User:Christian Niederauer/Notebook/Phgradients/2014/08/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==December 2013== | ==December 2013== | ||
===18.12.13 SNARF & NBA in H<math>_2</math>O without buffer=== | ===18.12.13 SNARF & NBA in H<math>_2</math>O without buffer=== | ||
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We fit a sigmoid-curve to the datapoints:<br> | We fit a sigmoid-curve to the datapoints:<br> | ||
<gallery>Image: | <gallery>Image:Chris_nie_calibration.PNG | Calibration Plot (Sigmoid-Fit) | ||
</gallery> | </gallery> | ||
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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>Image: | <gallery>Image:Chris_nie_Newpattern.png | Exposure Pattern | ||
</gallery> | </gallery> | ||
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| <gallery>Image:Chris_nie_Cc_focus.png | top focal plane | | <gallery>Image:Chris_nie_Cc_focus.png | top focal plane | ||
</gallery> || <gallery>Image: | </gallery> || <gallery>Image:Chris_nie_M_focus.png | mid focal plane | ||
</gallery> || <gallery>Image: | </gallery> || <gallery>Image:Chris_nie_C_focus.png | bottom focal plane | ||
</gallery> | </gallery> | ||
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<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> | ||
<gallery>Image: | <gallery>Image:Chris_nie_Ph85px.png | pH for various c[NBA] @ 85px </gallery> | ||
===24.02.14 Isoelectric Focusing=== | ===24.02.14 Isoelectric Focusing=== | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_05um_unten80.png | bottom focal plane</gallery>||<gallery>Image:Chris_nie_05um_mitte.png | mid </gallery>||<gallery>Image:Chris_nie_05um_oben80.png | top </gallery> | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_1um_unten80.png | bottom focal plane</gallery>||<gallery>Image:Chris_nie_1um_mitte.png | mid </gallery>||<gallery>Image:Chris_nie_1um_oben.png | top </gallery> | ||
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|<gallery>Image:Chris_nie_Nba_naoh_1.png | 0.2mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_2.png | 0.5mM NBA </gallery>||<gallery>Image:Chris_nie_0_7.png | 0.7mM NBA </gallery> || <gallery>Chris_nie_Nba_naoh_3.png | 1mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_4.png | 2mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_5.png | 4mM NBA </gallery> | |<gallery>Image:Chris_nie_Nba_naoh_1.png | 0.2mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_2.png | 0.5mM NBA </gallery>||<gallery>Image:Chris_nie_0_7.png | 0.7mM NBA </gallery> || <gallery>Image:Chris_nie_Nba_naoh_3.png | 1mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_4.png | 2mM NBA </gallery>||<gallery>Image:Chris_nie_Nba_naoh_5.png | 4mM NBA </gallery> | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_Ph10px.png | pH at 10px </gallery>||<gallery>Image:Chris_nie_Ph15px.png | pH at 15px </gallery>||<gallery>Image:Chris_nie_Ph25px.png | pH at 25px </gallery>||<gallery>Image:Chris_nie_Ph40px.png | pH at 40px </gallery> | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_Ares_with_expo.png |Exponential fits </gallery>||<gallery>Image:Chris_nie_Withouth_bleach.png | Normalized (without 2&22mer) </gallery> | ||
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'''1) pH-Dependencies of Beads | '''1) pH-Dependencies of Beads | ||
<gallery>Image: | <gallery>Image:Chris_nie_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> | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_Avgfram.png | pH-dependent Fluorescence </gallery>||<gallery>Image:Chris_nie_Freshold.png | Degradation of fluorescence after storage </gallery> | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_Lowno.png </gallery>||<gallery>Image:Chris_nie_Lowwith.png </gallery>||<gallery>Image:Chris_nie_Normalow.png </gallery> | ||
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<gallery>Image: | <gallery>Image:Chris_nie_Satura.png | </gallery> | ||
'''3) Should be checked: Is there a initial loss of intensity when having NBA present? | '''3) Should be checked: Is there a initial loss of intensity when having NBA present? | ||
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|<gallery>Image: | |<gallery>Image:Chris_nie_Expnormalized.png | Normalized for LED-Bleaching </gallery>||<gallery>Image:Chris_nie_Withexpnorm.png | Exponential Fits for 1mM NBA-Sample </gallery>||<gallery>Image:Chris_nie_Withoutexpnorm.png | Linear Fit for 0mM NBA (nearly constant)</gallery> | ||
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The fits only considers bleaching caused by LED!<br> | The fits only considers bleaching caused by LED!<br> |
Latest revision as of 00:11, 27 September 2017
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December 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 [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 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: SNARF-Manual
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. 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 These steps have to be followed:
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
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.
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
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: 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.
With decreasing NBA concentration we add less NaOH (around 40µl for the 4mM sample, to <10µl for 0.2mM NBA). 27.02.14 Analysis of 26.02.14The analyzed area is 45x90px 1) Ratios
"Appearently, 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.
#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. 2) pH-Dependencies of Cy-5
Change filter set to #1 and LED to 627nm. 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.
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?
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! |