O2k-Fluo LED2-Module

MitoPedia O2k and high-resolution respirometry: O2k-Open Support

Description

[[Description::

The O2k-Fluo LED2-Module is a component of the O2k-Fluorometer (O2k-Series D to G). It is an amperometric add-on module to the O2k-Core (O2k-Series D to G), adding a new dimension to high-resolution respirometry. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex® UltraRed), ATP production (Magnesium Green™), mt-membrane potential (Safranin, TMRM, Rhodamine 123), Ca²⁺ (Calcium Green™), and numerous other applications open for O2k-user innovation.]]

Reference: [[Info::Product details and purchase information]]

Additional resources

» O2k-Modules
» O2k-Fluo Manual
»O2k-Demo experiments
»Fluorescence-Sensor

» Keywords

Specific

» O2k-Fluorometer Series G

» Amplex® UltraRed

» Calcium Green™

» Magnesium Green™

» Safranin

» TMRM

» Fluorescence-Control Unit

» Fluorescence-Sensor Blue

» Fluorescence-Sensor Green

General

» HRR

» mt-membrane potential

» O2k-chamber

» O2k-Core

» O2k-Main Unit

» O2k output

» O2k signal

» Oroboros O2k

O2k signal and output

O2k signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
O2k output: type I, II, or III, or combinations.

Scope of applications

The scope of applications is defined by the excitation wavelengths of the Fluorescence-Sensor Green and Fluorescence-Sensor Blue and the selection of emission filters.

The O2k-Fluo LED2-Module is designed for measurements performed on samples placed in a stirred and temperature-controlled chamber. Fluorometric protocols based on measurements in cuvettes of various fluorometers can be used with the O2k-Fluo LED2-Module, and extended by simultaneous measurement of respiration and monitoring or regulation of oxygen concentration. The excitation and emission wavelengths of the fluorophore must be suitable for use with the O2k-Fluo LED2-Module. For examples see LED-intensity and amplification.

The O2k-Fluo LED2-Module is not designed to fulfill the functions of fluorescence microscopes or flow cytometers.

O2k-Manual: O2k-Fluo LED2-Module (for O2k series D-G)

Setup of the O2k-Fluo LED2-Module

LED-intensity and amplification

The light intensity of the LED (LED-intensity) and the signal amplification (Gain) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications.

The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex® UltraRed assay the fluorophore is formed during the experiment.
The recommendations apply to experiments at 37 °C. The Fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals.

Fluorophores

Application	Sensor	Filter set	O2k output	Light intensity (polarization voltage) - Note a	Gain	Comment
Amplex® UltraRed	Fluorescence-Sensor Green	AmR	Type II	100 - 500	1000	at c(AmR) = 10 µM
TMRM	Fluorescence-Sensor Green	AmR	Type III	200 - 500	1000	at c(TMRM) = 2 µM
Safranin	Fluorescence-Sensor Blue	Saf	Type III	100 - 200	1000	at c(safranin) = 2 µM
Magnesium Green™	Fluorescence-Sensor Blue	MgG / CaG	Type I and II	100 - 500	1000	at c(Mg Green) = 2 µM
Calcium Green™	Fluorescence-Sensor Blue	MgG / CaG	Type I	300 - 500	1000	at c(Ca Green) = 2 µM

Note a: Set the polarization voltage [mV] for the amperometric channel (Amp) in the DatLab menu [O2k-MultiSensor \ O2k Control \ Amp polarisation voltage]. Divide the polarisation voltage [mV] by 100 to obtain the current [mA] through the LED. For simple operation instructions, it is sufficient to refer to the polarization settings selected in DatLab.

The fluorescence signal

O2k signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [Amp]) as the primary signal.
O2k output: type I, II, or III, or combinations.

Graph / Select plots‎

Graph layout: Three plots are available in DatLab based on the recorded signal: Amp Raw Signal, Amp Calibrated, and Amp Slope. These plots can be selected from the drop-down lines and displayed with their check boxes either on the Y1 or Y2 [Graph layout / Select Plots].

Amp Raw Signal displays the raw voltage (including amplification) as recorded by the O2k at a given gain setting.

Amp Calibrated is the signal after calibration with the parameters set in the O2k-MultiSensor Calibration window.

Amp slope is the time derivative of the calibrated signal, multiplied by 1000, in units [mV(conc. Unit during calibration)/s], so if the signal was calibrated in µM [nmol/ml] the unit of the slope is pmol/(s ml). To obtain the slope of the raw signal check the appropriate box in the calibration window (DatLab 5.1.0.130 and above).

Graphs can be generated to display oxygen and fluorescence data, or several graphs can be added to display oxygen and fluorescence data separately. Layout templates are provided, which can be modified and saved as appropriate. All graph settings can be saved as user-defined layouts, see MiPNet19.01C DatLab Guide.

O2k-Fluorometry and the TIP2k

TIP2k: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the 2-mL O2k-chamber.

O2k-Fluorometry Workshops

Oroboros O2k-Workshops

» References

»O2k-Publications: Topics

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» O2k-Publications: O2k-Fluorometry - Abstracts

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Next step

MitoPedia methods: Fluorometry

MitoPedia O2k and high-resolution respirometry: O2k hardware, O2k-Open Support, O2k-FluoRespirometry