Talk:O2k-Fluo LED2-Module

Previous Product ID 34000-01

Measuring hydrogen peroxide

See Measuring H2O2 production with any brand similar to Amplex(R) REed see Amplex red and especially the accompanying Discussion page. For an overview of methods to measure H2O2 concentration or H2O2 production see Measuring hydrogen peroxide.

under construction: Spectral observables for fluorescence sensing / general fluorometry and related data processing

Reading recommendation:

Cchapter 19 of J.R. Lakowicz JR 2006 Principles of Fluorescence Spectroscopy 3ed or many introductory textbooks to fluorescence spectroscopy.

overview

• Intensity
  • Intensity - direct detection of analyte: analyte = fluorophore or fluorophore generated from analyte in stoechiometric reaction: e.g. resorufin generated by Amplex Red, TMRM, Safranin
  • Intensity -sensing: binding of analyte to a sensing probe changes fluorescence properties of probe, one excitation, one emission wavelength: Mg Green, Ca green, ....
• Intensity ratio: binding of analyte to a sensing probe (fixed concentration) changes fluorescence properties of probe, record two signal with different either excitation wavelength or detection wavelength
  • Excitation ratiometric: not possible with O2k-Fluo_LED2-Module
  • Emission ratiometric: not possible with O2k-Fluo_LED2-Module
• Anisotropy: not possible with O2k-Fluo_LED2-Module
• Fluorescence lifetime: time domain or phase modulation detected: not possible with O2k-Fluo_LED2-Module

Intensity: direct detection of the analyte

examples: e.g. TMRM, Safranin, NADH, resorufin generated by Amplex Red

Fundamentals

analyte = fluorophore or fluorophore is generated from analyte in stoechiometric reaction

Set up

• Select light intensity for one excitation wavelength

Data processing

For low analyte concentrations the analyte concentration [A] is directly and linear proportional to the fluorescence signal F: [A] = k * F + d

Especially for higher analyte concentrations and wide calibration ranges nonlinear calibrations may be necessary.

Intensity sensing

examples: Mg green, Ca green, DAN (NO), DAR (NO), DAF (NO), all ratiometric probes can also be used in this mode (with loss of specific benefits)

Fundamentals

The fluorescence intensity is maximal (or minimal) when all the fluorophore molecules are bound to analyte and minimal (or maximal) when no probe molecules are bound to the analyte. Binding of the analyte to the fluorophore probe is described by a bimolecular dissociation constant KD, therefore the resulting relationship between bound probe (and hence fluorescence intensity) and total analyte concentrations follows a relationship of the form y = x ( 1+x) resulting in a sigmoid curve. See also the fluorophores producers manual.

Set up

• Select light intensity for one excitation wavelength, select detection wavelength via filter set

Data processing

Correction for background fluorescence: not necessary.

The analyte concentration [A] is [A] = Kd * (F-Ffree)/ (Fbound - F) + intercept. with

F....fluorescence signal,

F bound: fluorescence signal of the completely analyte bound fluorophore e.g. at analyte saturation ,

F free: fluorescence signal of free fluorophore, e.g when analyte completely masked by an excess of chelating agent or analyte concentration is zero,

Kd dissociation constant of fluorophore - analyte complex.

This relationship can be used for calibration ( determining Kd and intercept) and for subsequent calculation of the analyte concentration. For calibration

• plot [A] against (F-Ffree)/ (Fbound - F), The term (F-Ffree)/ (Fbound - F) may be generated directly in DatLab as a calculated plot. (experimental feature!) or use an spreadsheet program (Excel)
• determine Kd and intercept from a linear regression
• use KD and intercept to determine unknown [A] via the equation above using a spreadsheet program (or plot in DatLab possible via experimental feature)

While a rough comparison of the obtained KD value with KD values known from literature is a valuable sanity check, the KD value determined in the calibration should primarily be considered an empirical calibration parameter. The intercept should theoretical be zero, and for this reason is sometimes omitted even in the fluorophores producers manual. However for practical purposes the intercept should always be determined and used for subsequent calculations. Frequently the terms "Fmin" for "Ffree" and "Fmax" for "Fbound" is found. If the fluorescence of the analyte bound fluorophore is observed these terms are indeed interchangeable. However, when the fluorescence of the unbound fluorophore is observed then the maximum fluorescence is reached when only the unbound form exists. Using (F-Fmin)/(Fmax -F) in this case does not work if max and min is interpreted as maxium and minimum fluorescence.

A logarithmic version of the equation can also be used.

So first of all, it is important to carry out an experiment where both Fbound and Ffree can be determined. Additionally determination of the fluorescence intensity (for a given concentration of fluorophore) measured at analyte concentration near and within the interested analyte concentration range is recommendable so that the linearity of the calibration in the interested range is given.

DatLab scripts

Sensing_calib: generates the plot (F-Ffree)/ (Fbound - F) used in the calibration procedure

In DatLab go to Advanced => calculate plot => generate plot (follow the instructions beneath and define the wanted fluorescence channel on the left side of the window as F)

• unit: u
• define input plots: F: fluorescence intensity plot (not background correction necessary)

{ copy from here downwards }

{ Biosensing, one wavelenght range, prepares plot for calibration }

{ uses [analyte concentration] = Kd * (F - Ffree) / (Fbound - F) + intercept }

{ calculates the term (F - Ffree) / (Fbound - F) }

{ for calibration: plot the analyte concentration versus that term, obtain Kd and intercept from a linear regression }

{ F ........Fluorescence signal, define desired Fluorescence channel as F}

{ N1F.......Ffree: minimal(maximal) Fluorescence signal, free analyte concentration = 0 }

{ N2F.......Fbound: maximal(minimal) Fluorescence signal, fluorophore indicator saturated with analyte }

var N1F, N2F: Float;

N1F:=104218;

N2F:=839162;

FuncResult:=(F - N1F) /(N2F - F);

Don´t copy from here downwards!

Set marks for a calculated analyte concentration and export the mark values of the newly created plot { (F - Ffree) / (Fbound - F) }. Next, plot the obtained values against the analyte concentration and make a linear regression (analyte concentration = y-axis, (F - Ffree) / (Fbound - F) = x-axis, K_D = slope).

sensing_results : calculates the analyte concentration, uses existing calibration parameters, rename to desired analyte e.g. [Mg]

In DatLab go to Advanced => calculate plot => generate plot (follow the instructions beneath and define the same fluorescence channel as F)

• unit: unit used for calibration, e.g. [mM]
• define input plots: F: fluorescence intensity plot (not background correction necessary)

{copy from here downwards}

{Biosensing, one wavelength range, calculates analyte concentration, based on existing calibration}

{ uses [analyte concentration] = Kd * (F - Ffree) / (Fbound -F) + intercept }

{ for calibration plot the analyte concentration versus (F - Ffree) / (Fbound -F), obtain Kd and intercept from a linear regression}

{ F .......Fluorescence signal, define desired Fluorescence channel as F}

{ N1F..... Ffree: Fluorescence signal for the free fluorophore, free analyte concentration = 0}

{ N2F......Fbound: Fluorescence signal for the fluorophore completely bound to analyte, fluorophore indicator saturated with analyte}

var N1F, N2F,Kd,intercept: Float;

N1F:=104218;

N2F:=839162;

Kd:=0.25995;

intercept :=0.15250;

FuncResult:= (Kd*(F - N1F) /(N2F -F)) + intercept;

Don´t copy from here downwards!

After generating the plot it represents the analyte concentration in the unit used for the calibration. For example if the concentrations for the calibration were entered in µM the K_D is also referring to µM and therefore the values of the generate plot display the analyte concentration in µM.

Simultaneous measurement of TPP and fluorescence

Question: Will it be possible to measure JO2, fluorescence AND TPP simultaneously, or just JO2/fluorescence vs JO2/TPP?

Answer: Background The main unknown is the chemical compatibility of the methods. We still have to find out whats the effect of Amplex red and its reaction product on the TPP signal. If this is not a big problem there are technical issues: a black TPP stopper will be required and then still light entering the chamber through the TPP electrode might be a problem. Producing the TPP electrodes in black is presumable not a very good idea because then it will no longer be possible to "see" the position of the membrane. I think the technical problems are solvable but first we have to check for chemical compatibility.

Update: First exploratory experiment:

A chamber was equipped with TPP electrodes (black stoppers) and a fluorescence module for measuring H2O2 via the Amplex Red method. A standard TPP calibration was carried out. Amplex red / HRP addition resulted only in a minor signal in the TPP channel, a further TPP calibration showed no obvious large negative effects on the performance of the TPP electrode. An H2O2 calibration of the fluorescence signal was carried out: The sensitivity for H2O2 was reduced by a factor of 3 to 4 as compared to measurements without TPP electrodes. At the same time the noise was drastically increased. In the comparative experiment (without TPP electrodes) no random noise was seen - digital resolution was the limiting factor. Based on this comparison, noise increased in the experiment with TPP electrodes at least by a factor of 20, probable more. It will be possible to estimate this factor with a higher precision after running an experiment without TPP electrodes at a higher gain (higher digital resolution).

It was further tested whether the presence of TPP+ alone (without electrodes) could explain this behavior but, if there were any effects at all, they were quite small.

Peroxide additions corresponding to a concentration change of 110 nM (110 pmol/ml) were still well visible but not additions corresponding to a 22 nM (22 pmol/ml) concentration change.

This was only one exploratory experiment (2 chambers)! (MF456)

Based on this preliminary data: An important point is why one would want to measure TPP and H2O2 simultaneously in the same chamber: If this is "only" for time / sample economy the results above strongly discourage this idea. Parallel measurement e.g one chamber TPP / one chamber H2O2 are preferable. If there is a very strong need to measure both parameters on the same sample (e.g. if strong in-homogeneity among samples in this respect is suspected) and a comparable high H2O2 flux is expected the simultaneous measurement might be principally doable, especially if further optimizations are possible. E.g.: Maybe with the TPP electrode in the chamber black stirrers become important again? However, this certainly would involve some methodological work and the results would be second class at the best.

What would be your driving interest for simultaneous measurements?

best greetings Mario

--Fasching Mario 10:49, 23 February 2012 (CET)

Question

How many parameters can be measured at once?

Answer

The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters: In both chambers oxygen concentration and oxygen flux (O2k-Core);

In both chambers fluorescence, with either identical fluorophores and identical optical probes, or different ones for different parameters in each chamber (H2O2, ATP production, Ca2+, mt-membrane potential, with the potential to extend these possibilities; O2k-Fluo LED2-Module);

Depending on fluorophore compatibility, additionally one potentiometric electrode can be inserted into each chamber (pH, TPP+, Ca2+; O2k-TPP+_ISE-Module);

Or in one chamber fluorescence and the other chamber NO (O2k-NO_Amp-Module)

Importantly, oxygen concentration is not only measured, but oxygen levels can also be controlled. This marks a new dimension in our evaluation of ROS production, with measurements spanning the entire ‘normoxic’, hyperoxic and deep hypoxic range.

Best wishes Erich

Oxygraph Series A to C

• The Oxygraph-2k, Series A to C, cannot be equipped with an O2k-Fluo LED2-Module. We thank you for your understanding.

Fasching Mario 11:55, 25 January 2012 (CET)

Other fluorophores

Question: In the brochure, specific mention is made of Amplex Red, Mg green, Safranin, and Ca green. Will other fluorophores work (e.g., MitoTracker or MitoSOX)? Is the module tuneable at all?

Answer: For some application no (or little) "tuning" might be necessary, see below for your examples.

a) General

The O2k-Fluo LED2-Module is "tunable" with two different levels of involvement: The detected emission wavelength can be changed totally by changing the filter in front of the photo diode. Similarly, some limited fine tuning of the effective excitation wavelength range is possible by changing the filters in front of the LED. Since filters (plastic films) are easily exchangeable this procedure is open to user innovation, maybe with a little help from us. In contrast, to drastically change the excitation wavelength a different sensor with a different LEd has to be used. At the moment we offer Fluorescence-Sensor Green and Fluorescence-Sensor Blue. There is limited opportunity for the customer to modify this part for herself, though we might offer a third sensors for UV excitation in the future.

b) Examples:

Remarks: a) Light intensity always has to be optimized to make sure the fluorophore considered is not affected significantly by photo bleaching. b) Many fluorophores are typically used for imaging applications. Be aware that the O2k-Fluo LED2-Module is NOT an imaging tool but quantifies fluorescence derived from the entire chamber.

MitoSOX: According to my tables, MitoSOX has an excitation maximum of 510 nm and an emission maximum of 772 nm. I do not have excitation or emission spectra right now to check if anything unusual or weird is going on with MitoSOX but just from the wavelengths this sounds easy. You could start with Fluorescence-Sensor Blue plus both the LED filter and photo diode filters from Filter Set Saf. Due to the huge separation of excitation and emission it is probably possible to use no LED filter at all. It should be checked if this affects the form of any calibration curve in any way.

Mitotracker: For me it would be interesting to learn the intended non-imaging application for Mitotracker? Also there are at least three different Mitotracker fluorophores: "green", orange", and "red". Which one do you want to use?--Fasching Mario 09:57, 21 May 2012 (CEST)

Coupling the O2k to optical instruments: Geometry

For various analytical techniques it is desired to couple optical sensors and light sources to the O2k chamber. The initial approach to do so was to insert the optical probe via a custom designed black stopper from above directly into the chamber, see … This approach (light and detection from above) requires also the use of black stirrer However, in Tony Hickey's (Hickey 2012 J Comp Physiol B) and in our own development of the fluorescence module (Fasching 2011 Abstract Berlin it became clear that for most applications in the visible range it is a better and easier strategy to introduce the light via the front window and detect the light via the same way. The Duran glass of the O2k chamber has very high transmission down to at least 350 nm and is probable usable even further down, see Duran optical properties The “via the window” approach has several important benefits:

1. The optical sensor does not “see” the stirrers. Therefore, for comparable small optical sensor diameters there was no need to use black stirrers. In fact using white stirrers increased overall sensitivity without introducing any disturbances.
2. No physical contact of the sample with the optical sensor necessary.
3. The space on top of the stopper is not taken up by the optical sensor, allowing for easier titrations and at least potentially for simultaneous use of additional electrodes introduced via the stopper. The black stopper is required to shield the chamber from outside light. Development with this approach was facilitated by the fact that the diameter of the O2k chamber window is identical to the chamber diameter, therefore a stopper with O-ring designed to be introduced into the O2k-Chamber from above, can also be placed in the chamber window for initial experiments. As a permanent, final sensor a more stable solution should be used. We used this configuration not only with the O2k-Fluo LED2-Module but also to couple a full spectrofluorometer to the O2k-Chamber.

We think this might be worth to try also for these groups that need a full spectrofluorometer or other light guide based detectors / sources attached to the oxygraph. --Fasching Mario 09:26, 17 February 2012 (CET)

Intensity of the light source

Question: What is the intensity of the light source (i.e. mW/cm2)?

We use a LED based approach which means that the user can vary the light intensity in a very wide range, see Fluorescence-Control_Unit#Control_of_LED-intensity. The current of the LED can be set to anything between 0.01 to 20 mA. Considering the current to voltage curve the total ELECTRICAL power consumption will therefore vary e.g. for the blue LED from 2 V* 0.01 mA = 0.02 mW to 3 V * 20 mA = 60 mW (similar for green). At 20 mA (setting = 2000, therefore at maxim power) the luminous intensity of the LED itself should be between 1400 to 2800 mcd according to specs, again for the blue light (similar for green). However, see O2k-Fluo_LED2-Module#Application-specific_settings for typical suggested light intensities that are far lower. We don't even try to pretend to be able to do any absolute luminous power measurements our-self. What we suggest at Fluorescence-Control_Unit#Control_of_LED-intensity is to use the minimum light intensity to reach the desired signal to noise in the fluorescence signal as a matter of principle. The simultaneous measurement of respiration of course provides an inherent quality control for any negative effects of the light on the biological sample. So far we have not seen any biological damage by blue and green light even for light intensities far higher than we recommend. Light stability of the used fluorophore may be more of a limiting factor and is complicated e.g by the observation that the non H2O2 dependent background oxidation of Amplex red seems to be light intensity dependent in some media (at least for very high light intensities) but not in others. Fasching Mario 10:30, 12 March 2015 (CET)

Homogeneity of Illumination

Question: Does the light source illuminate the whole sample or a small section of the chamber?

The truth is on middle ground: It dose illuminate a big section of the sample, i.e. in a clear solution there is a "light cone" typical for LEDs with half maximum viewing angles 25-35 deg. However, depending on your sample there may be very little or a lot of light scattering. The more light scattering, the more uniform will be the light distribution in the chamber. Fasching Mario 10:30, 12 March 2015 (CET)

Archive: Instructions for Older Models

O2k-Fluo LED2-Module, Series A

Setting the LED intensity directly at the Fluorescence-Control Unit Use the small supplied screwdriver to bring the switch on the front panel of the Fluorescence-Control Unit to the desired position using the table below:

Position	0	1	2	3	4	5	6	7	8	9
LED current [mA]	off	0.02	0.5	1	2	5	10	20	30	set by DatLab

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