What is the difference between fluorescence and phosphorescence?
Phosphorescence is a specific type of photoluminescence that is related to fluorescence. Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs. The excitation of electrons to a higher state is accompanied with the change of the spin state. Once they are in a different spin state, the electrons cannot relax into the ground state quickly, because the re-emission involves quantum-mechanically forbidden energy state transitions. As these transitions occur very slowly in certain materials, absorbed radiation may be re-emitted at a lower intensity for up to several hours after the original excitation.
Decay Time (Fluorescence): Decay Time (Phosphorescence):
τ < 10-5s τ >10-5s
What is your recommendation for the most accessible (easiest to establish) method for roughly estimating O2 concentration in 2D cell culture systems, ideally using standard lab equipment?
Time-resolved plate readers are very easy to use for initial measurements of O2concentrations in cell populations. If you want to have optical observation of the cells as well, the OPAL System is also simple to set up on standard microscopes.
Are applications in opaque organs/larger tissue chunks/in vivo possible? Can opaqueness somehow be overcome?
When working with living tissues/samples you cannot do any treatment to overcome opaqueness, as this would harm and influence the living samples. You have the same limitations with signal attenuation in depth, as with other fluorescence-based probes and techniques: approximately 200 µm with 1-photon excitation and up to 500 µm with 2-photon excitation.
How does OPAL compare to FLIM?
The OPAL System works in combination with wide-field microscopes. Therefore, the spatial resolution is lower when compared to the image-based FLIM microscope. The OPAL System is a complementary instrument for wide-field microscopes and costs approximately one tenth of the FLIM system price.
Since oxygen solubility depends on temperature, how does the OPAL System take this into account in the final measurement? Does the OPAL software do this automatically?
The calibration of the OPAL System needs to be done by the user for every experimental setup. The calibration must be performed under the same conditions (temperature, medium, microscope setup) as in the actual experiment. By calibrating the setup, the system will take the temperature into account.
Is the correlation between the decay time and the oxygen concentration linear from 0 to 21% O2?
No, it is not linear. To account for the nonlinearity, a third calibration point at 10.5 % is needed. In a three point calibration, the f-value defines the curvature of the calibration curve. However, in most cell culture situations, the OPAL System’s pre-calibrated f-value is sufficient.
How often do you need to calibrate the OPAL (at 0% and 21%)?
It is recommended that you always calibrate both points. A new calibration of the OPAL System is necessary for every change of the setup (e.g., medium, temperature, microscope setup, or sensor batch).
Is it possible to attach the OPAL System to a confocal microscope to observe the O2 distribution in 3D?
No, the OPAL System’s light source cannot be integrated into the confocal scanning illumination path. If the confocal system has a separate, wide-field illumination path, then the OPAL System can be integrated along that illumination path. It is impossible to combine the two systems to get oxygen measurements with confocal resolution along the z-axis.
How do you get an optical section of a spheroid using wide-field microscopy?
On a wide-field microscope, you can only create 2D images. Here, the O2 concentration is averaged over the entire z-stack.
There are so many brands of fluorescence microscopes. How can I be sure that the OPAL System adapts to mine?
ibidi provides standard adapters for Leica, Nikon, Olympus, and Zeiss microscopes. Additionally, ibidi can adapt the LED Light Source to liquid light guides of standard sizes.
Which units are used with the OPAL System for the output information? What accuracy can be achieved?
The OPAL System measures the decay times of the sensor particles. The measured values are converted by the OPAL System into a percentage oxygen concentration (% saturation or v/v). The values have an accuracy of ±0.2%.
Which oxygen sensing probes are compatible with the OPAL System?
CPOx from ibidi are suitable for extracellular oxygen measurements and Agilent MitoXpress® Intra is optimized for intracellular use.
Is it possible to measure reactive oxygen species (ROS) using the probes from ibidi?
No, our probes respond to molecular oxygen (O2), not to singlet oxygen. There is no cross-sensitivity to ROS.
Can CPOx beads also be used with a TRF reader?
Our CPOx sensor probes are compatible with TRF readers that have the following specifications:
The reader needs to be able to measure decay times in a range of microseconds.
The reader needs a detection technology that detects multiple photons after the excitation (e.g., Multi-Channel Scaling).
The excitation and detection wavelengths need to match those of the sensor probe.
Are your O2 probes photosensitive? Will the oxygen-sensitive probes / sensors sustain bleaching by lasers and other light sources when doing confocal or wide-field microscopy?
Yes, all O2 probes are photosensitive. The probes will be affected by light sources. To which extent the probes will bleach depends on the intensity of the light source that is used. To minimize bleaching effects, the CPOx beads have an integrated photo stabilizer in their matrix.
Is it possible to use the extracellular (CPOx) and intracellular (Agilent MitoXpress® Intra) probes together?
Yes, this is possible. You would need to use the CPOx-beads, orange for these kinds of experiments. Agilent MitoXpress® Intra and the CPOx-beads, red have very similar spectral properties. When using the OPAL system you need to calibrate the system for both probes before the actual experiment. While performing the measurement you need to switch between the two sensor types in the software.
Is 3D visualization with the ibidi oxygen-sensitive probes also possible if cells are seeded on a biomaterial (e.g., soft material like collagen sponges or hard materials like ceramics)?
Yes, it is possible. Microporous scaffolds have been used with FLIM without any problems. For thick scaffolds, an upright microscope would be more convenient.
How do you get the probes into a spheroid?
In order to integrate CPOx beads into a spheroid, the beads need to be coated. The optimal coating (e.g. collagen, fibronectin) depends on the cell type. Add the pre-coated beads to the medium during the formation of the spheroid.
Do you need a certain density of beads in a small area to get a good reading? Do the beads affect the growing cells?
For microbeads like CPOx (50 µm), more beads are needed for higher spatial resolution measurements. There were no observed effects on cell growth from these probes.
Can the probes from ibidi be used with suspension cells?
Yes, you can also stain suspension cells, but imaging of the suspension cells is difficult because of their constant movement.