK

Rietdorf, Jens

Re: Live cell imaging experiences, please

Dear Zoltan,

we have recently investigated this functionality for the Olympus FV1000,
in particular for the generation of large mosaic scans of up to
25x25x100 full frames. We found the multi area time lapse module of the
FV simple and intuitive. Data handling was easy, we exported into
several visualization and stitching software platforms. Long term
stability of the system is very good using a box incubator or the ZDC
autofocus.

Nikon has a very good autofocus unit as well, I have no experience with
the interface for multi-position acquisition tough.

(no commercial interest).

Best wishes,
jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Zoltan Cseresnyes
Sent: Monday, January 11, 2010 2:31 PM
To: [hidden email]
Subject: Live cell imaging experiences, please

Dear All,

We are planning to run a few tests, where we follow cell development
in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
min time interval, for a total of 14 hours at room temp, using GFP and
DsRed settings. We had lots of success with the Leica SP5 system for
the past 2 years. We are wondering if this community has more input
about how other systems would perfom here? We are most interested in
Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
the setting up procedure, the stability of the system during the long
scans, and the handling of the large data files.
All inputs will be much appreciated! Happy New Year to all! With
best wishes,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
Univ. Cambridge, UK

Christian Götze

Re: Live cell imaging experiences, please

In reply to this post by Zoltan

Commercial response!

Hi Zoltan,

exciting application! Please correct me, if I'm wrong but one of these
experiments would result in about 12.5 GByte of data (at 512x512 Pixels
per plane, 8 Bit intensity, 18 Planes per stack, 8 stacks per time
point, 1800 time points). What kind of software do you plan to use?

If you don't have a suitable candidate already, perhaps you should have
a look on our arivis Browser software. It is specialized in handling
very large experiments and could be the right solution for you.

(http://www.arivis.com/en/Produkte/arivis-Browser)

If you are interested or there are questions please don't hestitate to
contact me.

Christian

Zoltan Cseresnyes schrieb:

> Dear All,
>
> We are planning to run a few tests, where we follow cell development
> in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
> min time interval, for a total of 14 hours at room temp, using GFP and
> DsRed settings. We had lots of success with the Leica SP5 system for
> the past 2 years. We are wondering if this community has more input
> about how other systems would perfom here? We are most interested in
> Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
> the setting up procedure, the stability of the system during the long
> scans, and the handling of the large data files.
> All inputs will be much appreciated! Happy New Year to all! With
> best wishes,
>
> Zoltan
>

--
Christian Götze

arivis - Multiple Image Tools GmbH
Kröpeliner Straße 54, D-18055 Rostock

Tel : +49 381 461393 11
Fax : +49 381 46139399
Mail: [hidden email]
Web : http://www.arivis.com

Amtsgericht Rostock HRB 9732
Geschäftsführung: Raik Madla, Christian Goetze

Neeraj Gohad-2

Re: Live cell imaging experiences, please

Hi Zoltan,

I have done long term time lapse (24 hour) experiments looking at multiple stage points in 3 different channels. We have a Nikon TiE with a motorized stage, perfect focus system and a Sutter Lambda 10-3 mechanical shutter. Its fairly easy to setup multidimensional experiments in the Nikon elements software, you can setup pretty complicated configurations which can be saved and reloaded. Using the perfect focus you can set different Z offsets for different stage points, there is also a function which automatically optimizes the stage travel between the stage points.

As always, no commercial interests just a satisfied customer.

Neeraj V. Gohad, Ph.D.
Postdoctoral Fellow
Okeanos Research Group
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634

Please note my new email address: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Götze
Sent: Tuesday, January 12, 2010 5:36 AM
To: [hidden email]
Subject: Re: Live cell imaging experiences, please

Commercial response!

Hi Zoltan,

exciting application! Please correct me, if I'm wrong but one of these
experiments would result in about 12.5 GByte of data (at 512x512 Pixels
per plane, 8 Bit intensity, 18 Planes per stack, 8 stacks per time
point, 1800 time points). What kind of software do you plan to use?

If you don't have a suitable candidate already, perhaps you should have
a look on our arivis Browser software. It is specialized in handling
very large experiments and could be the right solution for you.

(http://www.arivis.com/en/Produkte/arivis-Browser)

If you are interested or there are questions please don't hestitate to
contact me.

Christian

Zoltan Cseresnyes schrieb:

George Peeters-2

Re: Live cell imaging experiences, please

In reply to this post by Zoltan

Papers describing very similar experiments and their technological methods have been published by Zena Werb's group (UCSF) and her colleagues, Andrew Ewald, now at Johns Hopkins Univ. and Mikala Egeblad, now at Cold Springs Harbor Labs. One paper that received a nod from Science Magazines editior's choice is listed below.

Best regards,

Solid Tumors in Living Color

The behavior of tumors is profoundly influenced

by the microenvironment in which they grow. In

addition to diffusible extracellular factors, this

environment harbors a complex and dynamic

population of stromal cells, including fibroblasts

and a variety of immune cells. Because different

types of stromal cells can have opposing effects

on tumor progression and responses to therapy,

it is important to understand how each cell type

behaves in actively growing tumors.

Egeblad et al. have combined confocal

microscopy with multicolor imaging techniques

to record in living mice the movement and localization

patterns of tumor-infiltrating stromal

cells during a 12-hour period. One feature

shared by several stromal cell types was greater

motility at the tumor periphery than within the

tumor mass. Regulatory T cells were found to

migrate near blood vessels, and their movement

was sensitive to tumor oxygen levels; in contrast,

the movement of myeloid cells (the most heterogeneous

group of stromal cells) was insensitive

to oxygen, and their localization patterns and

migration rates varied according to cell-surface

marker expression, probably reflecting important

functional differences. By helping to define

the contributions of specific stromal cells to

tumor growth, this imaging technology may lead

to more effective therapies. — PAK

Disease Models Mech. 1, 155 (2008).

George A. Peeters MD, MS

President, Solamere Technology Group Inc

1427 Perry Ave

Salt Lake City, UT 84103

www.solameretech.com

801 322-2645 office 801 322-2645 fax

801 232-6911 cell

On Jan 11, 2010, at 6:31 AM, Zoltan Cseresnyes wrote:

Dear All,

We are planning to run a few tests, where we follow cell development
in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
min time interval, for a total of 14 hours at room temp, using GFP and
DsRed settings. We had lots of success with the Leica SP5 system for
the past 2 years. We are wondering if this community has more input
about how other systems would perfom here? We are most interested in
Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
the setting up procedure, the stability of the system during the long
scans, and the handling of the large data files.
All inputs will be much appreciated! Happy New Year to all! With
best wishes,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
Univ. Cambridge, UK

Tim Feinstein-2

Re: Live cell imaging experiences, please

In reply to this post by Neeraj Gohad-2

_No commercial interest._

My experience with a Nikon A1 is similar to what Neeraj described. We almost never see drift in focus or XY during long time-lapse imaging of many (>20) independent XY positions, and when we do it is invariably a software bug rather than a limitation of the hardware. Elements software also has a convenient function for 'tiling' several XY points into one larger image.

The 'optimize' function in Elements makes a decent go of minimizing travel between points, but it optimizes for a single time point (e.g., the last point is usually the most distant from the starting position). That sometimes causes the focus control to miss on the return due to the long travel. I find it quite easy to modify the order by hand to eliminate any large distance gaps.

We have never had a problem managing files up to several GB.

Based on my experience with other microscopes, 'stability' during long-term imaging usually entails focus drift due to thermal expansion/contraction and/or vibration. With its current hardware Nikon simply does not have that problem. I cannot speak for other contemporary scopes, but my success rate for time lapse > 4 h. has gone from ~20% on a dated, non-motorized system to almost 100% using the A1. The only reason we lose a movie is rare software malfunctions (the Vista edition of Elements has benefitted from a year of bug fixes).

I believe that Nikon's PFS hardware uses a unique TIRF-like infrared mechanism to maintain a constant distance between objective and coverslip. This allows a refresh rate in the milliseconds and it does not depend on detecting fluorescence from the sample. PFS will stay locked even if the software crashes and the computer has to reboot. Elements even remembers XY positions entered just before a software crash, which is nice.

That said, if product demos are an option then I recommend that you try your experiment on several systems to see which best matches your particular needs.

All the best,

Tim

On Jan 12, 2010, at 11:04 AM, Neeraj Gohad wrote:

> Hi Zoltan,
>
> I have done long term time lapse (24 hour) experiments looking at multiple stage points in 3 different channels. We have a Nikon TiE with a motorized stage, perfect focus system and a Sutter Lambda 10-3 mechanical shutter. Its fairly easy to setup multidimensional experiments in the Nikon elements software, you can setup pretty complicated configurations which can be saved and reloaded. Using the perfect focus you can set different Z offsets for different stage points, there is also a function which automatically optimizes the stage travel between the stage points.
>
>
> As always, no commercial interests just a satisfied customer.
>
>
> Neeraj V. Gohad, Ph.D.
> Postdoctoral Fellow
> Okeanos Research Group
> Department of Biological Sciences
> 132 Long Hall
> Clemson University
> Clemson,SC-29634
>
> Please note my new email address: [hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Götze
> Sent: Tuesday, January 12, 2010 5:36 AM
> To: [hidden email]
> Subject: Re: Live cell imaging experiences, please
>
> Commercial response!
>
> Hi Zoltan,
>
> exciting application! Please correct me, if I'm wrong but one of these
> experiments would result in about 12.5 GByte of data (at 512x512 Pixels
> per plane, 8 Bit intensity, 18 Planes per stack, 8 stacks per time
> point, 1800 time points). What kind of software do you plan to use?
>
> If you don't have a suitable candidate already, perhaps you should have
> a look on our arivis Browser software. It is specialized in handling
> very large experiments and could be the right solution for you.
>
> (http://www.arivis.com/en/Produkte/arivis-Browser)
>
> If you are interested or there are questions please don't hestitate to
> contact me.
>
> Christian
>
> Zoltan Cseresnyes schrieb:
>> Dear All,
>>
>> We are planning to run a few tests, where we follow cell development
>> in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
>> min time interval, for a total of 14 hours at room temp, using GFP and
>> DsRed settings. We had lots of success with the Leica SP5 system for
>> the past 2 years. We are wondering if this community has more input
>> about how other systems would perfom here? We are most interested in
>> Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
>> the setting up procedure, the stability of the system during the long
>> scans, and the handling of the large data files.
>> All inputs will be much appreciated! Happy New Year to all! With
>> best wishes,
>>
>> Zoltan
>>
>
> --
> Christian Götze
>
> arivis - Multiple Image Tools GmbH
> Kröpeliner Straße 54, D-18055 Rostock
>
> Tel : +49 381 461393 11
> Fax : +49 381 46139399
> Mail: [hidden email]
> Web : http://www.arivis.com
>
> Amtsgericht Rostock HRB 9732
> Geschäftsführung: Raik Madla, Christian Goetze

Adrian Smith-6

Re: Live cell imaging experiences, please

In reply to this post by Zoltan

On 12/01/2010, at 12:31 AM, Zoltan Cseresnyes wrote:

Are you having particular problems with doing this on your SP5?

We've been doing overnight (and longer) multiposition z-stacks on our SP5 (with full enclosure) and it has been working very well.

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia

Richard Harris-6

Re: Live cell imaging experiences, please

In reply to this post by Zoltan

Hi Zoltan
We run a Zeiss Live 5 Duo (Vario 2) LSM system here and have had great
success collecting huge data sets on metamorphosis in drosophila - these are
typically 16 - 18 hour experiments collecting a Z-stack (20 or more slices)
every 2 1/2 minutes. The stage is very stable and we've been using the
stitching feature to generate large area, high resolution images - this
means we're collecting as many as 10 XY positions per critter every 2 - 3
minutes.
Results are impressive and we've been working at generating movies from
these HUGE 4D data sets.
The Zeiss system in Live mode is capable of generating up to 120 FPS (XY)
and/or fast Z-stacks (approx 8-10um/sec).
We are satisfied customers and have no commercial interest in Zeiss.
Rick,

Richard Harris, Manager - Integrated Microscopy @ Biotron
The Biotron - Center for Experimental Climate Change Research
University of Western Ontario,
London Ontario, CANADA.
N6A 5B7
Ph. 519-661-2111 ext. 86780
Fax 519-661-3935
e-mail [hidden email]
web: www.thebiotron.ca

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Zoltan Cseresnyes
Sent: Monday, January 11, 2010 8:31 AM
To: [hidden email]
Subject: Live cell imaging experiences, please

Dear All,

We are planning to run a few tests, where we follow cell development
in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
min time interval, for a total of 14 hours at room temp, using GFP and
DsRed settings. We had lots of success with the Leica SP5 system for
the past 2 years. We are wondering if this community has more input
about how other systems would perfom here? We are most interested in
Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
the setting up procedure, the stability of the system during the long
scans, and the handling of the large data files.
All inputs will be much appreciated! Happy New Year to all! With
best wishes,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
Univ. Cambridge, UK

Zoltan

Re: Live cell imaging experiences, please

Thank you all very much for the very valuable feedback on the
long-term stability (hardware and software) of the FV1000 and A1
systems! Just to clarify: we have been using the SP5 systems of our
Imaging Facility for these scans for 2 years now and are happy with
them. However, one of our users is now applying for a fellowship to
start his own lab outside the UK, and thus needed to get familiar with
the rest of the possible systems that are proven to be suitable for
such long scans (with large result files).
Thank you all for your feedback again! With best regards,

Zoltan

On Thu, Jan 14, 2010 at 6:48 PM, Richard Harris <[hidden email]> wrote:

> Hi Zoltan
> We run a Zeiss Live 5 Duo (Vario 2) LSM system here and have had great
> success collecting huge data sets on metamorphosis in drosophila - these are
> typically 16 - 18 hour experiments collecting a Z-stack (20 or more slices)
> every 2 1/2 minutes. The stage is very stable and we've been using the
> stitching feature to generate large area, high resolution images - this
> means we're collecting as many as 10 XY positions per critter every 2 - 3
> minutes.
> Results are impressive and we've been working at generating movies from
> these HUGE 4D data sets.
> The Zeiss system in Live mode is capable of generating up to 120 FPS (XY)
> and/or fast Z-stacks (approx 8-10um/sec).
> We are satisfied customers and have no commercial interest in Zeiss.
> Rick,
>
> Richard Harris, Manager - Integrated Microscopy @ Biotron
> The Biotron - Center for Experimental Climate Change Research
> University of Western Ontario,
> London Ontario, CANADA.
> N6A 5B7
> Ph. 519-661-2111 ext. 86780
> Fax 519-661-3935
> e-mail [hidden email]
> web: www.thebiotron.ca
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Zoltan Cseresnyes
> Sent: Monday, January 11, 2010 8:31 AM
> To: [hidden email]
> Subject: Live cell imaging experiences, please
>
> Dear All,
>
> We are planning to run a few tests, where we follow cell development
> in 8-9 XY positions, running 45 um Z stacks (step size 2.5 um), 2.5
> min time interval, for a total of 14 hours at room temp, using GFP and
> DsRed settings. We had lots of success with the Leica SP5 system for
> the past 2 years. We are wondering if this community has more input
> about how other systems would perfom here? We are most interested in
> Olympus FV1000 and Nikon A1. Our main concerns are the simplicity of
> the setting up procedure, the stability of the system during the long
> scans, and the handling of the large data files.
> All inputs will be much appreciated! Happy New Year to all! With
> best wishes,
>
> Zoltan
>
> --
>
> Zoltan Cseresnyes
> Facility manager, Imaging Suite
> Univ. Cambridge, UK
>

--

Zoltan Cseresnyes
Facility manager, Imaging Suite