User:Christian Niederauer/Notebook/Phgradients/2014/08/23: Difference between revisions
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'''5) Measurement'''<br> | '''5) Measurement'''<br> | ||
Background light in '''each''' wavelength channel is measured and subtracted from the absolute fluorescence values of the buffer samples (again, each channel separately). <br> | Background light in '''each''' wavelength channel is measured and subtracted from the absolute fluorescence values of the buffer samples (again, each channel | ||
Fluorescency in both SNARF channels is measured (approx. 10 pictures, time-evolution by bleaching etc. is not relevant) and we obtain the fluorescence ratio:<br> | |||
separately). <br> | |||
Fluorescency in both SNARF channels is measured (approx. 10 pictures, time-evolution by bleaching etc. is not relevant) and we obtain the fluorescence | |||
ratio:<br> | |||
<math> \dfrac{\text{pointa()}-\text{background(abs,a)}}{\text{pointb()}-\text{background(abs,b)}}=\dfrac{\text{pointa()}-107}{\text{pointb()}-100}</math><br> | <math> \dfrac{\text{pointa()}-\text{background(abs,a)}}{\text{pointb()}-\text{background(abs,b)}}=\dfrac{\text{pointa()}-107}{\text{pointb()}-100}</math><br> | ||
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We fit a sigmoid-curve to the datapoints:<br> | We fit a sigmoid-curve to the datapoints:<br> | ||
<gallery> | <gallery> | ||
Datei: | Datei:chris_ni_calib.curve.PNG|Calibration Plot (Sigmoid-Fit) | ||
</gallery> | </gallery> | ||
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'''7) Analysis: pK<math>_{\mathrm{A}}</math>-Value'''<br> | '''7) Analysis: pK<math>_{\mathrm{A}}</math>-Value'''<br> | ||
Originally, we wanted to obtain the coefficients for the equation which provides the pH-value of our sample by processing the relative fluoerescence intensities of SNARF. <br> | Originally, we wanted to obtain the coefficients for the equation which provides the pH-value of our sample by processing the relative fluoerescence | ||
We define <math>R=\dfrac{F_{\lambda 1}}{F_{\lambda 2}}</math> and it is important to normalize both fluorescence intensities (<math> F</math>) '''separately''' by substracting the background illumination.<br> | |||
intensities of SNARF. <br> | |||
We define <math>R=\dfrac{F_{\lambda 1}}{F_{\lambda 2}}</math> and it is important to normalize both fluorescence intensities (<math> F</math>) '''separately''' | |||
by substracting the background illumination.<br> | |||
<math>\lambda1=580</math>nm and <math>\lambda2=640</math>nm (in Labview: <math> \lambda X</math> is the upper channel).<br> | <math>\lambda1=580</math>nm and <math>\lambda2=640</math>nm (in Labview: <math> \lambda X</math> is the upper channel).<br> | ||
With <math>A</math> and <math>B</math> noting the ''a''cidic and ''b''asic endpoints of our titration it holds:<br> | With <math>A</math> and <math>B</math> noting the ''a''cidic and ''b''asic endpoints of our titration it holds:<br> | ||
<math>\text{pH}=\text{pK}_{\text{A}}-\log{\left[ \dfrac{R-R_B}{R_A-R}\cdot\dfrac{F_{B(\lambda2)}}{F_{A(\lambda2)}}\right] }</math> Source: [http://tools.lifetechnologies.com/content/sfs/manuals/mp01270.pdf SNARF-Manual] <br> | <math>\text{pH}=\text{pK}_{\text{A}}-\log{\left[ \dfrac{R-R_B}{R_A-R}\cdot\dfrac{F_{B(\lambda2)}}{F_{A(\lambda2)}}\right] }</math> Source: | ||
[http://tools.lifetechnologies.com/content/sfs/manuals/mp01270.pdf SNARF-Manual] <br> | |||
Now we plot that equation with the fixed values for <math>R_A, R_B, F_{B(\lambda 2)}, F_{A(\lambda 2)}</math> and receive pK<math>_\text{A}</math> as it is the | |||
y-intercept of a linear fit:<br> | |||
(<math>y=ax+b \Leftrightarrow \text{pH}=\text{pK}_{\text{A}}-x </math>) with slope of 1 while <math> x:= \log{\left[ \dfrac{R-R_B}{R_A-R}\cdot \dfrac{F_{B | |||
(\lambda2)}}{F_{A(\lambda2)}}\right] }</math><br> | |||
<gallery> | <gallery> | ||
Datei: | Datei:chris_ni_Pka.png|Linear Fit for pKA-value | ||
</gallery> | </gallery> | ||
We get <math> \text{pK}_{\text{A}}\approx 7.2</math>. Now we have a calibrated equation for our setup and we can calculate the pH value for the samples by only measuring the relative intensities. | We get <math> \text{pK}_{\text{A}}\approx 7.2</math>. Now we have a calibrated equation for our setup and we can calculate the pH value for the samples by only | ||
measuring the relative intensities. | |||
===22.01.14 Calibration with UV-Laser I === | ===22.01.14 Calibration with UV-Laser I === | ||
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An improved pattern for LED & Laser exposure to simplify background normalization procedure: <br> | An improved pattern for LED & Laser exposure to simplify background normalization procedure: <br> | ||
<gallery> | <gallery> | ||
Datei: | Datei:chris_ni_newpattern.png| Exposure Pattern | ||
</gallery> | </gallery> | ||
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|- | |- | ||
| <gallery> | | <gallery> | ||
Datei: | Datei:chris_ni_Ratio_led1.png | Radial dependency of the Fluorescence-Ratio generated by LED-Excitation | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_Ratio_laser1.png | Radial dependency of the Fluorescence-Ratio generated by Laser-"Excitation" (Crosstalk) | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_Ratio_laser_led1.png | Radial dependency of the Fluorescence-Ratio generated by LED&Laser-Excitation | ||
</gallery> | </gallery> | ||
|} | |} | ||
The first image clearly shows the pH-dependent emission-ratio of SNARF. The higher the ratio, the higher is the pH-value (violet curve is sample 17 with pH=8.72). As expected there is no radial distribution. <br> | The first image clearly shows the pH-dependent emission-ratio of SNARF. The higher the ratio, the higher is the pH-value (violet curve is sample 17 with | ||
The second image shows the ratio of SNARF's fluorescent emission by (uninentional) excitation with the UV-Laser. While the ratiometric analysis helps to avoid a lot of trouble (e.g bleaching of SNARF, varying SNARF concentration,...) the crosstalk of the Laser has to be substracted because SNARF has different pH-emission-curves for different excitation wavelenghts.<br> | |||
pH=8.72). As expected there is no radial distribution. <br> | |||
The second image shows the ratio of SNARF's fluorescent emission by (uninentional) excitation with the UV-Laser. While the ratiometric analysis helps to avoid | |||
a lot of trouble (e.g bleaching of SNARF, varying SNARF concentration,...) the crosstalk of the Laser has to be substracted because SNARF has different pH- | |||
emission-curves for different excitation wavelenghts.<br> | |||
The radial dependency of the ratio at the laser+led graphs is caused by the crosstalk and the spatial confinement of the laser beam.<br> | The radial dependency of the ratio at the laser+led graphs is caused by the crosstalk and the spatial confinement of the laser beam.<br> | ||
For greater radii, the laser-caused fluorescence merges with the emission caused by LED excitation.<br> | For greater radii, the laser-caused fluorescence merges with the emission caused by LED excitation.<br> | ||
If the emission ratio was independent of the excitation wavelength and only depending on the pH value the curves should be approximately constant for each pH, regardless of the laser being switched on or not. In the experiment, the UV-crosstalk shifts the ratio in the area of the laser beam. | If the emission ratio was independent of the excitation wavelength and only depending on the pH value the curves should be approximately constant for each pH, | ||
regardless of the laser being switched on or not. In the experiment, the UV-crosstalk shifts the ratio in the area of the laser beam. | |||
===29.01.14 SNARF pH Measurement NBA=== | ===29.01.14 SNARF pH Measurement NBA=== | ||
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|} | |} | ||
NBA stock concentration is 8mM. To each sample 2.5µl SNARF (@1mM Stock) and 2.5µl buffer ([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 8.0, sample #15]]) are added. SNARF concentration therefore is 50µM. | NBA stock concentration is 8mM. To each sample 2.5µl SNARF (@1mM Stock) and 2.5µl buffer ([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 8.0, | ||
sample #15]]) are added. SNARF concentration therefore is 50µM. | |||
'''2) Measurement <br> | '''2) Measurement <br> | ||
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'''3) General LabView Procedure <br> | '''3) General LabView Procedure <br> | ||
We want to subtract the background fluorescence from the signal. Because the background and the signal data originate from different measurements, and the capillaries aren't placed onto the exact same spot every time, we have to rearrange the areas where we take our data from every time. We have to do this for both channels of each capillary (we want the same area for each of the channels, too). | We want to subtract the background fluorescence from the signal. Because the background and the signal data originate from different measurements, and the | ||
capillaries aren't placed onto the exact same spot every time, we have to rearrange the areas where we take our data from every time. We have to do this for | |||
both channels of each capillary (we want the same area for each of the channels, too). | |||
These steps have to be followed:<br> | These steps have to be followed:<br> | ||
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# Remember the reference point coordinates | # Remember the reference point coordinates | ||
# Now change ''loop index'' to view the actual signal images | # Now change ''loop index'' to view the actual signal images | ||
# Move reference point to the center of laser spot and flip the picture area from the background reference point to the newly defined point with ''Switch reference''. | # Move reference point to the center of laser spot and flip the picture area from the background reference point to the newly defined point with ''Switch | ||
reference''. | |||
# Update the new picture area | # Update the new picture area | ||
# Readjust the moved reference point to the background image value and repeat (7)-(8) for the other channel | # Readjust the moved reference point to the background image value and repeat (7)-(8) for the other channel | ||
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'''1) NBA-Mixture-Induced pH-Drop for various Buffer Concentrations <br> | '''1) NBA-Mixture-Induced pH-Drop for various Buffer Concentrations <br> | ||
250µl NBA samples with mentioned buffer ([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 8.0, sample #15]]) volumes are filled up to 1000µl with water.<br> | 250µl NBA samples with mentioned buffer ([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 8.0, sample #15]]) volumes are filled up to 1000µl with | ||
water.<br> | |||
Impact of NBA is measured with pH-meter: | Impact of NBA is measured with pH-meter: | ||
{| class="wikitable" | {| class="wikitable" | ||
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The whole procedure is repeated with a freshly mixed 1mM NBA sample (stock: 8mM).<br> | The whole procedure is repeated with a freshly mixed 1mM NBA sample (stock: 8mM).<br> | ||
Furthermore, a sample without NBA (49µl Water, 1µl beads) is added.<br> | Furthermore, a sample without NBA (49µl Water, 1µl beads) is added.<br> | ||
The adjustment of the laser focus has to be done beforehand with a additional capillary containing some UV-fluorescent dye, since focussing the laser spot with the bead samples is difficult. The focus of the camera should be adjusted for every capillary, so that a nice amount of beads is in the focal plane.<br> | The adjustment of the laser focus has to be done beforehand with a additional capillary containing some UV-fluorescent dye, since focussing the laser spot with | ||
the bead samples is difficult. The focus of the camera should be adjusted for every capillary, so that a nice amount of beads is in the focal plane.<br> | |||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| <gallery> | | <gallery> | ||
Datei: | Datei:chris_ni_MAX_colored_0mM.jpg | Time-resolved (green-red) Projection of Bead Movement without NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_MAX_colored_0_32mM.jpg | Time-resolved (green-red) Projection of Bead Movement at 0.32 mM NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_MAX_colored_1mM_OLD.jpg | Time-resolved (green-red) Projection of Bead Movement at 1mM (old) NBA | ||
</gallery> | </gallery> | ||
|} | |} | ||
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'''1) NBA Concentration Array<br> | '''1) NBA Concentration Array<br> | ||
We prepare samples with different NBA concentrations in a pH 7.5 buffer. Since the NBA concentrations we used the last times seem to be already saturating, we chose to expand the sample array in the lower NBA concentrations this time. Also, the calibration curves we got last time, recommend a smaller pH value (7.5 instead of 8.0). <br> | We prepare samples with different NBA concentrations in a pH 7.5 buffer. Since the NBA concentrations we used the last times seem to be already saturating, we | ||
chose to expand the sample array in the lower NBA concentrations this time. Also, the calibration curves we got last time, recommend a smaller pH value (7.5 | |||
instead of 8.0). <br> | |||
We want to measure the pH value, too, so we are using 100µl sample volumes (otherwise the electrode would not fit in the sample). | We want to measure the pH value, too, so we are using 100µl sample volumes (otherwise the electrode would not fit in the sample). | ||
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|} | |} | ||
NBA stock concentration is 8mM. To each of the subsamples with 50µl desired volume, 2.5 µl SNARF (@1mM Stock) and 2.5µl buffer ([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 7.5, sample #14]]) are added. SNARF concentration therefore is 50 µM.<br> | NBA stock concentration is 8mM. To each of the subsamples with 50µl desired volume, 2.5 µl SNARF (@1mM Stock) and 2.5µl buffer | ||
([[ChristianNiederauer#22.01.14_Calibration_with_UV-Laser_I|pH 7.5, sample #14]]) are added. SNARF concentration therefore is 50 µM.<br> | |||
Additonally, a capillary without dye is measured for determination of background illumination.<br> | Additonally, a capillary without dye is measured for determination of background illumination.<br> | ||
The chopper's phase is adjusted in that way, that the laser spot is minimum visible. | The chopper's phase is adjusted in that way, that the laser spot is minimum visible. | ||
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|- | |- | ||
| <gallery> | | <gallery> | ||
Datei: | Datei:chris_ni_0c.png | 0mM NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_0.1.png | 0.1mM NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_0.5.png | 0.5mM NBA | ||
</gallery> ||<gallery> | </gallery> ||<gallery> | ||
Datei: | Datei:chris_ni_1g.png | 1mM NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_2df.png | 2mM NBA | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_4.png | 4mM NBA | ||
</gallery> | </gallery> | ||
|} | |} | ||
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Approximately: 0-120: LED on, 120-420: Laser and LED on, 420-750: LED on, 750 -820: Laser on<br> | Approximately: 0-120: LED on, 120-420: Laser and LED on, 420-750: LED on, 750 -820: Laser on<br> | ||
Further evaluation on bigger radii here: [[ChristianNiederauer#19.02.14_Evaluation_of_SNARF_Calibration_on_13.02.|19.02.14]]<br> | Further evaluation on bigger radii here: [[ChristianNiederauer#19.02.14_Evaluation_of_SNARF_Calibration_on_13.02.|19.02.14]]<br> | ||
See [https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/Februar2014#14.02.2014:_pH_Gradient_-_pH_Time_Traces_with_SNARF pH Time Traces with SNARF] | See [https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/Februar2014#14.02.2014:_pH_Gradient_-_pH_Time_Traces_with_SNARF pH Time Traces with | ||
SNARF] | |||
===18.02.14 Beads Variety=== | ===18.02.14 Beads Variety=== | ||
Carboxylated and fluorescent (BCECF-like) beads of various dimensions (2µm - 0.02µm diameter) will be used to determine the movement and flows of charged particles along the pH gradient.<br> | Carboxylated and fluorescent (BCECF-like) beads of various dimensions (2µm - 0.02µm diameter) will be used to determine the movement and flows of charged | ||
particles along the pH gradient.<br> | |||
'''1) 0.02µm Beads<br> | '''1) 0.02µm Beads<br> | ||
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|} | |} | ||
Two samples with 0.5mM NBA and two with 0.25mM NBA were used, beause the 0.5mM NBA samples already showed a great movement. With the smaller beads being less bright, the 1:100 dilution is more convenient. | Two samples with 0.5mM NBA and two with 0.25mM NBA were used, beause the 0.5mM NBA samples already showed a great movement. With the smaller beads being less | ||
bright, the 1:100 dilution is more convenient. | |||
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|- | |- | ||
| <gallery> | | <gallery> | ||
Datei: | Datei:chris_ni_100_a.png | laser off | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_100_b.png | laser on | ||
</gallery> | </gallery> | ||
|} | |} | ||
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|} | |} | ||
Each sample therefore has a NBA concentration of 1mM (NBA-Stock: 8mM). The mentioned bead volume is an already pre-diluted sample (1:10 with 20µl beads, 180 µl water).<br> | Each sample therefore has a NBA concentration of 1mM (NBA-Stock: 8mM). The mentioned bead volume is an already pre-diluted sample (1:10 with 20µl beads, 180 µl | ||
water).<br> | |||
The picture sequences show a inward stream superposed with an outward stream, appearently depending on which focal plane is observed. To determine this effect, three additional sequences are recorded. The lowest and highest focal plane and also one in between. <br> | The picture sequences show a inward stream superposed with an outward stream, appearently depending on which focal plane is observed. To determine this effect, | ||
three additional sequences are recorded. The lowest and highest focal plane and also one in between. <br> | |||
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| <gallery> | | <gallery> | ||
Datei: | Datei:chris_ni_Cc_focus.png | top focal plane | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_m_focus.png | mid focal plane | ||
</gallery> || <gallery> | </gallery> || <gallery> | ||
Datei: | Datei:chris_ni_c_focus.png | bottom focal plane | ||
</gallery> | </gallery> | ||
|} | |} | ||
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|- | |- | ||
|<gallery> | |<gallery> | ||
Datei: | Datei:chris_ni_Graph0_1_.png | Time-dependent Ratio for various c[NBA] | ||
</gallery>||<gallery> | </gallery>||<gallery> | ||
Datei: | Datei:chris_ni_Improved_data_analysis.png | Comparison of 10px and 85px Radii at 4mM NBA | ||
</gallery> | </gallery> | ||
|} | |} | ||
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===21.02.14 Evaluation of SNARF Calibration on 13.02. Part II=== | ===21.02.14 Evaluation of SNARF Calibration on 13.02. Part II=== | ||
With the [[https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/Februar2014#Calibration pH-conversion recipe]] the pH values can be calculated for the 85px radii.<br> | With the [[https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/Februar2014#Calibration pH-conversion recipe]] the pH values can be calculated | ||
for the 85px radii.<br> | |||
<math>pH=7.7309-1.4921\cdot\log_{10}(\frac{R}{1.5354-R})+\log_{10}(0.1295)</math> | <math>pH=7.7309-1.4921\cdot\log_{10}(\frac{R}{1.5354-R})+\log_{10}(0.1295)</math> | ||
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===24.02.14 Isoelectric Focusing=== | ===24.02.14 Isoelectric Focusing=== | ||
'''Paper''':<br> | '''Paper''':<br> | ||
[[Datei: | [[Datei:chris_ni_AES_IEF.pdf|AES Application Focus - Isoelectric Focusing]] <br> | ||
[[Datei: | [[Datei:chris_ni_BIO_SYSTEMS_Cell_isoelectric.pdf|BioSystems - Does a cell perform isoelectric focusing?]]<br> | ||
[[Datei: | [[Datei:chris_ni_SST_IEF_alternatives.pdf|Separation Science and Technology - A Brief Review of Alternative Electrofocusing | ||
Techniques]]<br> | Techniques]]<br> | ||
[[Datei: | [[Datei:chris_ni_PHYS_BIO_intracellular_transport.pdf|AES Application Focus - Isoelectric Focusing]]<br> | ||
[[Datei: | [[Datei:chris_ni_AES_IEF.pdf|IOP Science - pH-induced intracellular protein transport]] | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
|<gallery> 05um_unten80.png | bottom focal plane</gallery>||<gallery> | |<gallery> 05um_unten80.png | bottom focal plane</gallery>||<gallery> Datei:chris_ni_05um_mitte.png | mid </gallery>||<gallery> | ||
Datei:chris_ni_05um_oben80.png | top </gallery> | |||
|} | |} | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
|<gallery> 1um_unten80.png | bottom focal plane</gallery>||<gallery> | |<gallery> 1um_unten80.png | bottom focal plane</gallery>||<gallery> Datei:chris_ni_1um_mitte.png | mid </gallery>||<gallery> | ||
Datei:chris_ni_1um_oben.png | top </gallery> | |||
|} | |} | ||
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===26.02.14 NBA and SNARF with NaOH pH Adjustment=== | ===26.02.14 NBA and SNARF with NaOH pH Adjustment=== | ||
This time we want to measure the NBA induced change in pH value, without an additional buffer.<br> | This time we want to measure the NBA induced change in pH value, without an additional buffer.<br> | ||
Since NBA is acidic, and pH detection with SNARF doesn't work well in pH below 7 we are adding NaOH (5mM) to achieve a pH value of around 8. <br>The dye will decrease the pH a little bit, so the final pH before the measurement was meant to be safely determined to be between 7 and 8 (which appeared to be very wrong!).<br> | Since NBA is acidic, and pH detection with SNARF doesn't work well in pH below 7 we are adding NaOH (5mM) to achieve a pH value of around 8. <br>The dye will | ||
decrease the pH a little bit, so the final pH before the measurement was meant to be safely determined to be between 7 and 8 (which appeared to be very | |||
wrong!).<br> | |||
Sample volume for pH measurement is 10ml, because the electrode of the pH-meter won't fit in very small samples and pH regulation is easier for bigger volumes. | Sample volume for pH measurement is 10ml, because the electrode of the pH-meter won't fit in very small samples and pH regulation is easier for bigger volumes. | ||
{| class="wikitable" | {| class="wikitable" | ||
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With decreasing NBA concentration we add less NaOH (around 40µl for the 4mM sample, to <10µl for 0.2mM NBA).<br> | With decreasing NBA concentration we add less NaOH (around 40µl for the 4mM sample, to <10µl for 0.2mM NBA).<br> | ||
Afterwards, 2.5µl SNARF are added to a 47.5µl subsample, creating a 50µM SNARF concentration.<br> <br> | Afterwards, 2.5µl SNARF are added to a 47.5µl subsample, creating a 50µM SNARF concentration.<br> <br> | ||
Pure water with a 50µM SNARF concentration (475µl water, 25µl SNARF due to big pHmeter electrode) has pH 5.3,<br> so pH shift (-1) induced by SNARF has to be considered when measuring without a buffer.<br> | Pure water with a 50µM SNARF concentration (475µl water, 25µl SNARF due to big pHmeter electrode) has pH 5.3,<br> so pH shift (-1) induced by SNARF has to be | ||
considered when measuring without a buffer.<br> | |||
Appearently, adjusting the pH just with NaOH molecules does not generate stable pHs. | Appearently, adjusting the pH just with NaOH molecules does not generate stable pHs. | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
|<gallery> | |<gallery> chris_ni_Nba_naoh_1.png | 0.2mM NBA </gallery>||<gallery> chris_ni_Nba_naoh_2.png | 0.5mM NBA </gallery>||<gallery> chris_ni_0_7.png | 0.7mM NBA | ||
</gallery> || <gallery>chris_ni_Nba_naoh_3.png | 1mM NBA </gallery>||<gallery> | |||
chris_ni_Nba_naoh_4.png | 2mM NBA </gallery>||<gallery> | |||
chris_ni_Nba_naoh_5.png | 4mM NBA </gallery> | |||
|} | |} | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
|<gallery> | |<gallery> chris_ni_ph10px.png | pH at 10px </gallery>||<gallery> chris_ni_ph15px.png | pH at 15px </gallery>||<gallery> chris_ni_ph25px.png | pH at 25px | ||
</gallery>||<gallery> chris_ni_ph40px.png | pH at 40px </gallery> | |||
|} | |} | ||
[https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/ChristianNiederauer#26.02.14_NBA_and_SNARF_with_NaOH_pH_Adjustment "Appearently, adjusting the pH just with NaOH molecules does not generate stable pHs."]]<br> | [https://wiki.physik.uni-muenchen.de/Systems-Biophysics-Wiki/index.php/ChristianNiederauer#26.02.14_NBA_and_SNARF_with_NaOH_pH_Adjustment "Appearently, | ||
adjusting the pH just with NaOH molecules does not generate stable pHs."]]<br> | |||
Since the pH plots differed a lot from our expectations, the pHs were measured once again and as cited above, they dropped about 2 counts over night.<br> | Since the pH plots differed a lot from our expectations, the pHs were measured once again and as cited above, they dropped about 2 counts over night.<br> | ||
Since [http://www.lifetechnologies.com/order/catalog/product/C1270 SNARF] has a pKa of ~7.5, it is best to be used in the pH range between 7 and 8.<br> | Since [http://www.lifetechnologies.com/order/catalog/product/C1270 SNARF] has a pKa of ~7.5, it is best to be used in the pH range between 7 and 8.<br> | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
|<gallery> | |<gallery> chris_ni_Intensity0px.png |0px </gallery>||<gallery> chris_ni_Intensity10px.png | 10px </gallery> | ||
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|<gallery> | |<gallery> chris_ni_2mer_radial.png |2mer fits </gallery>||<gallery> chris_ni_5mer_radial.png | 5mer </gallery> || <gallery> chris_ni_10mer_radial.png | 10mer | ||
</gallery> || <gallery> chris_ni_80mer_radial.png | 80mer </gallery> | |||
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|<gallery> | |<gallery> chris_ni_140_radial.png | Frame 140 (~2frames after Laser ON) </gallery>||<gallery> chris_ni_155_radial.png | Frame 155 </gallery> || <gallery> | ||
chris_ni_200_radial.png | Frame 200 </gallery> | |||
|} | |} | ||
It seems that DNA is accumulating at the Laser spot (bleaching by laser is not accounted, but that effect would actually lead to reducing fluorescence instead of increasing). | It seems that DNA is accumulating at the Laser spot (bleaching by laser is not accounted, but that effect would actually lead to reducing fluorescence instead | ||
of increasing). | |||
==April 2014== | ==April 2014== | ||
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|<gallery> | |<gallery> chris_ni_Intenla.png | Intensity of 0.02µm Fluorescent pH-Sensitive Beads </gallery>||<gallery> chris_ni_Norma.png | Intensity of 0.02µm | ||
Fluorescent pH-Sensitive Beads without Bleaching </gallery> | |||
|} | |} | ||
Again, unusual strong bleaching occured (see above, left). Fitting the bleach curves exponentially and dividing by them should fix the problem.<br> | Again, unusual strong bleaching occured (see above, left). Fitting the bleach curves exponentially and dividing by them should fix the problem.<br> | ||
Since the cause for the bleaching still remains unknown (probably old NBA? NBA or NBA-photoproduct doing something with the dye?), another proton uncaging molecule is ordered.<br> | Since the cause for the bleaching still remains unknown (probably old NBA? NBA or NBA-photoproduct doing something with the dye?), another proton uncaging | ||
molecule is ordered.<br> | |||
The normalizing of the intensity is yet to be repeated.<br> | The normalizing of the intensity is yet to be repeated.<br> | ||
Igor Procedure for exp-fit:<br> | Igor Procedure for exp-fit:<br> | ||
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<gallery> phdepe.png | Intensity of 0.02µm Beads for varying pH </gallery> | <gallery> phdepe.png | Intensity of 0.02µm Beads for varying pH </gallery> | ||
The intensity varies about less than 10%. The two data waves presented were made from the same sample volumes, just different capillaries.<br> | The intensity varies about less than 10%. The two data waves presented were made from the same sample volumes, just different capillaries.<br> | ||
Still, there is a variation of about 100 counts, which means the error in focussing or placement of the capillary most probably is greater than the change in intensity due to pH.<br> | Still, there is a variation of about 100 counts, which means the error in focussing or placement of the capillary most probably is greater than the change in | ||
intensity due to pH.<br> | |||
Appearance of dark or bright spots after a local pH jump therefore can't be explained with pH dependent change in fluorescence only. | Appearance of dark or bright spots after a local pH jump therefore can't be explained with pH dependent change in fluorescence only. | ||
'''2) pH-Dependencies of Cy-5 | '''2) pH-Dependencies of Cy-5 | ||
Change filter set to #1 and LED to 627nm.<br> | Change filter set to #1 and LED to 627nm.<br> | ||
Cy5 appears to be unstable in acid regions "[http://www.nlv.ch/Molbiology/sites/Fluorescence1.htm Cy5 is physically unstable in acid conditions and should be stored and used in buffers above pH 7.]" | Cy5 appears to be unstable in acid regions "[http://www.nlv.ch/Molbiology/sites/Fluorescence1.htm Cy5 is physically unstable in acid conditions and should be | ||
stored and used in buffers above pH 7.]" | |||
===29.04.2014 Check: pH-Dependency of Cy5=== | ===29.04.2014 Check: pH-Dependency of Cy5=== | ||
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|} | |} | ||
Left picture is from yesterdays measurement. This time, pH 2.78 was measured at the beginning, but still is lower. Thererfore the fluorescence has to be pH dependent.<br> | Left picture is from yesterdays measurement. This time, pH 2.78 was measured at the beginning, but still is lower. Thererfore the fluorescence has to be pH | ||
dependent.<br> | |||
Degradation due to storage occurs in dark environment. | Degradation due to storage occurs in dark environment. | ||
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Checking if NBA uncaging still works sufficiently for lower laser powers, to aim for less cross-bleaching of the fluorescent dyes due to UV excitation.<br> | Checking if NBA uncaging still works sufficiently for lower laser powers, to aim for less cross-bleaching of the fluorescent dyes due to UV excitation.<br> | ||
Currently used standard current was 80mA at 1.6V, resulting in intensities of 3-6mW (depending on adjustment of the laser).<br> | Currently used standard current was 80mA at 1.6V, resulting in intensities of 3-6mW (depending on adjustment of the laser).<br> | ||
Reducing the voltage gives a lower current, and the resulting power has to be measured. Unfortunately, the measured intensity is strongly depending on the accuracy of the laser adjustment, and even measuring the laser power can change the initial adjustment greatly. Therefore, laser power is measured before and after the measurement to ensure accuracy.<br> | Reducing the voltage gives a lower current, and the resulting power has to be measured. Unfortunately, the measured intensity is strongly depending on the | ||
accuracy of the laser adjustment, and even measuring the laser power can change the initial adjustment greatly. Therefore, laser power is measured before and | |||
after the measurement to ensure accuracy.<br> | |||
One 50µl sample is setup with 1mM NBA (6.25µl), 2mM of the phosphate buffer (pH 7.4, 0.4mM -> 0.25µl) and 5µl of the small (0.02µm diameter) beads.<br> | One 50µl sample is setup with 1mM NBA (6.25µl), 2mM of the phosphate buffer (pH 7.4, 0.4mM -> 0.25µl) and 5µl of the small (0.02µm diameter) beads.<br> | ||
The sample is stored darkly during the measurements.<br> | The sample is stored darkly during the measurements.<br> | ||
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|<gallery> | |<gallery> chris_ni_lowno.png </gallery>||<gallery> chris_ni_lowwith.png </gallery>||<gallery> chris_ni_normalow.png </gallery> | ||
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Another aim is to see if the UV laser already bleaches all the abundant NBA at 1.6V. <br> | Another aim is to see if the UV laser already bleaches all the abundant NBA at 1.6V. <br> | ||
To check the existence of a saturation plateau, the laser power is reduced to slightly lower values and the signal is observed.<br> | To check the existence of a saturation plateau, the laser power is reduced to slightly lower values and the signal is observed.<br> | ||
As we can see, a different kinetic already appears for lower intensities. Thus, at 1mM NBA there's still plenty NBA present and higher intensities might uncage even more protons. | As we can see, a different kinetic already appears for lower intensities. Thus, at 1mM NBA there's still plenty NBA present and higher intensities might uncage | ||
even more protons. | |||
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|<gallery> | |<gallery> chris_ni_expnormalized.png | Normalized for LED-Bleaching </gallery>||<gallery> chris_ni_withexpnorm.png | Exponential Fits for 1mM NBA-Sample | ||
</gallery>||<gallery> chris_ni_withoutexpnorm.png | Linear Fit for 0mM NBA (nearly constant)</gallery> | |||
|} | |} | ||
The fits only considers bleaching caused by LED!<br> | The fits only considers bleaching caused by LED!<br> | ||
If there weren't the strange interaction between NBA or NBA residues, the UV-bleaching could be separated from actual bead movement easily by just doing a measurement with 0mM NBA at the same laser power.<br> | If there weren't the strange interaction between NBA or NBA residues, the UV-bleaching could be separated from actual bead movement easily by just doing a | ||
measurement with 0mM NBA at the same laser power.<br> | |||
In the left picture the yellow curve shows the effect of UV bleaching. There is no NBA in this sample, so the kinetics must be caused by UV bleaching.<br> | In the left picture the yellow curve shows the effect of UV bleaching. There is no NBA in this sample, so the kinetics must be caused by UV bleaching.<br> | ||
But it is likely that not only the LED bleaching, but also UV induced bleaching is amplified when NBA is abundand which can't be easily separated by the bead flows this way.<br> | But it is likely that not only the LED bleaching, but also UV induced bleaching is amplified when NBA is abundand which can't be easily separated by the bead | ||
At least it is known, that the kinetics shown in the graph are not result of UV bleaching only, since we already could watch single bigger beads being carried away (and into) the region of the laser spot.<br> | |||
flows this way.<br> | |||
At least it is known, that the kinetics shown in the graph are not result of UV bleaching only, since we already could watch single bigger beads being carried | |||
away (and into) the region of the laser spot.<br> | |||
<br> | <br> | ||
The jump around frame 600 in the green wave is an artefact, the data points actually connect smoothly. | The jump around frame 600 in the green wave is an artefact, the data points actually connect smoothly. |
Revision as of 00:35, 23 August 2014
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Christian NiederauerDecember 201318.12.13 SNARF & NBA in H[math]\displaystyle{ _2 }[/math]O without buffer
SNARF stock concentration: 1mM NBA stock concentration: 8 mM Volume: 50µl SNARF concentration: 20 µM (V/V_sample = c/c_stock) January 201408.01.14 Determination of buffer impacts on NBA-induced pH dropPreparation of buffer sequence:
Measuring pH of each sample yielded values around 7.9 to 8.1 pH-values of samples after adding NBA
Now 47.5µl-size samples are taken for testing them in the laser setup. 16.01.14 SNARF Calibration1) Preparing missing Sorensen buffers with pH in the ranges of 5.4, 6.8 and 7
2) Remeasurement of remainig Sorensen buffers
3) Remeasurement of non-Sorensen buffers
Halved capillaries (0.30x3.00mm) are filled with samples and put under excitation LED 5) Measurement separately). ratio: [math]\displaystyle{ \dfrac{\text{pointa()}-\text{background(abs,a)}}{\text{pointb()}-\text{background(abs,b)}}=\dfrac{\text{pointa()}-107}{\text{pointb()}-100} }[/math]
6) Analysis: Calibration Curve We fit a sigmoid-curve to the datapoints:
In blue: datapoints for ferric and sulfuric samples mit estimated pH-value (just for seeing if the points could lie on the curve somehow) 7) Analysis: pK[math]\displaystyle{ _{\mathrm{A}} }[/math]-Value intensities of SNARF. by substracting the background illumination. With [math]\displaystyle{ A }[/math] and [math]\displaystyle{ B }[/math] noting the acidic and basic endpoints of our titration it holds: [math]\displaystyle{ \text{pH}=\text{pK}_{\text{A}}-\log{\left[ \dfrac{R-R_B}{R_A-R}\cdot\dfrac{F_{B(\lambda2)}}{F_{A(\lambda2)}}\right] } }[/math] Source:
y-intercept of a linear fit:
We get [math]\displaystyle{ \text{pK}_{\text{A}}\approx 7.2 }[/math]. Now we have a calibrated equation for our setup and we can calculate the pH value for the samples by only measuring the relative intensities. 22.01.14 Calibration with UV-Laser I1) Remeasuring pH-Values of Buffer Samples
2) New Routine An improved pattern for LED & Laser exposure to simplify background normalization procedure:
23.01.14 Calibration with UV-Laser II3) Data Analysis
Radial dependency of the absolute fluorescence (minus background) is evaluated for LED exposure, LASER exposure and LASER+LED exposure.
The first image clearly shows the pH-dependent emission-ratio of SNARF. The higher the ratio, the higher is the pH-value (violet curve is sample 17 with pH=8.72). As expected there is no radial distribution. a lot of trouble (e.g bleaching of SNARF, varying SNARF concentration,...) the crosstalk of the Laser has to be substracted because SNARF has different pH- emission-curves for different excitation wavelenghts. regardless of the laser being switched on or not. In the experiment, the UV-crosstalk shifts the ratio in the area of the laser beam. 29.01.14 SNARF pH Measurement NBA1) Sample Preparation
NBA stock concentration is 8mM. To each sample 2.5µl SNARF (@1mM Stock) and 2.5µl buffer (pH 8.0, sample #15) are added. SNARF concentration therefore is 50µM. 2) Measurement 3) General LabView Procedure capillaries aren't placed onto the exact same spot every time, we have to rearrange the areas where we take our data from every time. We have to do this for both channels of each capillary (we want the same area for each of the channels, too). These steps have to be followed:
reference.
After every Analyze process LabView has to be stopped and restarted (reload pictures) to clear the memory. 4) Evaluation to be continued 30.01.14 SNARF pH Measurement at various Buffer Concentrations1) NBA-Mixture-Induced pH-Drop for various Buffer Concentrations water.
2) UV-Induced pH-Drop of NBA
February 201410.02.14 Improving the SetupThe Thorlab Optical Chopper System MC2000 is added to the setup. 11.02.14 NBA Effects on Fluorescent pH-Sensitive Beads PartThe movement of specific pH-sensitive, fluorescein-marked beads is measured.
To each sample of 49µl, a 1µl drop of bead+fluorescein is added.
the bead samples is difficult. The focus of the camera should be adjusted for every capillary, so that a nice amount of beads is in the focal plane.
12.02.14 SNARF Calibration with Chopper SetupWith the new Setup, the calibration of SNARF has to be redone as the effects of the laser are reduced in the measurements. 13.02.14 SNARF Calibration1) NBA Concentration Array chose to expand the sample array in the lower NBA concentrations this time. Also, the calibration curves we got last time, recommend a smaller pH value (7.5 instead of 8.0).
NBA stock concentration is 8mM. To each of the subsamples with 50µl desired volume, 2.5 µl SNARF (@1mM Stock) and 2.5µl buffer (pH 7.5, sample #14) are added. SNARF concentration therefore is 50 µM.
Ratio of the radial average of the two channels versus time: SNARF] 18.02.14 Beads VarietyCarboxylated and fluorescent (BCECF-like) beads of various dimensions (2µm - 0.02µm diameter) will be used to determine the movement and flows of charged particles along the pH gradient. 1) 0.02µm Beads
Two samples with 0.5mM NBA and two with 0.25mM NBA were used, beause the 0.5mM NBA samples already showed a great movement. With the smaller beads being less bright, the 1:100 dilution is more convenient.
2) 2µm Beads
Each sample therefore has a NBA concentration of 1mM (NBA-Stock: 8mM). The mentioned bead volume is an already pre-diluted sample (1:10 with 20µl beads, 180 µl water). The picture sequences show a inward stream superposed with an outward stream, appearently depending on which focal plane is observed. To determine this effect, three additional sequences are recorded. The lowest and highest focal plane and also one in between.
2µm beads with 1:100 dilution and 0.5mM NBA (timescale is black-red black-green again)
19.02.14 Evaluation of SNARF Calibration on 13.02.
Taking a bigger area improves our signal a lot. Also, the greater radius shows a decreased pH effect (as assumed).
21.02.14 Evaluation of SNARF Calibration on 13.02. Part IIWith the [pH-conversion recipe] the pH values can be calculated for the 85px radii.
24.02.14 Isoelectric FocusingPaper:
25.02.14 0.5µm & 1µm Diameter Bead Movement1) 0.5µm Beads
2) 1µm Beads
26.02.14 NBA and SNARF with NaOH pH AdjustmentThis time we want to measure the NBA induced change in pH value, without an additional buffer. decrease the pH a little bit, so the final pH before the measurement was meant to be safely determined to be between 7 and 8 (which appeared to be very wrong!).
With decreasing NBA concentration we add less NaOH (around 40µl for the 4mM sample, to <10µl for 0.2mM NBA). considered when measuring without a buffer. 27.02.14 Analysis of 26.02.14The analyzed area is 45x90px 1) Ratios
adjusting the pH just with NaOH molecules does not generate stable pHs."]] March 201405.03.14 DNA1) Sample Preparation
2) Measurement
06.03.14 DNA AnalysisThe intensity of 5x5 pixel areas centered on the laser spot and 10px away from the laser spot are averaged and plotted against time.
To account for the bleaching of the fluorescent labeled DNA(which is considerably larger with NBA, which is yet to be investigated, too)
Radial profiles for different -mers are plotted with set time.
It seems that DNA is accumulating at the Laser spot (bleaching by laser is not accounted, but that effect would actually lead to reducing fluorescence instead of increasing). April 201423.04.14 DNA+NBA in Different Buffer ConcentrationsA new NBA solution (50ml) is mixed with 8mM concentration (151 g/mol). New 2-component phosphate buffers (each 200ml) are made, too. 0mM NBA
4mM NBA
24.04.14 DNA+NBA in Different Buffer Concentrations: ResultsExceptionally strong bleaching ocurred and actual movement of DNA seemed questionable. 24.04.14 Beads in NBA+Buffer
Again, unusual strong bleaching occured (see above, left). Fitting the bleach curves exponentially and dividing by them should fix the problem. molecule is ordered.
#pragma rtGlobals=1 // Use modern global access method. 28.04.14 pH-Dependencies1) pH-Dependencies of Beads
The intensity varies about less than 10%. The two data waves presented were made from the same sample volumes, just different capillaries. intensity due to pH. 2) pH-Dependencies of Cy-5
Change filter set to #1 and LED to 627nm. stored and used in buffers above pH 7.]" 29.04.2014 Check: pH-Dependency of Cy5Repeating yesterdays measurment with samples freshly mixed directly before the measurement, and 10 minutes after (dark) storage.
Left picture is from yesterdays measurement. This time, pH 2.78 was measured at the beginning, but still is lower. Thererfore the fluorescence has to be pH dependent.
29.04.14 Reducing Laser Power1) Minimum Laser Power for Sufficient Uncaging? Checking if NBA uncaging still works sufficiently for lower laser powers, to aim for less cross-bleaching of the fluorescent dyes due to UV excitation. accuracy of the laser adjustment, and even measuring the laser power can change the initial adjustment greatly. Therefore, laser power is measured before and after the measurement to ensure accuracy.
Measurements performed with 4x4 binning and 30ms exposure.
Left picture shows the fluorescence intensities over time for some laser powers without NBA. 2) Saturation of NBA Uncaging? even more protons.
3) Should be checked: Is there a initial loss of intensity when having NBA present? 30.04.2014 Dividing by Exponential Fit to Account for Bleaching
The fits only considers bleaching caused by LED! measurement with 0mM NBA at the same laser power. flows this way. away (and into) the region of the laser spot. |