Overnight scan of human fibroblasts

[Zoltan]

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

[Leigh Silvester]

One answer, although not helpful in the short run is to use a tuneable infra red laser with multiphoton capability.

This can actually excite a wide range of fluorochromes. The excitation wavelength is approximately half that of the set wavelength.

Hence an IR laser pulsing nicely at 750 ish nm will excite DAPI beautifully.

 

As a result of the wavelength you don't get any DNA damage - just slightly warmed cells.

 

However, these lasers do not come cheap, and they are hard work/expensive to keep in running condition - although newer ones may be better.

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 12:19
To: [hidden email]
Subject: Overnight scan of human fibroblasts

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.

[Armstrong, Brian]

Zoltan, in my experience the 2P/Ti:Sa lasers are still quite hard on the cell that you are imaging. I think the real benefit of 2P in live-cell is that you do not bleach the cells that are out of the focal spot of the 2P laser.

 

How to help you now: image less often, the total amount of energy you put into the sample can be greatly reduced by acquiring less often (I’m sure you must have thought of this already). Another idea is to use dyes that are Far-red (such as Draq5?) instead of the dyes that excite at UV or at V (405nm). I assume you are imaging nuclear staining of some kind. UV wavelengths are always going to damage your tissues more than the Red/IR wavelengths used to excite dyes in the far red range.

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 Leigh Silvester
Sent: Friday, March 27, 2009 6:07 AM
To: [hidden email]
Subject: Re: Overnight scan of human fibroblasts

 

One answer, although not helpful in the short run is to use a tuneable infra red laser with multiphoton capability.

This can actually excite a wide range of fluorochromes. The excitation wavelength is approximately half that of the set wavelength.

Hence an IR laser pulsing nicely at 750 ish nm will excite DAPI beautifully.

 

As a result of the wavelength you don't get any DNA damage - just slightly warmed cells.

 

However, these lasers do not come cheap, and they are hard work/expensive to keep in running condition - although newer ones may be better.

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 12:19
To: [hidden email]
Subject: Overnight scan of human fibroblasts

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

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[Sylvie Le Guyader-2]

Hi Zoltan

 

I think using a short wavelength could be the problem. Not only will you induce more phototoxicity compared with longer wavelengths but you will also bleach your FRET acceptor and donor which presumably absorb blue light. One solution if you have a 633/640/647 laser is to label your nuclei with a far red fluorophore for live cells. You could use Draq5 http://www.biostatus.com/product/draq5/.

 

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 13:19
To: [hidden email]
Subject: Overnight scan of human fibroblasts

 

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

[xavier Sanjuan]

Hi list,

 

One of my users is using Kaede in a zebrafish model to run in vivo experiments. Once they have finished he fixes the embryo to do further imaging (also to attempt FISH experiments), but whereas the green signal can be imaged without any problem, the red fluorescence in the photoconverted areas vanishes 1-2 days after mounting. He is doing a standard 4%PFA fixation, and he mounts the zebrafish embryos with glycerol.

 

Is he doing something wrong that is bleaching/destroying the photoconverted Kaede?

 

Thanks in advance for your help!

 

Best,

 

Xavi.

 

___________________________________

Xavier Sanjuan
Servei de Microscòpia Confocal
Departament de Ciències Experimentals i de la Salut
Universitat Pompeu Fabra
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88 - Sala 309
08003 Barcelona - Spain

Tel.:  + 34 93 316 08 64
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web: http://www.upf.edu/sct

[Julio Vazquez]

Zoltan, 

As was pointed out, the shorter the wavelength, the more the damage, and therefore 405 nm is probably not the best choice for long term imaging., although certainly better than UV. Shifting to a red nuclear dye would make a big difference. 

This being said, you also need to exclude trivial reasons. I don't know how much power 20% is on your system (depends on the lasers you are using and the overall efficiency of the system), but 20% power for a 514 line seems very high to me, if you are using "standard" lasers with about 10 mW output. With our Argon laser, we typically use 2%-3% power for live imaging, and that is generally plenty. Even with our wimpy 1 mW 543 nm HeNe (basically a laser pointer), we often need no more than 10%. Therefore, I would recommend you check the  efficiency of your system and maybe the alignment of your optics. It's amazing how a slight misalignment of the pinhole can lead people to crank up lasers to lethal levels...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

==

 

On Mar 27, 2009, at 5:18 AM, Zoltan Cseresnyes wrote:

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

[Fred Mast]

I agree with Julio, while it's difficult to judge how much 20% laser power is, it does seem excessive.  With such a low z-resolution I would open the pinhole to collect the maximum amount of light. This would help with reducing the laser power. Or you could implement a rectangular region of interest for your experiment (long on the x-axis and short on the y-axis) as reducing the the number of scans does improve speed of acquisition. 

Cheers,

Fred

On 27-Mar-09, at 10:28 AM, Julio Vazquez wrote:

Zoltan, 

As was pointed out, the shorter the wavelength, the more the damage, and therefore 405 nm is probably not the best choice for long term imaging., although certainly better than UV. Shifting to a red nuclear dye would make a big difference. 

This being said, you also need to exclude trivial reasons. I don't know how much power 20% is on your system (depends on the lasers you are using and the overall efficiency of the system), but 20% power for a 514 line seems very high to me, if you are using "standard" lasers with about 10 mW output. With our Argon laser, we typically use 2%-3% power for live imaging, and that is generally plenty. Even with our wimpy 1 mW 543 nm HeNe (basically a laser pointer), we often need no more than 10%. Therefore, I would recommend you check the  efficiency of your system and maybe the alignment of your optics. It's amazing how a slight misalignment of the pinhole can lead people to crank up lasers to lethal levels...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

==

 

On Mar 27, 2009, at 5:18 AM, Zoltan Cseresnyes wrote:

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

[Zoltan]

Dear All,

Thank you all for the excellent ideas!!  To be more precise about our requirements:  we have to use either 405 or 458 (much preferably 405) because of the FRET pair that we are trying to image within the nucleus.  The 405 nm exc. is thus used not to label the nucleus but to excite the donor.   The time resolution is critical, we have to image every selected location every 3-4 minutes at least, otherwise we miss the time course of the process that we are trying to monitor.  The cells sometimes crawl over each other, hence the need to do a 6-um Z stack.
  Overall, we can right away try to lower the laser power and up the gain (we are currently using gains in the 750-900 range,  max. is 1250) to see if we can still resolve the events (the critical areas are only 1-3 um diameter), and we'll try the vitamins as well (C, E).  Our user is also trying to increase the expressed signal levels in the cells.
  Thanks very much everyone!!!

Zoltan

On Fri, Mar 27, 2009 at 4:10 PM, Sylvie Le Guyader <[hidden email]> wrote:

Hi Zoltan

 

I think using a short wavelength could be the problem. Not only will you induce more phototoxicity compared with longer wavelengths but you will also bleach your FRET acceptor and donor which presumably absorb blue light. One solution if you have a 633/640/647 laser is to label your nuclei with a far red fluorophore for live cells. You could use Draq5 http://www.biostatus.com/product/draq5/.

 

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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

Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes
Sent: 27 March 2009 13:19

To: [hidden email]
Subject: Overnight scan of human fibroblasts

 

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

--

Zoltan Cseresnyes
Facility manager, Imaging Suite

[Zoltan]

Hi Fred (and All),

Thanks for these ideas!! We are already using 1.5 or 2 AU pinholes, and that helped in the beginning.  It is indeed likely that the damage is related to the 405 at a fairly high power; unfortunately, the objects that we are following within the nuclei are very small (1-3 um diameter) and not very bright, so we need the best resolution that we can get (currently 118 nm per pixel).  We need to keep the area of scan fairly large (appr. 80x80 um) because the cells crawl a bit during such a long period of time, even when plated at high density, and with smaller/tighter scan areas we'd loose them from our view.  We'll try higher gain/lower laser still, and the vitamins.
  Thanks again,

Zoltan

On Fri, Mar 27, 2009 at 5:06 PM, Fred Mast <[hidden email]> wrote:

I agree with Julio, while it's difficult to judge how much 20% laser power is, it does seem excessive.  With such a low z-resolution I would open the pinhole to collect the maximum amount of light. This would help with reducing the laser power. Or you could implement a rectangular region of interest for your experiment (long on the x-axis and short on the y-axis) as reducing the the number of scans does improve speed of acquisition. 

Cheers,

Fred

On 27-Mar-09, at 10:28 AM, Julio Vazquez wrote:

Zoltan, 

As was pointed out, the shorter the wavelength, the more the damage, and therefore 405 nm is probably not the best choice for long term imaging., although certainly better than UV. Shifting to a red nuclear dye would make a big difference. 

This being said, you also need to exclude trivial reasons. I don't know how much power 20% is on your system (depends on the lasers you are using and the overall efficiency of the system), but 20% power for a 514 line seems very high to me, if you are using "standard" lasers with about 10 mW output. With our Argon laser, we typically use 2%-3% power for live imaging, and that is generally plenty. Even with our wimpy 1 mW 543 nm HeNe (basically a laser pointer), we often need no more than 10%. Therefore, I would recommend you check the  efficiency of your system and maybe the alignment of your optics. It's amazing how a slight misalignment of the pinhole can lead people to crank up lasers to lethal levels...

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org

==

 

On Mar 27, 2009, at 5:18 AM, Zoltan Cseresnyes wrote:

Dear All,

One of our users would like to scan cultured human fibroblasts (focusing only on the nucleus) for about 15-17 hours at a time, 6 um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.  We have done a few very successful experiments this way already, using our SP5 inverted with an environmental chamber (The Box).  We used 458 nm or 488 nm excitation in those successful experiments at low laser power (<=20%).  Last night we tried 405 nm excitation (20% laser power) as well because we were attempting to do a live FRET scan; the scanned cells all died after about 2 hours.  Do you have any suggestions about how to protect these cells from photodamage for 15-17 hours?  We are trying to keep our lasers low, we were doing 20% power on both the 405 and 514 nm lines.  The temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is okay as judged by the colour of the phenol red and the excellent survival of the cells in the non-scanned regions.    In one experiment last week we used 458 nm at 48% power (we had a poor fluorescence signal that time but we tried anyway) and then the scanned cells also died after about 6 hours.  These findings seem to point towards photodamage, hence my enquiry to this excellent forum of experts!
  Thanks very much,

Zoltan

--

Zoltan Cseresnyes
Facility manager, Imaging Suite
University of Cambridge, UK

Fred D. Mast

Department of Cell Biology

Medical Sciences Building Room 5-14

University of Alberta

Edmonton, Alberta, T6G 2H7

Canada

Tel: [phone]

[hidden email]

--

Zoltan Cseresnyes
Facility manager, Imaging Suite