• ibidi OPAL - Optical O<sub>2</sub> Measurement System
  • ibidi OPAL - Optical O<sub>2</sub> Measurement System

ibidi OPAL - Optical O2 Measurement System

Versatile system for measuring the oxygen concentration in cell cultures or tissues

  • Fast measurement within seconds
  • High spatial resolution
  • Non-invasive and real-time measurements
  • Ideal for in vitro hypoxia conditions in 2D or 3D cell culture (e.g., tumor models or metabolism studies)

ibidi OPAL Controller

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OPAL Detector Unit, Filter Cube, and
LED Light Source

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Beads and nanoparticles for
quantitative O2 measurement


in cooperation with

PRACTICAL COURSE

Register for a Laboratory Course at ibidi Munich / Germany:
OPAL Real-Time Oxygen Measurement in Live Cell Imaging

Contact ibidi for a free demo of the ibidi OPAL System.

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Cat. No. Description Pcs./Box
74001 ibidi OPAL - Optical O2 Measurement System: Optoelectronic hardware and software for generating and processing of oxygen-dependent phosphorescence lifetime signals. Controller, Detector Unit, LED Light Source, Adapter Set, Filter Cube, PC software 1
74051 Adapter Set: for mounting OPAL to different microscopes, customized for your microscope and your requirements (1 x adapter for light source, 1 x mounted filter set) 1
74002 OPAL Mirror Box 1

Applications

  • Exact measurement of absolute oxygen concentration in cell cultures or tissue
  • Ideal for in vitro hypoxia conditions in 2D or 3D cell culture (e.g., tumor models or metabolism studies)
  • Measurement of oxygen consumption or production


The ibidi OPAL Optical O2 Measurement System allows for quantitative, real-time monitoring of absolute oxygen values in both the extracellular and intracellular environments of cells, tissues, and spheroids. The system uses phosphorescence lifetime to measure the concentration of oxygen by way of oxygen sensitive fluorophores in the form of cell-impermeable polystyrene beads or cell-permeable nanoparticles.

During live-cell imaging assays, a stage-top incubation system is used to control the oxygen and carbon dioxide levels; however, oxygen concentration is typically lower near or in cell clusters than in the incubation chamber (Figure 1). Knowledge of the precise oxygen levels in or near an experimental sample is often imperative for an accurate understanding of cellular activity.

 

Controlling and measuring of oxygen levels are critical factors for many research applications as the levels are much lower in the physiological environment of cells and tissues than in air.

Technical Specifications

Detector Unit:
Photocathode Area Size Dia.8 mm (round shaped area)
Wavelength 230 nm (short); 630 nm (peak);
920 nm (long)
Luminous Sensitivity Min. 350 μA/lm (Cathode);
3.5 x 107 V/lm (Anode)
Luminous Sensitivity Typ. 500 μA/lm (Cathode);
1.0 x 108 V/lm (Anode)
Radiant Sensitivity Typ. 78 mA/W (Cathode);
15 V/nW (Anode)
Ripple Noise (peak to peak) Max. 0.5 mV
Settling Time Max. 10 s
LED Light Source:
Radiation Class Incoherent optical radiation
LED Color green (530nm)
Power 300 mW

Filter Set:
Emission 630 / 92 nm or 607/70 nm
Excitation 531 / 40 nm
Dichroic Mirror 555 nm

OPAL Controller:
Temperature Range  Ambient temperature to +55°C
Lifetime Range  1 – 1000 µs

Working Principle and Components of the ibidi OPAL System

The ibidi OPAL System is easily connected to your fluorescence microscope.


Compatibility to Microscopes

The ibidi OPAL System can be connected to all modern inverted fluorescence 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 specialized 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 by using an eyepiece adapter.

   


The selected objective defines the measured area. An iris is used to reduce the field of measurement down to 20 µm. The density of the CPOx beads or NanO2-labeled cells can be selected to further specify the sample area.


Extracellular O2 Monitoring

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

  • CPOx-Beads have an oxygen-reactive fluorophore
  • The 50 μm polystyrene beads are non-permeable and remain in the cell media for monitoring extracellular oxygen levels
  • Results are obtained optically using a fluorescent microscope with an integrated OPAL System
  • CPOx-Beads are also compatible with phosphorescence-lifetime imaging microscopy (PLIM)
 


Intracellular O2 Measurement

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

  • NanO2 Nanoparticle Reagent contains an oxygen-reactive fluorophore
  • Cell-permeable nanoparticles enable monitoring of intracellular oxygen levels via endocytosis
  • Results are obtained optically using a fluorescent microscope with an integrated OPAL System
  • NanO2 Nanoparticle Reagent is also compatible with PLIM
 


Overview: O2 Monitoring and O2 Imaging

ibidi OPAL
on Fluorescence Microscope

(Phosphorescence Lifetime Measurement)
CPOx Beads
NanO2
Quantitative
Extracellular
Not Image-Based
Quantitative
Intracellular
Not Image-Based
PLIM Microscope
(Phosphorescence Lifetime Imaging):
 
CPOx Beads
NanO2
Quantitative
Extracellular
Image-Based
Quantitative
Intracellular
Image-Based
Fluorescence Microscope
(Intensity-Based Imaging):
 
MM2
Semi-Quantitative
Intracellular
Image-Based

Experimental Examples

Oxygen Consumption in Cell Culture Medium

The CPOx-Beads were seeded with cells (3x105 cells/ml) in a µ-Slide VI 0.4 (Cat. No. 80606). The oxygen concentrations in the incubation system were set from 5.1 kPa to 19.5 kPa. Site-resolved measurements with the OPAL System revealed that the oxygen level in the cell culture media did not equal the set values of the incubation system. The oxygen consumption of the cells in the culture media was too great for equilibrium to be established between the gaseous and liquid phases.

 

Response of the NanO2 Nanoparticle Reagent

The oxygen-sensitive nanoparticles have an inverse optical response to oxygen concentration. Low sample oxygenation produces a high response, and a high oxygen level produces a low response.
Red: Response at 0% oxygen.
Blue: Response at 21% oxygen.

Advantages:

  • Passive cell staining – simply add the reagent to the medium, incubate, wash, and measure
  • Low toxicity, long retention in cells, bright signal
  • Optimized for multi-parametric (multi-color) analyses



Control the Cell Imaging Environment with the ibidi Heating & Incubation System

To provide the optimal conditions to perform cellular oxygen level studies, the ibidi system consists of the following components:

  • Gas mixer for optimal CO2 and O2 levels
  • Humidifying column humidifies the mixed gas as it exits the controller
  • Temperature controller for stable and uniform consistency
  • Stage-top chamber with heated plate eliminates condensation for excellent phase-contrast
    imaging
 


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