Hello all! We are using confocal to introduce DNA damage followed by recruiting DNA repair proteins to the nuclear. We used two methods to introduce DNA damage but had inconsistent results. I’d appreciate it if someone can give me some suggestion.
Here’s the setup. We have one GFP-labeled and one mcherry-labeled DNA repair proteins co-expressed in the cells. We acquired two-channel (green and red) baseline confocal image before photobleaching. Since we don’t have a UV laser, we used two following methods to introduce DNA damage in the nuclear
1st method: 40x/1.3 water lens. Use Ti:Sapphire pulse laser tuned to 780nm (8% of ~1W average power ), 15us to photobleach a strip area in the nuclear. We were able to see dramatic increase (500% or more) in red, and green signal at the bleached site inside nuclear. And it happens instantaneously (1 frame per second for two channels), stayed at this intensity for over 15 min.
2nd method: 63x/1.3 oil lens. we first incubated cells with 10um brdu to sensitize the DNA overnight. On 2nd day, use 405nm laser (100% of 50mW laser power, 30/pixel us), 10 iteration to photobleach a strip area in the nuclear. We were only able to see 50% increase in red but no green signal increase at the bleached site inside nuclear. And it takes more than 5 frames to show up and quickly went back to base line intensity.
Can someone shed some light on the discrepancy on the cell behavior? Is it because of different extent of DNA damage introduced? We cannot do a head-to-head comparison due to different microscope setup. Thank you for your help in advance.
I’m sure there are others who can provide actual numbers to go with this, but here’s my intuitive interpretation:
There is certainly a difference in the power being delivered because of the different types of laser technology for the Ti:Sapph and 405. As you may know, the Ti:Sapph is a pulsed laser, so the photons come as a series of photon packets and it acts like a jackhammer with a repetition rate in the pico- or femto-second range (depending on your system). The 405 is a continuous wave laser, so it’s more like just pushing really hard. To break things, jackhammers are really handy.
A “sanity check” for this explanation would be to lower the % of the Ti:Sapph to see if you can replicate what you see with the 405, with the caveat that the pulsed nature of the Ti:Sapph might always be more impactful than the continuous wave 405.
If you’re stuck with the 405, you want the scanning for the photobleaching event to be as slow as possible so you’re “pushing” with as much power as you can. Why slower instead of faster? The fastest pixel dwell time I’ve seen on a commercial confocal is in the 0.5us range, so even if you iterated in just one spot, the repetition rate would be several orders of magnitude slower than the Ti:Sapph so the 'jackhammer" effect doesn’t really take effect.
Thanks for the reply. The pulsed laser is 120fs, which I no longer have access to. Thus, I used the longest dwell time for 405nm which is ~30us. I totally agree the laser dosage is much less in the case of 405nm.
I’m trying to compensate for the low laser power by using more iterations, longer dwell time, etc. Would the lens magnification make a difference? Or anything else I can use to increase the DNA damage by using just 405nm CW laser? Thanks.
That’s right. More iterations can increase the laser dosage, but I also hope to catch the onset of the signal increase since the pulsed laser induced the signal increase instantaneously. Thanks for your input.