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MitoImage® – NanO2 / MM2

Image 1

  • Image 2Exact oxygen measurement in cell cultures or tissue
  • Fast measurement within seconds
  • High spatial resolution
  • Non-invasive and real-time measurement
  • Ideal for in vitro hypoxia conditions, like 3D cultures, spheroid models, and tissue

The oxygen concentration can be measured optically and directly in a 2D cell culture dish, a compact 3D cell spheroid, or even in a piece of tissue. Oxygen-sensitive beads or cell-permeable fluorophores are utilized to measure the fluorescence lifetime.

 

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Compatibility to Microscopes

The iBidi OPAL System can be connected to all merdern inversted microscopes from Nikon, Olympus, Leica, and Zeiss. Small adaptations need to be made, and during this process, iBidi will comprehensively support you. These adaptations include a specialised OPAL Filter set and an LED light source coupler for the microscope (included in the ibidi OPAL System). The OPAL Detector Unit fits the standard C-Mount camera port, or can also be mounted on an ocular.

Note: The OPAL System measures lifetimes in the range of 1-1000 µsec (i.e. phosphorescence). To keep things simple, ibidi uses the more common term fluorescence, instead of phosphorescence.

 

Extracellular O2 MonitoringImage 4

Oxygen monitoring is defined as the measurements of of O2 in the direct neighbourhood of cultured cells. Oxygen sensitive beads, with a diameter of 50 µm (CPOx), are used in combination with the ibidi OPAL system or a FLIM microscope to measure the oxygen concentration.

 

CPOxImage 13

  • 50 µm polystyrene beads with an O2 sensitive fluorophore (fluorescence lifetime)
  • Quantitative results with OPAL and FLIM microscopes
  • Biocompatible in cell culture, spheroids or tissue
  • Not cell permeable
  • For extracellular O2 monitoring

 

Intracellular O2 MeasurementImage 6

For intracellular oxygen measurements, an oxygen sensitive nanoparticle reagent is brought into the cells. The ibidi OPAL
System or FLIM microscopes reveal quantitative O2 concentrations by identifying a change in the lifetime of the fluorophore NanO2.

 

 

 

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  • Image 8Monitoring in situ oxygenation of cell culture
  • Measuring the 3D distribution of oxygen in spheroids or tissue
  • Studying metabolic processes in cancer research studies
  • Investigating infections in cell cultures, like bacteria and fungi

 

Principle of FLIM – Fluorescence Lifetime Imaging

Some fluorophores stay in their excited state for a relatively long time. This time period is called the fluorescence lifetime. FLIM microscopes and OPAL measure this lifetime, instead of measuring the intensity.

Some long-lifetime fluorpohores show a strong dependency on the surrounding oxygen level. By measuring the fluorescence lifetime, you are able to optically quantify the oxygen concentration.

In the example on the left, a 3D cell spheroid is measured optically for oxygen concentration. All cells were labelled with NanO2 and imaged with a widefield FLIM microscope. O2 concentration can be easily calculated from the fluorescence lifetime. The higher the oxygen concentration is, the shorter the fluorescence lifetime.

 

Image 9

 

Intracellular O2 ImagingImage 6

For intracellular oxygen imaging, an oxygen sensitive nanoparticle reagent is brought into the cells. FLIM microscopes image quantitative O2 concentrations by identifying a change in the lifetime of the fluorphore NanO2. With a standard fluorescence (or a confocal) microscope, a quantitative O2 concentration can also be determined by measuring the fluorescence intensity of the ratiometric fluorophore reagent MM2.

 

NanO2Image 10

  • O2 sensitive fluorophore (fluorescence lifetime)
  • Quantitative results with OPAL and FLIM microscopes
  • Biocompatible in cell culture, spheroids, or tissue
  • Directly cell permeable, self-loading
  • For Intracellular image based O2 measurement

 

MM2Image 11

  • O2 sensitive fluorophore (fluorescence lifetime)
  • Quantitative results with widefield or confocal fluorescence microscopes (independent of OPAL and FLIM systems)
  • Biocompatible in cell culture, spheroids, or tissue
  • Directly cell permeable, self-loading
  • For Intracellular image based O2 measurementImage 12
  • Ratiometric measurement based on the fluorescence intensity ratio between emission 1 (reference signal) and emission 2 (O2 sensitive signal)
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