Talk:MiPNet17.05 O2k-Fluo LED2-Module

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

Recommended reading:
Lakowicz JR (2006) Principles of fluorescence spectroscopy. 3ed, Chapter 19.

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
Setup
  • Select light intensity for one excitation wavelength
Data processing
For low analyte concentrations the analyte concentration, cA, is directly and linear proportional to the fluorescence signal F: cA = 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.
Setup
  • Select light intensity for one excitation wavelength, select detection wavelength via filter set
Data processing
Correction for background fluorescence: not necessary.
The analyte concentration cA is cA = Kd * (F-Ffree)/ (Fbound - F) + intercept.

with

F....fluorescence signal,
Fbound: fluorescence signal of the completely analyte bound fluorophore e.g. at analyte saturation,
Ffree: 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 cA against (F-Ffree)/ (Fbound - F). The term (F-Ffree)/ (Fbound - F) may be calculated directly in DatLab and shown as a plot, or use a spreadsheet program (Excel).
  • Determine Kd and intercept from a linear regression;
  • Use Kd and intercept to determine ca.
While a rough comparison of the obtained Kd value with Kd known from the literature is valuable, the Kd 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.
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.


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 does 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°. However, depending on the sample type, there is more or less light scattering. The more light scattering, the more uniform is the light distribution in the chamber.