It would be good to have a better idea of what it is you are trying to do and where the indicators you mentioned are failing. Are you working in culture, in tissue explants or in vivo? Also, do you load your indicators via AM (which I suspect given the indicators you list), via a patch pipette, injection or electroporation?

In general: Indeed, red indicators are hard. Red fluorophores tend to be less polar than green ones, leading to faster leaking from cells. They have a stronger tendency to compartmentalize (mainly to mitocondria), which also causes problems. One way around both of these issues is to use dextran conjugates. In most indicators the conjugation will change the binding properties of the calcium indicator though, so this might also be a problem. Depending on your experiment (e.g. if you are using AM-loading), dextran conjugates might not be an option for you, or would require a change in loading method. To add a shameless plug, we discuss several aspects of this in a recent publication on a new red-shifted indicator, which I have attached below. 

Depending on your specific experiment, the new red-shifted genetically encoded calcium indicators (jRCaMP from Janiela and R-CaMP-2 from the Bito lab) might also be worth considering. These are both substantial improvements over previous red GECIs. Given that your signals are "big and slow", you should have no problems at all detecting them with either indicator.

It would be good to have a better idea of what it is you are trying to do and where the indicators you mentioned are failing. Are you working in culture, in tissue explants or in vivo? Also, do you load your indicators via AM (which I suspect given the indicators you list), via a patch pipette, injection or electroporation?

In general: Indeed, red indicators are hard. Red fluorophores tend to be less polar than green ones, leading to faster leaking from cells. They have a stronger tendency to compartmentalize (mainly to mitocondria), which also causes problems. One way around both of these issues is to use dextran conjugates. In most indicators the conjugation will change the binding properties of the calcium indicator though, so this might also be a problem. Depending on your experiment (e.g. if you are using AM-loading), dextran conjugates might not be an option for you, or would require a change in loading method. To add a shameless plug, we discuss several aspects of this in a recent publication on a new red-shifted indicator, which I have attached below. 

Depending on your specific experiment, the new red-shifted genetically encoded calcium indicators (jRCaMP from Janiela and R-CaMP-2 from the Bito lab) might also be worth considering. These are both substantial improvements over previous red GECIs. Given that your signals are "big and slow", you should have no problems at all detecting them with either indicator.

Dear Damian,

We have been characterizing some new dyes recently. Like you, we need to multiplex different fluorophores and are particularly keen to find good red-shifted calcium indicators. To cut to the chase; we're quite impressed with the Cal590 and Cal620 (AAT Bioquest). We use Fluo4 and Fura2 mostly, but when we need to used a red-shifted indicator we switch to one of Cal590 or Cal620. In our hands, they are quite leakage resistant and have a decent dynamic range.

We've found Calcium Orange to have poor signal-to-noise. Rhod2 loads into mitochondria in most cell types because it is positively charged. Fura-red is interesting as it can be used for ratiometric measurements in combination with Fluo4 as its emission decreases with increasing calcium concentration.

Slow and small calcium changes can be a challenge for single wavelength indicators as focal drift, cell movement etc can give similar changes in fluorescence emission that have nothing to do with a calcium change. However, 'big and slow' calcium signals, as you described, should be fine.

I've attached a simple poster showing some of our preliminary findings. I hope this helps.

Best wishes,

Martin

I endorse everything Martin and Christian say. As you are transfecting, there are also some other new red/orange GECI from the Campbell lab (available on Addgene). The dynamic range of their O-GECO1 is pretty staggering, though we find it is very dim at basal (partly because its Kd is quite modest, about 1uM), but the cells light up like a Christmas tree upon stimulation.

We did try Cal590 too, but I think we must've had a bad batch because I saw no loading (whereas fluo4 was fine). Martin's success is more likely to be a better reflection of the dye's properties.

Regards,

Ant

Dear all,

Thanks so much for this, it is all very helpful. I would like to do a couple of things: Puffing a AMPAR agonist onto cultured neurons (GFP+) to assess Ca2+ handling (measured from the soma) and also to determine spontaneous action potential firing in similar cultures (i.e. detect single spikes), if possible. Unfortunately, I can't cuse GECIs (these indicators are getting really good!) , so have to rely on bulk loading.

@Christian

Thanks. The aRuby-Nano looks very promising. Did the AM conjugate show significant change in the binding properties? Do you know if it commercially available? A perfunctory Google search didn't return any useful results.

@Martin

Thanks for the poster. I suffered the same problems with Rhod-2 and poor S/N with Calcium Orange and Fura-red. I'll definitely give the Ca-590 a try. 

@Ant

Thanks for the confirmation  - We know each other from Oxford! 

Somewhat related, I'm looking for an automated method for carrying out  analysis of action potential driven Ca fluctuations in neuronal networks (cultured cells). Template matching doesn't work too well for my data. Any recommendations? I was wondering about the Paninski method (below) or something similar. Their method is pretty complex and I''m not sure whether it is worth taking the time to learn about 'constrained nonnegative matrix factorization' etc.

Thanks again!

Damian

Article Simultaneous Denoising, Deconvolution, and Demixing of Calci...

Hi Damian,

Sorry I didn't twig it was you, it's been many years ! Greetings, hope all well Stateside.

Sorry I misunderstood that you were transfecting when you just have a transgenic (I guess).  I too would love a good Red dye. Tried Asante Red too, but that was pants too.

One day our prince will come...

All best to you,

Ant

Damien, 

Unfortunately, Ca-Ruby Nano is not commercially available. Yet. The first author of the paper is working on it.

We did not specifically test the binding properties of the AM version. I would not expect a large change, though.

Do try Cal-590. I have heard and read good things about the whole indicator family. I would definitely stay away from Asante-Red. Unless the redesigned the indicator from scratch in the past five years. When I tried it, it was very dim.

Regarding spike inference: Eftychios' method is pretty much the best there is at the moment. My understanding is that it is very straightforward to use. I haven't tried it myself, but students I were supervising as well as several colleagues faired well with it.

Just checking in to let you know that I tried Cal-590. It works really well and I would thoroughly recommend it!

Thanks for all your help!

Hi,

Really glad Cal-590 worked for you.

Best of success with the experiments.

Martin

Dear Martin, We met more than 25 years ago while I was working with Jean J. Nordmann in Bordeaux, Strasbourg and during my visiting Babraham Institute at John Bicknell's lab; and when I was in Human Anatomy at Oxford Univ (1987-1989)..Happy to get back to you via RG and interesting discussion on choosing an appropriate calcium-sensitive dye. In recent years, I am using genetically modified rats for vasopressin (AVP-eGFP) and measure spontaneous calcium oscillations for several minutes (some times up to 60-90 mins) using Fura-2 AM in freshly isolated green AVP neurones from the SON. We use both fast fluorescence photometry and CCD camera imaging as well. For more details you can please check with our recent papers in Cell Calcium (Kortus S et al 2016; uploaded in the RG) I have no experience with Cal-590. Very interesting poster. Thank you. I should attempt. Look forward to seeing you/discussing with you not in far future. With Best regards, Daya

Dear Dr. Williams, Good luck with the experiments. You have already nice discussions on this issue. Best regards, Daya

Hi Daya,

It's great to hear from you. Good memories from such a long time ago! John Bicknell left BI many years ago, and I have moved on too. I don't think you would recognize BI now.

Great papers! Evidently, things are going well with you; I'm very glad. I've downloaded your Cell Calcium paper and will read with interest. It would be great to catch up.

Best wishes,

Martin

Dear Martin, Very glad to read your quick response. Thank you. True, its hard to recognize your RG photo! Feel please free to visit Prague (a beautiful city) at your convenience. I am in Prague over 8 years, first time I spent long years in one laboratory. But I am very independent to run my department and research programs in Prague. I have permanent home in Montpellier and a research laboratory but visit only a few times. Look forward to seeing/meeting you.

With Best regards,

Daya