Controlled DNA damage by lasers
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Michal Jarnik |
Controlled DNA damage by lasers
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Listers,
We would need to perform experiments including controlled DNA damage to cells in culture. One idea was to use the LSCM and expose an area in a way similar to the bleaching part of a FRAP experiment. We do have a LED 405 nm laser (I would assume that would be the right wavelength, we do not have any really UV laser), Nikon C1 can do quite a lot of zooming to concentrate the energy in a small area. I have no idea, though, about the power necessary/available and other conditions. Any recommendations would be very much appreciated. Thanks, M. -- Michal Jarnik, Ph.D. Cell Imaging Facility Electron Microscope Facility Fox Chase Cancer Center 333 Cottman Ave. Philadelphia, PA 19111 215-728-5675 (v.) 215-728-2770 (v.) 215-728-2412 (f.) |
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Michal,
It would help to know what you mean by controlled DNA damage. # Strand breaks, repair errors, cell killing? A 405 nm laser is by itself not particularly specific for DNA whose bases absorb much more strongly from 240-280 nm and not much at all above 320 nm (as far as I remember). However, dye molecules located near or bound to DNA can absorb longer wavelengths generating photo-toxic products such as singlet oxygen, superoxide, *OH, phenoxyls, etc. You might be able to create a dye/pigment enhanced system using the 405 nm laser. Multiphoton excitation might work, e.g., three photon absorption ~800 nm. But that means an entirely different sort of system and the chances of frying your cells becomes very probable. As for zooming, you must mean this is in the most generic sense since for any given objective its numerical aperture will always create the same energy density profile given the same amount of illumination, unless you mean to move it out of focus on the target. I am not sure that this is the best way to provide even illumination. Anyway, more details could be helpful. Mario >Listers, > >We would need to perform experiments including controlled DNA damage >to cells in culture. One idea was to use the LSCM and expose an area >in a way similar to the bleaching part of a FRAP experiment. We do >have a LED 405 nm laser (I would assume that would be the right >wavelength, we do not have any really UV laser), Nikon C1 can do >quite a lot of zooming to concentrate the energy in a small area. I >have no idea, though, about the power necessary/available and other >conditions. Any recommendations would be very much appreciated. > >Thanks, M. > >-- > >Michal Jarnik, Ph.D. >Cell Imaging Facility >Electron Microscope Facility >Fox Chase Cancer Center >333 Cottman Ave. >Philadelphia, PA 19111 >215-728-5675 (v.) >215-728-2770 (v.) >215-728-2412 (f.) -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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In reply to this post by Michal Jarnik
Hi Michal,
I didn't really follow the entire link but I induce DNA damage to cells for Tunnel analysis by flow cytometry by simply exposing our cells to the UV lamp of the tissue culture hood. Normally 30 minutes is sufficient and after 48 hours our cells (A549) show very high levels of apoptosis. The only drawback it that it is not very controlled - other than time. Hopefully this helps. Tej. > Date: Mon, 21 Jun 2010 12:20:17 -0400 > From: [hidden email] > Subject: Controlled DNA damage by lasers > To: [hidden email] > > Listers, > > We would need to perform experiments including controlled DNA damage to > cells in culture. One idea was to use the LSCM and expose an area in a > way similar to the bleaching part of a FRAP experiment. We do have a LED > 405 nm laser (I would assume that would be the right wavelength, we do > not have any really UV laser), Nikon C1 can do quite a lot of zooming to > concentrate the energy in a small area. I have no idea, though, about > the power necessary/available and other conditions. Any recommendations > would be very much appreciated. > > Thanks, M. > > -- > > Michal Jarnik, Ph.D. > Cell Imaging Facility > Electron Microscope Facility > Fox Chase Cancer Center > 333 Cottman Ave. > Philadelphia, PA 19111 > 215-728-5675 (v.) > 215-728-2770 (v.) > 215-728-2412 (f.) The New Busy think 9 to 5 is a cute idea. Combine multiple calendars with Hotmail. Get busy. |
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Craig Brideau |
Re: Controlled DNA damage by lasers
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I agree with the other posters. 405nm is not short enough; it's
really more 'violet' than UV. You would be better off with Tej's suggestion of a good arc lamp with appropriate UV pass filters. Just focus the lamp onto a section of your cells and let them 'bake' a while. You will need a good filter to only let the appropriate UV band through, otherwise the excess energy at other wavelengths will do other forms of damage to your cells. Craig On Mon, Jun 21, 2010 at 11:45 AM, Tej Hiran <[hidden email]> wrote: > Hi Michal, > > I didn't really follow the entire link but I induce DNA damage to cells for > Tunnel analysis by flow cytometry by simply exposing our cells to the UV > lamp of the tissue culture hood. Normally 30 minutes is sufficient and > after 48 hours our cells (A549) show very high levels of apoptosis. The > only drawback it that it is not very controlled - other than time. > Hopefully this helps. > > Tej. > >> Date: Mon, 21 Jun 2010 12:20:17 -0400 >> From: [hidden email] >> Subject: Controlled DNA damage by lasers >> To: [hidden email] >> >> Listers, >> >> We would need to perform experiments including controlled DNA damage to >> cells in culture. One idea was to use the LSCM and expose an area in a >> way similar to the bleaching part of a FRAP experiment. We do have a LED >> 405 nm laser (I would assume that would be the right wavelength, we do >> not have any really UV laser), Nikon C1 can do quite a lot of zooming to >> concentrate the energy in a small area. I have no idea, though, about >> the power necessary/available and other conditions. Any recommendations >> would be very much appreciated. >> >> Thanks, M. >> >> -- >> >> Michal Jarnik, Ph.D. >> Cell Imaging Facility >> Electron Microscope Facility >> Fox Chase Cancer Center >> 333 Cottman Ave. >> Philadelphia, PA 19111 >> 215-728-5675 (v.) >> 215-728-2770 (v.) >> 215-728-2412 (f.) > > ________________________________ > The New Busy think 9 to 5 is a cute idea. Combine multiple calendars with > Hotmail. Get busy. |
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Julio Vazquez |
Re: Controlled DNA damage by lasers
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In reply to this post by Michal Jarnik
Michal, DNA damage has been achieved with a 405 laser on a laser scanning confocal (such as in Bergink et al, Genes Dev 20: 1343-1352, 2006). People have also had success with a Photonic Instruments Micro-Point laser (Ayoub et al, Nature 453, 682-686, 2008). A FRAP of photokinesis laser module from a variety of vendors will also work fine. If you check the literature, you will see that different wavelengths may induce different types of DNA damage, also depending on which sensitizers you add to your cells, so doing a review of the literature would be a good idea. A PubMed search for " laser DNA damage" or similar will give you plenty to read. One review on different laser irradiation methods was published recently (Kong et al, NAR 37(9): e68-2009). Exact conditions will depend on your samples and experiment... as usual for this type of work, I would recommend using the most gentle settings that allow you to see a response. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Jun 21, 2010, at 9:20 AM, Michal Jarnik wrote:
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Z.J. Zhang |
Re: Controlled DNA damage by lasers
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In reply to this post by Tej Hiran
Hi Michal: Did you consider using DNase? It would cause “DNA damage” in a
more controlled way. Zhaojie Zhaojie Zhang, Ph. D. Director, Microscopy Core Facility University of Wyoming Laramie, WY 82071 PHONE: 307-766-3038 FAX: 307-766-5625 From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Tej Hiran Hi Michal, The
New Busy think 9 to 5 is a cute idea. Combine multiple calendars with Hotmail. Get busy. |
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Adams,Henry P |
Re: Controlled DNA damage by lasers
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In reply to this post by Michal Jarnik
Michal,
We have a Micropoint targeted illumination setup from Photonic Instruments for inducing DNA damage. It uses a nitrogen 365nm laser that is directly coupled to the epifluorescence path on a Nikon TE2000 inverted microscope. The laser power, number of pulses and ROI are controlled by a Metamorph plug-in. Investigators draw a line through the nucleus with the ROI tool and lase it. They usually lase 20 to 50 cells in one session, fix and stain. Or they use the timelapse feature when their proteins are coupled to FPs. I have no stake in the company. Hank Adams Genetics Department Rm S14.8316A U.T. M.D.Anderson Cancer Center Houston, Tx 77030 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michal Jarnik Sent: Monday, June 21, 2010 11:20 AM To: [hidden email] Subject: Controlled DNA damage by lasers Listers, We would need to perform experiments including controlled DNA damage to cells in culture. One idea was to use the LSCM and expose an area in a way similar to the bleaching part of a FRAP experiment. We do have a LED 405 nm laser (I would assume that would be the right wavelength, we do not have any really UV laser), Nikon C1 can do quite a lot of zooming to concentrate the energy in a small area. I have no idea, though, about the power necessary/available and other conditions. Any recommendations would be very much appreciated. Thanks, M. -- Michal Jarnik, Ph.D. Cell Imaging Facility Electron Microscope Facility Fox Chase Cancer Center 333 Cottman Ave. Philadelphia, PA 19111 215-728-5675 (v.) 215-728-2770 (v.) 215-728-2412 (f.) |
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Hank, Michal
Do you know the power per pulse and the lifetime of the
Micropoint system (2-20 nsec is common for N2
lasers, I think)? As I recall, nitrogen lasers have a number of UV
lines including 337 and 357 nm; the former might be particularly
useful for Michal's DNA study. When I worked on creating lanthanide
labels for x-ray microscopy, I considered using a 337 nm nitrogen
laser as the excitor for part of a test platform. However, I developed
an alternative especially as the nitrogen laser I was considering was
particularly suited to ablation, and was sold specifically for
that purpose.
This makes me think that unless one uses a sensitizer enabling
longer wavelengths to be used (including 357 or 365 nm lines), it may
be very difficult to produce anything that would be considered
reproducible dose dependent DNA damage. What I imagine is DNA being
surrounded by a poisonous soup of photolysis products. Even if you had
quartz objectives enabling the use of selective Hg UV lines readily
absorbed by DNA bases (e.g., 253 nm) or a noble gas flash lamp (1
usec. pulses), the surrounding nuclear proteins will also absorb the
same light producing DNA damaging products in difficult to predict
ways. Then again maybe this is what Michal wants.
Photodynamic therapy (PDT) has been around for nearly a century
and there is a large body of literature pertaining to the use of
sensitizers and long wavelength visible light. That would probably be
a good place to look. Of newer approaches, soft x-ray illumination
using the "water window" of the nucleus might be
particularly interesting, as the cells can be targeted down from whole
nuclei to very tiny sub volumes (< 50 nm x 50 nm x 10 um thick).
This can be done with cells at cryogenic temperatures such that the
primary damage would occur via direct targeting of DNA. Non DNA free
radical damage would be minimized.
Mario
Michal, -- ________________________________________________________________________________
Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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Adams,Henry P |
Re: Controlled DNA damage by lasers
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Mario, It is actually a nitrogen laser-pumped dye laser. The nitrogen
laser is ~120 ujoules of 337nm with a 2 to 6 nsec pulse length. The pulsed beam
is directed through a low loss multimode fiber to a dye laser. The dye laser
beam is 364nm and exits the objective. Hank From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Mario Hank, Michal Do you know the power per pulse and the lifetime of the
Micropoint system (2-20 nsec is common for N2
lasers, I think)? As I recall, nitrogen lasers have a number of UV lines
including 337 and 357 nm; the former might be particularly useful for Michal's
DNA study. When I worked on creating lanthanide labels for x-ray microscopy, I
considered using a 337 nm nitrogen laser as the excitor for part of a test
platform. However, I developed an alternative especially as the nitrogen laser I
was considering was particularly suited to ablation, and was sold
specifically for that purpose. This makes me think that unless one uses a sensitizer
enabling longer wavelengths to be used (including 357 or 365 nm lines), it may
be very difficult to produce anything that would be considered reproducible
dose dependent DNA damage. What I imagine is DNA being surrounded by a
poisonous soup of photolysis products. Even if you had quartz objectives
enabling the use of selective Hg UV lines readily absorbed by DNA bases (e.g.,
253 nm) or a noble gas flash lamp (1 usec. pulses), the surrounding nuclear
proteins will also absorb the same light producing DNA damaging products in
difficult to predict ways. Then again maybe this is what Michal wants. Photodynamic therapy (PDT) has been around for nearly a
century and there is a large body of literature pertaining to the use of
sensitizers and long wavelength visible light. That would probably be a good
place to look. Of newer approaches, soft x-ray illumination using the
"water window" of the nucleus might be particularly interesting, as
the cells can be targeted down from whole nuclei to very tiny sub volumes (<
50 nm x 50 nm x 10 um thick). This can be done with cells at cryogenic
temperatures such that the primary damage would occur via direct targeting of
DNA. Non DNA free radical damage would be minimized. Mario
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Gert van Cappellen |
Re: Controlled DNA damage by lasers
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In reply to this post by Michal Jarnik
With the combination of Hoechst33342 and a 405 laser you can make
controlled DNA damage. We compared this with multi-photon and 266 laser
induced DNA damage.
<a href="javascript:AL_get(this, 'jour', 'J Cell Sci.');" title="Journal of cell science." style="border-bottom: 1px dotted black; color: black; font-weight: normal; text-decoration: none;">J Cell Sci. 2007 Aug 1;120(Pt 15):2731-40. Activation of multiple DNA repair pathways by sub-nuclear damage induction methods.Dinant C, de Jager M, Essers J, van Cappellen WA, Kanaar R, Houtsmuller AB, Vermeulen W. Department of Pathology, Josephine Nefkens Institute, ErasmusMC, Rotterdam, The Netherlands. AbstractLive cell studies of DNA repair mechanisms are greatly enhanced by new developments in real-time visualization of repair factors in living cells. Combined with recent advances in local sub-nuclear DNA damage induction procedures these methods have yielded detailed information on the dynamics of damage recognition and repair. Here we analyze and discuss the various types of DNA damage induced in cells by three different local damage induction methods: pulsed 800 nm laser irradiation, Hoechst 33342 treatment combined with 405 nm laser irradiation and UV-C (266 nm) laser irradiation. A wide variety of damage was detected with the first two methods, including pyrimidine dimers and single- and double-strand breaks. However, many aspects of the cellular response to presensitization by Hoechst 33342 and subsequent 405 nm irradiation were aberrant from those to every other DNA damaging method described here or in the literature. Whereas, application of low-dose 266 nm laser irradiation induced only UV-specific DNA photo-lesions allowing the study of the UV-C-induced DNA damage response in a user-defined area in cultured cells. on 21-06-2010 18:20 Michal Jarnik said the following: Listers, -- Dr. W.A. (Gert) van Cappellen Room Ee914; Tel. +31-10-7043578 Mob. +31 6 22544785 Optical Imaging Centre http://www.erasmusmc.nl/oic Reproduction & Development http://www.erasmusmc.nl/rede Erasmus MC, Dr. Molenwaterplein 50 3015 GE ROTTERDAM, The Netherlands |
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