Embryo time lapse

[Valeria Berno-2]

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

[Armstrong, Brian]

Hello, IMHO, I would use a widefield system, an LCD, light engine, or Exfo illumination system, an EMCCD camera, and a quality incubation system that covers the entire scope. You should also consider the laser “focus systems” offered from Zeiss, Nikon, and coming soon Leica (sorry don’t know if Olympus has one yet).

You should be able to accomplish ALL the tasks you outlined.

One thing, if you want to do low mag (5X, 10X) time-lapse imaging you will want a camera with small pixels. The fast EMCCD cameras for high mag have rather large pixels (~16um).

I can talk specs “off-line” if you like.

 

Cheers,

 

Brian D Armstrong PhD

Light Microscopy Core Manager

Beckman Research Institute

City of Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, June 21, 2010 8:19 AM
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

---------------------------------------------------------------------
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[Alice Rodriguez Diaz]

Dear Valeria,

A spinning disk does the less damage to embryos for long term imaging,

Best

Alice Rodriguez Diaz

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, June 21, 2010 10:19 AM
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

[leoncio vergara]

I understand Olympus has just released or is about to release a fast autofocus system as well... we want to test it as soon is available

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: Monday, June 21, 2010 10:44 AM
To: [hidden email]
Subject: Re: Embryo time lapse

Hello, IMHO, I would use a widefield system, an LCD, light engine, or Exfo illumination system, an EMCCD camera, and a quality incubation system that covers the entire scope. You should also consider the laser “focus systems” offered from Zeiss, Nikon, and coming soon Leica (sorry don’t know if Olympus has one yet).

You should be able to accomplish ALL the tasks you outlined.

One thing, if you want to do low mag (5X, 10X) time-lapse imaging you will want a camera with small pixels. The fast EMCCD cameras for high mag have rather large pixels (~16um).

I can talk specs “off-line” if you like.

 

Cheers,

 

Brian D Armstrong PhD

Light Microscopy Core Manager

Beckman Research Institute

City of Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, June 21, 2010 8:19 AM
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

---------------------------------------------------------------------
SECURITY/CONFIDENTIALITY WARNING:
This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
---------------------------------------------------------------------

[Jim Beacher]

* Commercial Interest *

 

While considering the best overall microscope package, it is well worthwhile to consider an LED-based illumination system.

 

Benefits for your time-lapse requirements are that the LEDs will photobleach significantly less.  LEDs are narrow-band and will be centred around your desired wavelengths - GFP,Venus,dsRed.  Fast-switching in microseconds is also available for any dual staining.  LEDs are solid-state so offer instant on/off and stable, repeatable (over seconds, hours, months and years) illumination.  CoolLED products are integrated with most leading imaging software packages.

 

Good luck with you deliberations.

 

Best Regards

 

JIM Beacher / CoolLED

[hidden email]

www.coolled.com

 

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: 21 June 2010 15:44
To: [hidden email]
Subject: Re: Embryo time lapse

Hello, IMHO, I would use a widefield system, an LCD, light engine, or Exfo illumination system, an EMCCD camera, and a quality incubation system that covers the entire scope. You should also consider the laser “focus systems” offered from Zeiss, Nikon, and coming soon Leica (sorry don’t know if Olympus has one yet).

You should be able to accomplish ALL the tasks you outlined.

One thing, if you want to do low mag (5X, 10X) time-lapse imaging you will want a camera with small pixels. The fast EMCCD cameras for high mag have rather large pixels (~16um).

I can talk specs “off-line” if you like.

 

Cheers,

 

Brian D Armstrong PhD

Light Microscopy Core Manager

Beckman Research Institute

City of Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, June 21, 2010 8:19 AM
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

---------------------------------------------------------------------
SECURITY/CONFIDENTIALITY WARNING:
This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
---------------------------------------------------------------------

[Adams,Henry P]

Valeria,

We have been using a laser based spinning disc for live cell imaging, C.elegans and mouse embryo development for 4 years and have been quite satisfied with the results. Obviously, there are certain limitations such as imaging depth with spinning discs, so most of the mouse development studies involved removing the specific tissue/organs of interest and growing  them in modified chambers.  C.elegans does amazingly well with the spinning disc (taking stacks every 20-30 secs) for 20 minutes or more).

Hank Adams, Microscopy Core Manager

Genetics Department

U.T.M.D.Anderson Cancer Center

Houston, Tx

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: Monday, June 21, 2010 10:19 AM
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

[Sylvie Le Guyader-2]

Hi Valeria

I have never imaged mouse embryos but only mouse skin and zebrafish and C.e. embryos. However it seems to me that if you want to follow cells in a mouse embryo, a 2-photon system would be best as you can image rather deep. We find that to follow labelled cells among non labelled cells and know what is going on, it is crucial to acquire an excellent DIC image. I would recommend oblique illumination (Dodt contrast) which is available with several commercial 2-photon systems. I would also say that if you have no need to image intracellular events, an excellent 20x objective will work just fine for your purpose while giving you the field of view and the NA needed.

Med vänlig hälsning / Best regards

 

Sylvie

 

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader

Live Cell Imaging Unit

Dept of Biosciences and Nutrition

Karolinska Institutet

Sweden

office: [phone]

mobile: [phone]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Valeria Berno
Sent: 21 June 2010 17:19
To: [hidden email]
Subject: Embryo time lapse

 

Dear all,

I am running a facility with many different users and I am trying to pull together their needs on imaging in order to improve the equipments.

Right now the biggest demand is about time lapse imaging (long term time lapse (24-48hours with a 15-30min step)) specifically on mouse embryo (E7-E8) with fluorescence (GFP,Venus,dsRed).

So we need very high sensitivity system, weak photo damage, both high magnification (to follow single cells) and possibility to follow movement in the entire embryo.

Too many tasks for a single system?

Which do you think would be the best system to achieve these goals?
a confocal? a multiphoton? the new confocal-stereomicroscope from Nikon AZ-C1? a regular inverted widefield with a good camera?

Which system do you use for these time lapse?

Thanks for all your always helpful input

Valeria

-- 

Valeria Berno,PhD

Microscopy Facility Manager

MRC Centre for Regenerative Medicine

Institute for Stem Cell Research

University of Edinburgh

Roger Land Building

West Mains Road

Edinburgh

EH9 3JQ

Rm 273

Tel Office 0131 6517265

[Martin Spitaler]

Dear Valeria,

 we have done embryo development studies in our facility for 72 hours on a
standard widefield system with standard CCD camera (although a
higher-sensitivity camera would have been desirable, to avoid
phototoxicity). The main problem is definitely the focus, but for fish
embryo development it's not so much the microscope, it's the actual embryo
that starts moving around like crazy after a day, and the thickness of the
sample, so an infrared autofocus will be of limited help (but I would still
go for it for other long time-lapse samples). We just did a few stacks and
picked the interesting ones after completion of time lapse. Just don't
forget to calculate the expected file size before you start, it's going to
be huge! Data storage and transfer is definitely an important consideration.

Good luck,

Martin

======================================
Martin Spitaler, PhD

FILM - Facility for Imaging by Light Microscopy
- Facility Manager -
Sir Alexander Fleming Building, desk 401
Imperial College London / South Kensington
Exhibition Road
London SW7 2AZ
UK

Tel. [phone]
E-mail [hidden email]
Website: http://imperial.ac.uk/imagingfacility

[phil laissue]

Hi Valeria,

Nikon A1R with fast resonant scanner to reduce photobleaching (see Borlinghaus RT: MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching. Microsc Res Tech. 2006 Sep;69(9):689-92) would be my choice.

Best wishes

Philippe
______________________________
Philippe Laissue, PhD - Bioimaging Manager
Dept. of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / [phone] 2464
[hidden email]