Hi Julius,

I used to do calcium measurements with fast video imaging. The stimulus was applied by two platinum electrodes approximately 1 mm appart. First of all the voltage pulse was alternating in polarization. Secondly the pulse was rectangular with a pulse length of 5 ms. And last but not least the voltage was 6 V with this setup. With mouse ventricular myocytes I used 1 Hz at room temperature and 4 Hz at 37 degree. Rat myocytes were usually stimulated at 0.5 Hz at room temperature. Our recording protocoll was the following. Start of recording, then 10 seconds later start of stimulation. 40 s of measuring 3 minutes pacing without recording and then again 40 s of recording. This way we could get post rest behaviour and steady state behaviour.

I hope this helps.

Best regards

Benjamin

PS: I used 1 µM Fluo 4 AM on a 1 ml well with a 1 inch coverslip.

The recording looks like the changes in amplitude may be physiological. You may consider change in the cell condition slower than each individual response. 

Hi Julius.

According to my experience, your setup looks very fine. We have used these settings for CaT measurements of guinea pig cardiomyocytes. 

These disturbances could be due to technical problems like as suggested by Benjamin Sauer. You may try to decrease concentration of Fluo 4 AM to 0.5 to 1 µM. Also, decrease in Voltage from 20 V to 5-10 V and increase in pulse length might help.

Second reason could be the quality of cardiomyocytes. As Alexey Semyanov suggested, such response might indicated (patho)physiological condition. We faced some problems in CaT recording when we used high concentrations of Liberase or Collagenase for isolating cardiomyocytes. 

Try to record a cell for longer time (e.g. 5 min or longer) and then cell should be stabilized against stimulation. If it does then you may use recordings from last traces for analysis.

All the best and regards.

Chintan

There are several possibilities why:

- your recording is too long, which would damage the cell, producing some free radicals or side products;

- if solution is not changed during the recording it could lead to the change in pH, which will influence the cell state;

- finally, because of contraction the cell could go out of the focus, which will influence the obtained signal...

Dr. Sauer,

Hello! Thank you for the sharing your protocol in detail; however, I am the first one to do this in my laboratory and have no previous experience with a confocal microscope and a field stimulator so I do not understand some of the terms. So I was able to change the pulse wave form from biphasic to alternating, and the duration from 2 ms to 5 ms; but how can i make the pulse rectangular? I am using an IonOptix field stimulator. Also, I do my staining in an eppendorf tube, should that be a problem (hypoxia perhaps)? Anyway, this has been great help! Thanks again!

Dr. Semyanov, Dr. Koyani, and Dr. Gusev

Thank you all for your responses. So for my experiment I use 0.01% collagenase only in calcium free tyrode for about 9-15 minutes. Actually I get various cell yield and they usually experiece hypercontracture in physiological calcium levels. Also, now I am using 10 V. Sometimes, the cells just show calcium transients but without contraction. But I will try all of these tips you have given today. All of these were beyond helpful. Thank you!!!

Hi Julius.

0.01% collagenase for 9-15 min sounds fine according to me. Cell yield variability is unavoidable. If you see calcium transient without contraction then cells are facing contractile dysfunction and cell quality is bad. However, it depends on your aim whether you are focusing on only CaT or also contractility of cardiomyocytes.

I would suggest to play around with collagenase concentration, time of perfusion as well as force of perfusion.

Also make sure that perfusion of NT is running while measuring CaT. I would suggest to maintain temperature. Also, the cell which you are measuring, should not be surrounded by any viable cell or touched to dead cell bcos they influence recordings too.

Hi Julius,

I think the reason you might be getting this variability in contracture is due to the cell quality, you say that they are hypercontractile in physiological calcium. That is a bad sign. I agree with Chintan, reducing collagenase concentration might result in lower yields but better quality myocytes.

Hi Julius,

no Doctor on my side, yet.

We used some self built pulsing devices I guess it is not really necesary to have rectangular pulses. In my former lab we used to plate out the cells on coverslips before experiment. The protocoll for our way of isolation can be found in.

Viero, C., Kraushaar, U., Ruppenthal, S., Kaestner, L., & Lipp, P. (2008). A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes. Cell Calcium, 43(1), 59–71. doi:10.1016/j.ceca.2007.04.001

The loading is done in a homebuilt chamber to hold the coverslip on an inverted microscope. And yes like Davor and Chintan pointed out contraction of the myocytes without stimulation happens but hyperactive cells are a bad sign. Perfusion is also important to wash away residues of the cells metabolic products as well as photo derived toxic byproducts and providing fresh glucose and so on.

Good luck with your further work.

Best reagards

Benjamin

Hi Julius. What do you mean by "inconsistent"?

Hello Professor Oron!

I am sorry but for the lack of a more appropriate word I just said inconsistent. I just said so because I could not get a steady-state/baseline recording. As you can see, the peaks are of different amplitudes. I thought it was also because of the sampling frequency, which is 1 ms, but they told me here that it should be 0 ms, to get more sampling points for analysis. Sorry for the ambiguity!

I agree with the comments made by Davor and Alexey.  From the CaT trace you posted, it does seem real, physiological or pathophysiological (in response to Iso?).  I'm puzzled by your comment about baseline CaT, though.  You said you couldn't get a baseline recording, but the cells responded to Iso.  Does it mean you couldn't detect baseline CaT?  If that is the case, it could be simply due to poor cell quality as mentioned by some comments here.  Besides, Iso is really powerful in terms of inducing Ca overload and hyper-contraction.  Even the good cells don't last long with Iso treatment.  I'd say, follow the suggestions by others and focus on detecting baseline CaT before going to do the drugs.  Good luck.

Hi Julius. I had limited experience with cardiomyocytes. Nevertheless, some general remarks that might be of value. Your rapid line scan has three inherent problems: the amount of info in a single pixel is very small and, therefore, variable; the dye is sensitive to shape change and you will have results that underestimate Ca in non-contracted and overestimate in contracted cells; with consecutive scans photobleaching will lower the signal and produce toxic breakdown products. Additionally, and seldom considered, is the dye/Ca binding kinetics and intracellular diffusion kinetics. The former limit the time resolution to ~5 ms for Fura2! Fluo 4 is faster, but not by that much, so here is another source of variability.

in my opinion you are measuring approximate changes in Ca, but the relative effects are still valid for drug research. The best would be to run a few scans and smooth the results by running averaging with a bin of 3-5. See if it helps with variability. If you are not into measuring Ca sparks, I would still opt for the slower but more accurate ratiometric Fura2 method. Just in case you missed it-Methods in Molecular Biology Volume 689, 2011, pp 205-214.

Above all, remember that the data you see in papers are usually the best, while the ones you get are the average.

I hope my input might be of use.

Professor Oron,

By shape change, do you mean that the cell starts to bend with prolonged scanning? Because that it what I observe when I do line scanning at 0 ms. I make a line longitudinal to the cell, almost always encompassing the entire cell (0.19-30 microns pixel size), this is the correct way right? Or can I do it at shorter pixel sizes and do it transverse to the cell? Thank you very much for sharing this!!!

Dr. Wang,

Thank you for taking the time to answer! I treat the cells with 200 nM isoproterenol for 5 minutes, but see effects at 3 minutes (I record the transients for only 10 seconds with every treatment). However, sometimes, the isoproterenol works only after washout, or sometimes, the cells experience arrhythmia, but only after isoproterenol, which is indeed just because of poor cell condition. As for the baseline recording, I was able to remedy it by decreasing the line scan interval time to 0 ms; but what happens is that the cell bends after some time of line scanning.

Hi Julius,

if the cells bend this hints to inhomogeneous Calcium distribution inside the cell, probably due to poor quality. But also patho-/physio-logical parameters can influence this. Calcium alternans, inhomogeneous Calcium along the long axis of the cell might occure if the cell is paced faster than it can handle it. The Calcium homeostasis is perturbed and no longer "physiological". On the other hand the pacing frequency the cell can tolerate depends on parameters like temperature, medium and intrinsic patho-/physio-logical parameters. If the reactions to Isoproteronol are this inconsitent you might need to think about how you do the perfusion during experiment. In my old group we used small rubber tubes connected to a perfusion manifold if multiple solutions with and without hormonal stimulation were to be used. This perfusion manifold consits of multiple in- and one out-put hoses. This allowed to connect several reservoirs to the perfusion system. The output hose was then placed in between the stimulation electrodes, close to the cells that needed to be measured. But be carefull the flow and pressure of the solution might influence the cell and therefor the measurement if its placed too close.

Best regards

Benjamin

Mr. Sauer,

I tried measuring the post-rest behavior of contraction but I followed the protocol from the reference you shared with me earlier, thank you very much for that! I got one question, though. Does it mean then that I should expect, and only use, the transient recordings showing the same steady state recording? I've attached a file of my previous recording, just in case you would like to see it. With regard to the perfusion system, we are using something like that too. As for the bending of the cell, I only observe it during prolonged line scanning at 0 ms intervals, but if I use 1 ms or longer, the cell is fine. Is it possibly photobleaching? Because after line scanning, I can also see a concentration of the dye at the area subjected to line scanning. Really grateful for your input, sir!

Sincerely,

Julius

Hi Julius,

the recordings you added look like those I'm used to. Concerning steady state and post rest you should know that only the very first transient after you started the stimulation is the post rest transient and if you want this for your data you really need to catch it. Best is you start your recording, the scanning, some seconds before you start electrical stimulation and have an eye on not exciting the cell accidentally before that. Also take out cell which show frequent auto contractions. As you can see the amplitude decreases meaning the SR Calcium load is higher after rest than in the first few seconds after the stimulationg started. There are cases in which this is the oposite way. Both behaviours have a name but I don't remember them at the moment.

Maybe this paper helps.

Gingerol, isoproterenol and ouabain normalize impaired post-rest behavior but not force-frequency relation in failing human myocardium.

Maier LS1, Schwan C, Schillinger W, Minami K, Schütt U, Pieske B. Cardiovasc Res. 2000 Mar;45(4):913-24.

The steady state amplitude, or whatever parameters you determine from the recordings, is calculated as the mean of all transients you recorded. We usually recorded only 40 or so but if you add isoproterenol you will need to record more and probably some time (1 min+) after you increased the hormone concentration in the bathsolution. First because you want a homogeneous concentration around your cells and second because you might see how the cell adapts slowly to the hormonal stimulus. There unfortunatly i have not as much experience becaus I never investigated hormonal influence on our mouse models

What confocal microscope do you use? What does scanning at 0 ms intervall mean?

The bright line might hint to a too high excitation power. It might be this is not Fluo 4 Ca dependend fluorescence but autofluorescence of the cell or what is known to some people as anti-bleaching which is a Calcium independent fluorescence of Fluo 4.

Best regards

Benjamin

Dear Julius

It depends on the aperture of the pinhole or the slit. If you're doing a real confocal scan (0.5-1.0 um depth) then you have no problem with shape but very little signal from small volume. If, however, you choose to open the slit, you will be collecting from a crosssection that is either larger or smaller than the cell depth at each point. When the cells contract, the optical pathway increases, and therefore the amount of the dye in the pathway increases and the relative signal on non-ratiometric dye increases. With relaxation it is the opposite.

Hope it helps

Yoram Oron

Hi Mr Sauer!

We are using LSM 700 (Zeiss). So with line scanning, it's a time series experiment right, and there are 2 measures: cycles and interval. So what I do is I set the interval time to 0 ms. However, there is also the scan speed, and usually I set that to 1.53 ms. Actually I am confused too when I read: The line scan was performed at a sampling rate of, say, 1.54 ms. So does sampling rate pertain to the scan speed or interval time of scanning? 

Hi Julius,

I guess both. As I would interpret it the scan speed tell you sth. about the dwell time, the time that collected photons are adjected to one pixel. Lets say you have the scanning spped that that it takes 1.52 ms to scan a line. And you have 512 pixel then the pixel dwel time is approximately 1.52 ms /512. The intervall is the the between two linescans. So even with the intervall set to zero the galvo scnner takes some time to move back to the start of line. This so called flyback time is nonzero usually. So the sampling rate is limited by those parameters. Intervall set to zero means the galvo has no time to rest after the fly back. This might, if the steering of the glavo is not implemented good enough, influence the measurement because the galvo isn't doing what it's supposed to do. You might try putting in a very small intervall to give the galvo some settling time before a new scan. And as I mentioned before try to increase detector gain 750 to 900 e.g. and reduce the laser power. To get rid of additonal noise you can smooth the measurement mathematically or by binning in time  you still should be fast enough.

Best regards

Benjamin