Different florecent probs available i used H2DFDA and cehcked role of calcium in ROS .

@shazia, do you mean H2DCFDA? I think this probe could not provide the spatial information with sufficient resolution. But thank you all the same!

Maybe you could use the roGFP that is a redox biosensor. I used in plants, a roGFP expressed in the mictochondria.

Dear Xiaoxiao Sun,

for the future it would be good if you could provide more detailed information about the organism you work in as well as the compartments you're thinking about. This will defenitely improve the answers you'll get.

Mr. Bosch made a very good suggestion, roGFP is a very good redox biosensor. The nice thing in addition is, that you can target it to any compartment you like as there are all the signal peptides you may need.

There is this paper that did in vivo mapping in Drosophila:

http://www.sciencedirect.com/science/article/pii/S1550413111004190

And this is the method paper:

http://www.nature.com/nmeth/journal/v5/n6/full/nmeth.1212.html

Regarding the problem autofluorescence, this depends on your sample and many other things. Maybe you can tell us a little bit more.

Dear Falco,

Thank you very much for your suggestion. I work with human cancer cells. And the compound I study could somehow produce localized ROS -- probably not within mitochondria but adjacent to plasma membrane. So I wonder whether there is a probe could show me the precise localization of my drug-induced ROS accumulation.

Since, for example, the fluorescence of H2DCFDA could be strongly induced when it is exposed to the laser light of confocal, microscope-based detection using this probe would not be suitable in this case. Maybe you have any ideas of solving this problem?

First of all I have to say that I'm working with plants, so I have no experience with human cell lines etc. However, if possible and if you have access to such a device, you should try to use a spinning disc microscope for your experiments. It has some advantages compared to a point-scanning CLSM regarding speed (with exceptions) and phototoxicity. Still, the acquired images are confocal.

If this is also not working, you could try a widefield microscope since you are working in a single cell layer. If you acquire z-stacks or even single images, there SNR is not nice but these images can be deconvolved and gain enough quality for you to see the ROS accumulation.

If you only have access to a CLSM, you could try to accumulate images. With this you can decrease the excitation laser power accordingly. Image acquisition will take longer and you scan your image more often, but maybe the laser-induced ROS stress you have a problem with has a treshold? If the speed is not an issue you could accumulate a lot of images with a really low laser power.

Thank you, Falco. According to my case, speed matters, because the cells are alive. Still, maybe I can try and see whether there is a threshold for laser-induced oxidative stress.

If speed really matters, try to get hands on a spinning disc microscope.

Dear Xiaoxiao,

I agree with Falco that a spinning disc is the faster solution. I use a cell culture of kidney cells to study transport processes and mitochondrial function as add on to ex vivo and in vivo two photon measurements in the kidney. With the spinning disc I record 4 different marker with 1 frame/sec in a good quality. With a point scanner (Leica SP8 resonant scanner) I need at least 3 seconds with less good quality.

I partly disagree with the accumulation at the point scanner because you still have a accumulated excitation which is also toxic for the cells if you record for longer time. But this is also dependent on your available equipment (like the detectors at the CLSM or the sensitvity of the camera at the spinning disc)

My phd student and I used a ROS detector called CellRox from molecular probes. The substance is not fluorescent until its reaction with ROS species. We use the deep red Cellrox with abs/em ~644/665 which should be less phototoxic than a green or red one. The substance is not reversible like roGFP but easy to use and you don't have to transfect it.

About the speed, you should have an idea how fast the ROS is produced and if your dye or reporter is sensitive enough to show this differences within seconds. I think one frame/ 30 sec or 1 frame/min could be fast enough. Speed matters in living cells but it strongly depends on your question and the used dyes. If your reporter is not sensitive enough or ROS production slow it is not helpfull to record every second like you would do it in calcium imaging.

I would adress this question with a staining like this: a marker for the membrane (like cellmask by molecular probes), a mitochondrial marker (there are many of them) and the cellrox. Thereby you would hopefully see the ROS and you can analyze if it the ROS production is located to the mitochondria or the membrane. But this approach is not the cheapest. I would record with 1 fr/ 30 sec.

I hope you find a good solution.

Best,

Claus