This work was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1A6A3A01011832), by the Ministry of Science, ICT &#38; Future Planning (NRF-2016M3C1A6936606) and by the Ministry of Trade, Industry &#38; Energy (10068076).

All experimental protocols were approved by the local institutional animal care and use committee.
1. Solution preparation
<ol>
	<li>Prepare single freshly isolated cardiac myocytes11. 
	NOTE: Each laboratory might have a different cell storage solution. Here, the myocytes are stored in culture medium (DMEM).</li>
	<li>Prepare 100 mL of Ca2+-free solution (Table 1).</li>
	<li>Prepare 50 mL of culture medium in a 50 mL beaker. Aliquot 5 mL and put it in a water bath at 37 &#176;C. Keep the remaining solution at room temperature.</li>
	<li>Prepare 50 mL of the saponin solution by adding 5 mg of saponin to 50 mL of Ca2+-free solution. 
	NOTE: Saponin is used to permeabilize cardiac myocytes, to remove cytosolic compartments, and to visualize the mitochondrial fluorescence only.</li>
	<li>Prepare 16 &#956;L of 1 mM fura-2-FF-AM dissolved in dimethyl sulfoxide (DMSO). 
	NOTE: Make 1 mM stock solution of fura-2-FF-AM dissolved in DMSO and aliquot 16 &#956;L in a 2 mL tube. Store them at -20 &#176;C until use.</li>
	<li>Prepare 50 mL of NADH-free Ca2+-free solution (Table 1) and 50 mL of NADH-free Ca2+-saturated solution (Table 1) when isosbestic points are to be measured. Adjust pH to 7.0 with KOH. 
	NOTE: NADH-free Ca2+-free solution (Table 1) contains 10 &#181;M FCCP and 100 &#181;M ADP without any mitochondrial substrates to minimize NADH in mitochondria.</li>
	<li>Prepare 50 mL of Ca2+-free solution, 50 mL of malate solution, 50 mL of pyruvate solution, 50 mL of malate-pyruvate solution, and 50 mL of rotenone solution to be used for NADH correction factor measurements (Table 1).</li>
</ol>
2. Fluoroprobe loading procedure into the mitochondria
<ol>
	<li>Prepare the dye-loading solution by adding 2 mL of the culture medium to 16 &#181;L of 1 mM fura-2-FF-AM. 
	NOTE: Fluorescent dye is fragile under the light. Prepare the solution just before use. Keep the solution containing the fluorescent dye in a dark place. The final concentration of fura-2-FF-AM is 8 &#181;M. If carboxy-SNARF-1 was used, prepare the dye loading solution with 2 &#181;M carboxy-SNARF-1-AM.</li>
	<li>Take 2 mL of the isolated cells and place in a 5 mL test tube in an upright position.</li>
	<li>Wait 15 min for myocytes to sink to the bottom and remove the supernatant. 
	NOTE: The supernatant may contain cell debris. Do not centrifuge the tube to avoid cell damage.</li>
	<li>Add 2 mL of the dye-loading solution.</li>
	<li>Incubate the dye-loading solution with cells for 60 min at 4 &#176;C.</li>
	<li>Then, put the test tube in a 37 &#176;C water bath for 30 min in an upright position.</li>
	<li>Remove the supernatant, and add 4 mL of the prewarmed culture medium of 37 &#176;C. Incubate the cells for 60 min in a 37 &#176;C water bath.</li>
	<li>Finally, remove the supernatant, add 4 mL of culture medium at room temperature and keep the tube at room temperature.</li>
</ol>
3. Introduction of the multiparametric measurement system
NOTE: Figure 1 shows a diagram of the whole system.
<ol>
	<li>For an excitation light source, use a fast monochromator (polychrome II) that can change the light within 3 ms.</li>
	<li>Use an oil immersion lens (40x, NA 1.3) with an inverted microscope to increase the signal intensity.</li>
	<li>Use a near-infrared filter and a charge-coupled device (CCD) camera to monitor the object field without fluorescent signal interference.</li>
	<li>Capture the object field image to get the area.</li>
	<li>Adjust the object field in monitor screen with a field diaphragm just to show the cell for reducing the background.</li>
	<li>Use four photomultiplier tubes with each band-pass filter (450, 500, 590, and 640 nm) to detect emission wavelengths with photon counting method. Use the appropriate dichroic mirrors to split and to redirect the emission light. 
	NOTE: The excitation light is very strong compared to the emission light. Thus, choose the band-pass filter with the highest blocking characteristics to reduce the background. A photon counting system comprises a combination of PMTs, photon counter units, and a high-speed counter. To control the system and to sample the data, a custom-made driving software was used. Finding a way to apply this method with other systems is necessary.</li>
</ol>
4. NADH correction methods with a multiparametric measurement system
<ol>
	<li>Identification of the isosbestic points of Fura-2-FF in situ. 
	NOTE: Many reports have stated that fluorescent characteristics are changed in cells. Therefore, perform all procedures to obtain the parameters to correct the interference in situ.
	<ol>
		<li>Mount the dye-loaded cell on the microscope and wait for 3 min to sink the cells to the bottom. 
		NOTE: Adjust cell numbers to see around one cell per one objective field with 40x objective lens.</li>
		<li>Perfuse the NADH-free Ca2+-free solution at 37 &#176;C.</li>
		<li>After targeting cell, measure the cell-free background and the cell area as shown in section 4.1. Calculate the cell background from the cell area. 
		NOTE: Both background signals need to be corrected in each experiment.</li>
		<li>Perfuse the saponin solution for 60 s and return to the NADH-free Ca2+-free solution.</li>
		<li>Measure Fura-2-FF-emitted signals at 450 nm and 500 nm simultaneously by the excitation scan from 350 nm to 365 nm with the 0.1 nm step.</li>
		<li>Perfuse the NADH-free Ca2+-saturated solution and repeat step 4.2.5.</li>
		<li>Subtract the signals in the Ca2+-saturated solution from the signals in the Ca2+-free conditions.</li>
		<li>Repeat the procedures from 4.2.2 to 4.2.7 with other single cardiac myocytes. 
		NOTE: If the signal intensity become weaker, repeat from 4.2.1. Repeat the procedure for, at least, 5 different cells.</li>
		<li>From all obtained signals, calculate the standard deviations of the emission at each excitation and choose the excitation wavelength showing the minimum standard deviation (SD) value as an isosbestic point. 
		NOTE: The representative figures are shown in Figure 2.</li>
	</ol>
	</li>
	<li>The background signal detection and the correction methods with the cell area 
	NOTE: There are two kinds of the backgrounds. One comes from the cells and the other comes from the reflection on the cover slip (the cell-free background). Both backgrounds need to be corrected in each experiment.
	<ol>
		<li>Mount the dye-free cells in the bath on the microscope and wait for 3 min to sink the cells to the bottom. Perfuse NADH-free Ca2+-free solution for around 5 mins. 
		NOTE: All solution perfusion rate is 2-3 mL/min at 37 &#176;C. 
		NOTE: Adjust cell numbers to see around one cell per one objective field with 40x objective lens.</li>
		<li>Set the object field to cover the targeted cell.</li>
		<li>After moving the cell out of the field, measure the background signals of the cell-free window and set them as offsets. 
		NOTE: The signal means the light signal to be detected in the photon counting system.</li>
		<li>Return the cell to the initial position and measure the cell background signals and the cell area. 
		NOTE: Even though the excitation light is filtered with the bandpass filter, it still contains quite large amount of the filtered light. This light is dispersed when hitting the cells and causes a considerable background signal because the photon counting system is highly sensitive. It needs to be corrected. 
		NOTE: The cell area may be calculated with a captured cell image and an available imaging software. The unit of the cell area can be any unit including pixel count. Just standardization is necessary.</li>
		<li>Repeat from 4.1.1 to 4.1.4 for 10 times to obtain the relationship between the cell area and the cell background signals. 
		NOTE: Later, the cell background signals can be calculated from the cell area from the relationship. Since the excitation light bulb become aging, this procedure needs to be repeated, at least, every month.</li>
	</ol>
	</li>
	<li>Measurement of R factors
	<ol>
		<li>Calculate Rf with the equation 4 from the signals obtained in section 4.2.</li>
		<li>Mount the dye-free cells on the microscope and perfuse the Ca2+-free solution.</li>
		<li>Measure the signals such as F361, 450, NADH, F400, 500, NADH, F361, 450, NADH, and F353, 500, NADH.</li>
		<li>Perfuse the malate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the pyruvate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the malate-pyruvate solution and repeat 4.3.3. and measure the signals.</li>
		<li>Perfuse the rotenone solution and repeat 4.3.3. and measure the signals. 
		NOTE: The example of the NADH signal recorded on 5 mM pyruvate, 5 mM malate plus 5 mM pyruvate, and 10 &#181;M rotenone addition is shown in Figure 3.</li>
		<li>Calculate each slope of F361, 450, NADH vs. F400, 500, NADH and F361, 450, NADH vs. F353, 500, NADH. As shown in Figure 3. Each slope indicates RN1 and RN2.</li>
	</ol>
	</li>
</ol>
5. Selection of the excitation and the emission light for TMRE or carboxy-SNARF-1
<ol>
	<li>If TMRE for measuring the mitochondrial potential was used in addition, use the 530 nm excitation wavelength and the 590 nm emission wavelength.</li>
	<li>If carboxy-SNARF-1 for measuring the mitochondrial potential was used in addition, use the excitation wavelength of 540 nm and emission wavelengths of 590 nm and 640 nm12.</li>
</ol>
6. Selection of Kd value of fura-2-FF
<ol>
	<li>The change of pH can affect Kd values for Ca2+ binding on fura-2-FF10. Use the Kd value of 5.28 at pH 7.5 for the mitochondria.</li>
</ol>

<ol><li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+signalling%3A+dynamics%2C+homeostasis+and+remodelling%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nature Reviews Molecular Cell Biology. 4 (7), 517-529 (2003).</li>
<li>Miyata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+mitochondrial+free+Ca2%2B+concentration+in+living+single+rat+cardiac+myocytes%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes.</a> American Journal of Physiology. 261 (4 Pt 2), H1123-H1134 (1991).</li>
<li>Allen, S. P., Stone, D., McCormack, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+loading+of+fura-2+into+mitochondria+in+the+intact+perfused+rat+heart+and+its+use+to+estimate+matrix+Ca2%2B+under+various+conditions%5BTitle%5D)+AND+%22Journal+of+Molecular+Cellular+Cardiology%22%5BJournal%5D)">The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca2+ under various conditions.</a> Journal of Molecular Cellular Cardiology. 24 (7), 765-773 (1992).</li>
<li>Griffiths, E. J., Halestrap, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyrophosphate+metabolism+in+the+perfused+heart+and+isolated+heart+mitochondria+and+its+role+in+regulation+of+mitochondrial+function+by+calcium%5BTitle%5D)+AND+%22Biochemical+Journal%22%5BJournal%5D)">Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium.</a> Biochemical Journal. 290 (Pt 2), 489-495 (1993).</li>
<li>Crompton, M., Moser, R., Ludi, H., Carafoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+interrelations+between+the+transport+of+sodium+and+calcium+in+mitochondria+of+various+mammalian+tissues%5BTitle%5D)+AND+%22European+Journal+of+Biochemistry%22%5BJournal%5D)">The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.</a> European Journal of Biochemistry. 82 (1), 25-31 (1978).</li>
<li>Chance, B., Schoener, B., Oshino, R., Itshak, F., Nakase, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidation-reduction+ratio+studies+of+mitochondria+in+freeze-trapped+samples.+NADH+and+flavoprotein+fluorescence+signals%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.</a> The Journal of Biological Chemistry. 254 (11), 4764-4771 (1979).</li>
<li>Eng, J., Lynch, R. M., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nicotinamide+adenine+dinucleotide+fluorescence+spectroscopy+and+imaging+of+isolated+cardiac+myocytes%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.</a> Biophysical Journal. 55 (4), 621-630 (1989).</li>
<li>Brandes, R., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+measurements+of+mitochondrial+NADH+and+Ca%282%2B%29+during+increased+work+in+intact+rat+heart+trabeculae%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Simultaneous measurements of mitochondrial NADH and Ca(2+) during increased work in intact rat heart trabeculae.</a> Biophysical Journal. 83 (2), 587-604 (2002).</li>
<li>Jo, H., Noma, A., Matsuoka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium-mediated+coupling+between+mitochondrial+substrate+dehydrogenation+and+cardiac+workload+in+single+guinea-pig+ventricular+myocytes%5BTitle%5D)+AND+%22Journal+of+Molecular+Cellular+Cardiology%22%5BJournal%5D)">Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes.</a> Journal of Molecular Cellular Cardiology. 40 (3), 394-404 (2006).</li>
<li>Lee, J. H., Ha, J. M., Leem, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Nicotinamide+Adenine+Dinucleotide+Correction+Method+for+Mitochondrial+Ca2%2B+Measurement+with+FURA-2-FF+in+Single+Permeabilized+Ventricular+Myocytes+of+Rat%5BTitle%5D)+AND+%22Korean+Journal+of+Physiology+and+Pharmacology%22%5BJournal%5D)">A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.</a> Korean Journal of Physiology and Pharmacology. 19 (4), 373-382 (2015).</li>
<li>Powell, T., Terrar, D. A., Twist, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+properties+of+individual+cells+isolated+from+adult+rat+ventricular+myocardium%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Electrical properties of individual cells isolated from adult rat ventricular myocardium.</a> Journal of Physiology. 302, 131-153 (1980).</li>
<li>Sun, B., Leem, C. H., Vaughan-Jones, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+chloride-dependent+acid+loader+in+the+guinea-pig+ventricular+myocyte%3A+part+of+a+dual+acid-loading+mechanism%5BTitle%5D)+AND+%22Journal+of+Physiology%22%5BJournal%5D)">Novel chloride-dependent acid loader in the guinea-pig ventricular myocyte: part of a dual acid-loading mechanism.</a> Journal of Physiology. 495 (Pt 1), 65-82 (1996).</li>
<li>Page, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+ultrastructural+analysis+in+cardiac+membrane+physiology%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">Quantitative ultrastructural analysis in cardiac membrane physiology.</a> American Journal of Physiology. 235 (5), C147-C158 (1978).</li>
<li>Page, E., McCallister, L. P., Power, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sterological+measurements+of+cardiac+ultrastructures+implicated+in+excitation-contraction+coupling%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Sterological measurements of cardiac ultrastructures implicated in excitation-contraction coupling.</a> Proceedings of the National Academy of Sciences of the United States of America. 68 (7), 1465-1466 (1971).</li>
<li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+generation+of+Ca2%2B+indicators+with+greatly+improved+fluorescence+properties%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> The Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li>
</ol>

The authors have no conflicts of interest to disclose.

The interference correction method was successfully developed for measuring the signals of NADH and fura-2 analogs. Exact measurement of the signals is essential for exact correction. However, the inherent nature of the fluorescent device produces a background signal unrelated to that of NADH of fura-2. The highest quality band-pass filter can only pass up to 10&#8722;8 of the unwanted wavelengths of the light. However, the fluorescent signal from a single cell is very small, and the reflection of the excitation light after the band-pass filter is still strong enough to contaminate the actual fluorescent signals. Therefore, careful correction of the background signal is necessary.
Fura-2 has a loading problem to measure mitochondrial Ca2+. First, it is not easy to load the dye specifically into the mitochondria, and nonspecific loading into another organelle could be erroneous. Mitochondrial Ca2+ concentration is generally higher than that of the cytosol, and the use of fura-2-FF with a high Kd value could avoid the contamination of cytosolic Ca2+ changes. The other problematic organelle is the sarcoplasmic reticulum (SR). However, the distribution volume differences (SR 3.5% vs. mitochondria 34%&#8722;36% in rat ventricular myocytes)13,14 and the removal of ATP in experiments could compensate for the contamination from SR.
Our calibration equation (Equation 14 and 15) has many advantageous characteristics over Grynkiewicz&#8217;s equation15 as follows: 
1) It requires only three parameters: Kd, F400,500,max/F353,500,max, and Rmin. 
2) There is linearity of the ratio value to the Ca2+ concentration at a constant pH. 
3) There is a relative error-free parameter in F400,500,max/F353,500,max compared with Sf2/Sb215. 
4) In Equation 15, only Kd and Rmin are required if isosbestic excitation is used. 
5) The calibration procedure to obtain the parameter is much simpler with Equation 15.
However, there is a limitation because Ca2+-saturated fura-2-FF generates a very small emission. It causes an error. The new equation can be applied to [Ca2+] concentrations up to 50x that of Kd.
In conclusion, a protocol was developed to successfully solve the existing problem of NADH and fura-2-FF interference. This method can measure Ca2+ dynamics more accurately. Multiparametric measurement system, particularly in the mitochondria, will help understand the mitochondrial physiology in a quantitative way.

The significant role of intracellular Ca2+ is widely known1. The quantification of [Ca2+] is essential to understand the processes of the cellular physiological functions. Fura-2 analogs are quite useful because they are excited in the UV range (&#60;400 nm), and the ratiometric method can be applied for the quantitative measurement. Therefore, other physiological parameters such as pH, membrane potential, etc., can be measured with other fluorescent dyes. The mitochondrial Ca2+ concentration ([Ca2+]m) range was reportedly 0.08&#8722;20 &#956;M2,3,4,5. Among fura-2 analogs, fura-2-FF is appropriate for measuring this range of [Ca2+]. However, the live cells unfortunately contain NADH/NADPH for their metabolic processes, and NADH generates signal interference because of the overlapping excitation and emission spectra with the fura-2 analog. This interference greatly limits the use of fura-2 analogs. Specifically, if the analog is applied to measure mitochondrial [Ca2+], this interference is the biggest obstacle because the highest amount of NADH is in the mitochondria. This is further complicated by NADH changes being related to the mitochondrial membrane potential (&#936;m) and the change of &#936;m affects [Ca2+]m6,7,8,9. Furthermore, for studying [Ca2+]m dynamics, it is essential to know the status of other mitochondrial parameters, such as NADH, &#936;m, and pH.
The emissions at 450 nm and 500 nm with excitations at 353 nm, 361 nm, and 400 nm contain the signals from NADH and fura-2-FF, and the equations are as follows. Herein, 353 nm and 361 nm are the isosbestic points of fura-2-FF for emissions at 450 nm and at 500 nm, respectively.
F361,450 = F361,450,NADH + F361,450,Fura&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Equation 1 
F353,500 = F353,500,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 2 
F400,500 = F400,500,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 3
where Fx,y is the measured emission intensity at y-nm by x-nm excitation, Fx,y,NADH represents the pure NADH-dependent emission intensity, and Fx,y,Fura represents the pure fura-2-FF-dependent emission intensity. Under the same concentration of the fluorescent dye, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Ca2+ and fura-2 did not affect NADH fluorescence characteristics; therefore, the ratio of the emission at 450 nm and at 500 nm of NADH was constant at any excitation wavelength. The same rule can be used for fura-2-FF based on the assumption that NADH or [Ca2+] does not affect the emission and excitation spectra of fura-2-FF. However, Ca2+ caused a spectral shift of the fura-2-FF emission. Therefore, to remove the effect of Ca2+, isosbestic excitation, which is independent of Ca2+, needs to be used. Each emission wavelength (i.e., 450 nm and 500 nm) has a different isosbestic point, and from our experimental setup, 353 nm at 500 nm and 361 nm at 450 nm were chosen. From these, the following equations are valid10.
Rf = F361,450,Fura/F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 4 
RN1 = F400,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 5 
RN2 = F353,500,NADH/F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 6
With these constants, the following equations from (Equation 1) (Equation 2), and (Equation 3) are valid.
F361,450 = F361,450,NADH + Rf &#215; F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 7 
F353,450 = RN2 &#215; F361,450,NADH + F353,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 8 
F400,500 = RN1 &#215; F361,450,NADH + F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 9
From these equations, if Rf, RN1, and RN2 are known, pure signals of NADH and fura-2 can be obtained as follows.
F361,450,NADH = (F361,450 - Rf &#215; F353,500)/(1 &#8722; Rf &#215; RN2) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 10 
F353,500,Fura = (RN2 &#215; F361,450 &#8722; F353,500)/(Rf &#215; RN2 &#8722; 1) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 11 
F400,500,Fura = F400,500 &#8722; RN1 &#215; F361,450,NADH &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; Equation 12 
RFura = F353,500,Fura/F400,500,Fura &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 13
The Ca2+-bound form of fura-2-FF was practically non-fluorescent at the 400 nm excitation wavelength. Based on this property, the following new calibration equation can be derived.
[Ca2+] = Kd &#8729; (F400,500,max/F353,500,max) &#215; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 14
where Kd is a dissociation constant, F400,500,max and F353,500,max are the maximum values of the emitted signals at 500 nm with excitations at 400 nm and 353 nm, respectively, and Rmin is the minimum RFura in Ca2+-free condition. Since the isosbestic excitations were used, the equation can be simplified further as follows.
[Ca2+] = Kd &#8729; (1 / Rmin) &#8729; (RFura &#8722; Rmin) &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;Equation 15
Therefore, only Kd and Rmin values are required to calculate [Ca2+].

<table><tbody><tr><td>&lt;strong&gt;2 mL eppendorf tube&lt;/strong&gt;</td><td>Axygen</td><td>MCT-200-C</td><td>2 mL Tube</td></tr><tr><td>&lt;strong&gt;AD/DA converter&lt;/strong&gt;</td><td>Instrutech</td><td>ITC-18</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;ADP, Adenosine 5&amp;prime;-diphosphate monopotassium salt dihydrate&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>A5285</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Band pass filter&lt;/strong&gt;</td><td>Ealing Electro-Optics, Inc</td><td>35-3920</td><td>Equipment, 640&amp;plusmn;11nm</td></tr><tr><td>&lt;strong&gt;Band pass filter&lt;/strong&gt;</td><td>Omega Optical</td><td>690-9823</td><td>Equipment, 590&amp;plusmn;15nm</td></tr><tr><td>&lt;strong&gt;Band pass filter&lt;/strong&gt;</td><td>Omega Optical</td><td>500DF20-9916</td><td>Equipment, 500&amp;plusmn;20nm</td></tr><tr><td>&lt;strong&gt;Band pass filter&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>60685</td><td>Equipment, 450&amp;plusmn;30nm</td></tr><tr><td>&lt;strong&gt;Calcium chloride solution&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>21114</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;carboxy-SNARF-1(AM)&lt;/strong&gt;</td><td>Invitrogen</td><td>C1272</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Charge-coupled device (CCD) camera&lt;/strong&gt;</td><td>Philips</td><td>FTM1800NH/HGI</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Dichroic mirror&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>86009</td><td>Equipment, Multiband dichroic mirror, Reflection : &amp;lt;400nm, 490&amp;plusmn;10, 560&amp;plusmn;10, Transmission : 460&amp;plusmn;15, 510&amp;plusmn;20, &gt;580nm</td></tr><tr><td>&lt;strong&gt;Dichroic mirror&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>567DCXRU</td><td>Equipment, Reflection : &amp;lt;560nm, Transmission : &gt; 580 nm</td></tr><tr><td>&lt;strong&gt;Dichroic mirror&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>480dclp</td><td>Equipment, Reflection : &amp;lt;470nm, Transmission : &gt; 490 nm</td></tr><tr><td>&lt;strong&gt;Dichroic mirror&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>20728</td><td>Equipment, Multiband dichroic mirror, Reflection : &amp;lt;405nm, 470&amp;plusmn;30, Transmission : 430nm~520nm, &gt; 640 nm</td></tr><tr><td>&lt;strong&gt;Dimethyl sulfoxide(DMSO)&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>154938</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;DMEM, Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>D5030</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;EGTA, Egtazic acid, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, Glycol ether diamine tetraacetic acid&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>E4378</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;FCCP, Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>21857</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;field diaphragm&lt;/strong&gt;</td><td>Nikon</td><td>86506</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Fura-2-FF(AM)&lt;/strong&gt;</td><td>TEFLABS</td><td>137</td><td>chemicals</td></tr><tr><td>&lt;strong&gt;Green tube&lt;/strong&gt;</td><td>DWM</td><td></td><td>test tube</td></tr><tr><td>&lt;strong&gt;HEPES,&amp;nbsp; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&amp;prime;-(2-ethanesulfonic acid)&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>H3375</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;High-speed counter&lt;/strong&gt;</td><td>National Instruments</td><td>NI-6022</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Hot mirror&lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>21002</td><td>Equipment, 50:50</td></tr><tr><td>&lt;strong&gt;Inverted microscope &lt;/strong&gt;</td><td>Nikon</td><td>TE-300</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Malate&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>27606</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Near infrared filter &lt;/strong&gt;</td><td>Chroma Technology Corp.</td><td>D750/100X</td><td>Equipment, 750&amp;plusmn;100nm</td></tr><tr><td>&lt;strong&gt;Oil immersion lens &lt;/strong&gt;</td><td>Nikon</td><td>MRF01400</td><td>40x, NA 1.3; Equipment</td></tr><tr><td>&lt;strong&gt;Photon counter unit&lt;/strong&gt;</td><td>Hamamatsu</td><td>C3866</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Photon multiplier tube&lt;/strong&gt;</td><td>Hamamatsu</td><td>R2949</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Polychrome II&lt;/strong&gt;</td><td>Till Photonics</td><td>SA3/MG04</td><td>Equipment</td></tr><tr><td>&lt;strong&gt;Potassium chloride&lt;/strong&gt;</td><td>Merck</td><td>1.04936</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Potassium hydroxide solution&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>P4494</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Pyruvate&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>107360</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Rotenone&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>R8875</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;Saponin&lt;/strong&gt;</td><td>Sigma-aldrich</td><td>S4521</td><td>Chemicals</td></tr><tr><td>&lt;strong&gt;TMRE, Tetramethylrhodamine, ethyl ester &lt;/strong&gt;</td><td>Molecular probes</td><td>T669</td><td>Chemicals</td></tr></tbody></table>

a novel nicotinamide adenine dinucleotide correction method for intracellular ca2+ measurement with fura-2-analog in live cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in live cells because of autofluorescence interference, mainly from nicotinamide adenine dinucleotide (NADH). More specifically, this is a major obstacle when measuring the mitochondrial [Ca2+] quantitatively using fura-2 analogs because the majority of NADH is in the mitochondria. If the fluorescent dye concentration is the same, a certain excitation intensity should produce the same emission intensity. Therefore, the emission intensity ratio of two different excitation wavelengths should be constant. Based on this principle, a novel online correction method of NADH signal interference to measure [Ca2+] was developed, and the real signal intensity of NADH and fura-2 can be obtained. Further, a novel equation to calculate [Ca2+] was developed with isosbestic excitation or excitation at 400 nm. With this method, changes in mitochondrial [Ca2+] could be successfully measured. In addition, with a different set of the excitation and emission wavelengths, multiple parameters, including NADH, [Ca2+], and pH or mitochondrial membrane potential (&#936;m), could be simultaneously measured. Mitochondrial [Ca2+] and &#936;m or pH were measured using fura-2-FF and tetramethylrhodamine ethyl ester (TMRE) or carboxy-seminaphtorhodafluor-1 (carboxy-SNARF-1).

Mitochondrial Ca2+ changes due to correction10 
Figure 4 shows the changes in [Ca2+]m before and after the correction. The results clearly showed the substantial changes in [Ca2+]m. The mitochondrial resting calcium concentration without cytosolic Ca2+ ([Ca2+]c) was 1.03 &#177; 0.13 &#181;M (mean &#177; S.E., n = 32), and the maximum [Ca2+]m at 1-&#181;M [Ca2+]c was 29.6 &#177; 1.61 &#181;M (mean &#177; S.E., n = 33) (Figure 5).
Simultaneous measurement of NADH, [Ca2+], and &#936;m10 
A positively charged TMRE can be distributed in a membrane potential-dependent manner. Membrane potential can be calculated using the Nernst&#8217;s equation with the concentration in each compartment. The mitochondrial TMRA was monitored with the perfusion of 2-nM TMRE. The initial &#936;m was assumed to be &#8722;150 mV, and the change of &#936;m was calculated based on that. The application of Ca2+ decreased NADH but affected &#936;m only negligibly (Figure 6).
Mitochondrial pH changes by the change in [Ca2+]m10 
The mitochondrial pH with the additional loading of carboxy-SNARF-1 was monitored following Ca2+ changes (Figure 7). The mitochondrial pH was not affected by the increase in [Ca2+]m. The resting mitochondrial pH was 7.504 &#177; 0.047 (mean &#177; S.E., n = 13). From these results, 5.28 &#181;M was the chosen Kd value of fura-2-FF at pH 7.5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59881/59881fig1.jpg" /> 
Figure 1: A microfluorometry system for multiparametric measurement 
The schematic diagram of the microfluorometry system was shown. The mounted cells were visualized via a CCD camera. Four different emission lights were detected with four PMTs via a photon counting system. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59881/59881fig2.jpg" /> 
Figure 2: Identification of isosbestic points 
(A) The red arrow points to the isosbestic point at the 450 nm emission wavelength. Fura-2 FF in the non-bound state is shown with a dotted line and in the Ca2+ bound state with a solid line. (B) The red arrow is pointed to the isosbestic point at the 500 nm emission wavelength. (C) The subtracted data of the signal at 450 nm in Ca2+-free conditions from Ca2+-free saturated conditions are shown. (D) The subtracted data of the signal at 500 nm in Ca2+-free conditions from Ca2+-free saturated conditions are shown. (E) Standard deviation data from graph C are shown. (F) Standard deviation data from graph D are shown. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59881/59881fig3.jpg" /> 
Figure 3: Measurement of RN factors. 
(A) Changes in the NADH signal without fluorescent dye by applying various mitochondrial substrates were measured at 361 nm excitation and 450 nm emission wavelengths. (B) The NADH interference in the fura-2-FF signals, F400,500 (&#8729;&#8729;&#8729;) and F353,500 (&#8212;), were simultaneously monitored. (C) The relationships between F361,450,NADH and F400,500,NADH (&#9675;)and between F361,450,NADH and F353,500,NADH (&#9679;) are shown. The obtained slopes are represented as RN1 and RN2, respectively. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59881/59881fig4.jpg" /> 
Figure 4: Results of NADH and fura-2-FF interference correction 
The change of the signals from before the correction (shown in the left panels) to after the correction (shown in the right panels). (A) NADH signals at the 450 nm emission wavelength. (B) Fura-2-FF signals at the 500 nm emission wavelength. The figure shows F400,500 (&#8722; &#8722;), F353,500 (----), and the ratio of fura-2-FF (&#8212;). (C) The mitochondrial calcium concentration. The red dotted line indicates the zero. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59881/59881fig5.jpg" /> 
Figure 5: Resting [Ca2+]m without cytosolic Ca2+ and maximal steady state [Ca2+]m at 1 &#181;M cytosolic Ca2+ 
Mitochondria were energized with the perfusion of malate-pyruvate solution. The steady state [Ca2+]m in a Ca2+-free conditions and in 1 &#181;M Ca2+ conditions were shown. The addition of 5 mM Na+ recovered NADH and reduced [Ca2+]m to the baseline. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59881/59881fig6.jpg" /> 
Figure 6: Simultaneous measurement of NADH, [Ca2+]m, and &#936;m 
Mitochondria were energized with the perfusion of malate-pyruvate solution. The changes of NAHD, [Ca2+]m and &#936;m were shown. The addition of 1 &#181;M Ca2+ decreased NAHD and increased [Ca2+]m but &#936;m was not changed significantly. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59881/59881fig7.jpg" /> 
Figure 7: Simultaneous measurement of NADH, [Ca2+]m, and pH 
The repeated application of Ca2+ could induce the decrease of NADH and the increase of [Ca2+]m but the mitochondrial pH was not affected by the application of Ca2+. The addition of Na+ could return the NADH and [Ca2+]m to the baseline. This figure has been reproduced with permission from The Korean Journal of Physiology &#38; Pharmacology10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59881/59881fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Name of Solutions</td>
			<td colspan="9">Concentration (mM)</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>HEPES</td>
			<td>EGTA</td>
			<td>CaCl2</td>
			<td>M</td>
			<td>P</td>
			<td>R</td>
			<td>FCCP</td>
			<td>ADP</td>
			<td>Saponin</td>
		</tr>
		<tr>
			<td>Ca2+-free </td>
			<td>150</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NADH-free</td>
			<td rowspan="2">150</td>
			<td rowspan="2">10</td>
			<td rowspan="2">1</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">0.01</td>
			<td rowspan="2">0.1</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Ca2+-free</td>
		</tr>
		<tr>
			<td>NADH-free</td>
			<td rowspan="2">135</td>
			<td rowspan="2">10</td>
			<td rowspan="2"></td>
			<td rowspan="2">1</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">0.01</td>
			<td rowspan="2">0.1</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Ca2+-Saturated</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>150</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>0.1mg/ml</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>145</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>145</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td></td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Malate-pyruvate</td>
			<td>140</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td>5</td>
			<td>5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>140</td>
			<td>10</td>
			<td>1</td>
			<td></td>
			<td>5</td>
			<td>5</td>
			<td>0.01</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Medium</td>
			<td colspan="10">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
		</tr>
		<tr>
			<td>Dye-loading </td>
			<td colspan="10">Add an 1mM Fura-2-FF-AM stock(16 mL) in the Culture medium (2 mL)</td>
		</tr>
	</tbody>
</table>
Table 1: Solutions

Watch this Scientific Journal Video about A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells at JoVE.com

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Due to the spectral overlapping of the excitation and emission wavelengths of NADH and fura-2 analogs, the signal interference from both chemicals in live cells is unavoidable during quantitative measurement of [Ca2+]. Thus, a novel online correction method of NADH signal interference to measure [Ca2+] was developed.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in ...

a-novel-nicotinamide-adenine-dinucleotide-correction-method-for

Nanyang Technological University

Research

JoVE Journal

Biology

6.7K Views.  University of Ulsan College of Medicine/Asan Medical Center. UV prikkelbare meervoudsondes, hebben een inherente signaalverontreiniging, vanwege de auto-meervoud geluiden. Het grootste deel van het probleem met NADH, is het beperken van het gebruik van een meervoud sonde. Een methode kan worden gebruikt om de buitenste florescentie van NADH nauwkeurig te corrigeren, om een echt, niet-verontreinigd meervoudsondesignaal te extraheren.Het aantonen van de procedure zal Jeong Hoon Lee zijn, onze assistent-professor in mijn laboratorium. Nadat de myoc...

Video: A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Neuroscience

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Bioengineering

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Single Cell Calcium Imaging for Channel Activation Studies

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca2+ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca2+. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca2+) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca2+. By measuring the 340/380 ratio, the Ca2+ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca2+ changes in multiple cells. The results are stored in &#8220;.XLSX&#8221; format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca2+ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca2+ imaging.

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single cell Ca2+ imaging is essential for the study of Ca2+ channels activated by various stimulations like temperature, voltage, native compound and ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

Education

JoVE Science Education

Advanced Biology

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca²⁺ Measurement with Fura-2-Analog in Live Cells

Samenvatting

Meer video's verkennen