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
Which specifications of a FLIM system are necessary to use it with ibidi probes?
The key, additional specification for FLIM detection of the oxygen sensitive probes is the ability of the system to measure phosphorescence lifetimes in the range of 10-60 microseconds. Standard FLIM systems are usually set up for ps-ns lifetimes, so they may require some modifications to the hardware and software. Additional specifications are essentially the same as for other imaging platforms:
Spectral compatibility with NanO2/MM2 probes, i.e.:
Excitation source (laser or LED): 390-405 nm
Red-sensitive photodetector (up to 750-800 nm): PMT or camera-based. The main emission band is 650+/-30 nm. The secondary band for the MM2 probe is 430-450 nm.
Optimal filters and dichroics to match a) and b).
The microscope should be configured for live imaging, so it should have active control over sample temperature and atmosphere (O2, humidity, and CO2). High sensitivity and reduced photo bleaching (e.g., spinning disk, camera-based, or TCSPC)
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%.
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. The CPOx beads are less sensitive to light exposure than the nanoparticles because of the integrated photo stabilizer in their matrix.
Do your intracellular O2 probes have side effects that can affect cells?
At high illumination intensities, singlet oxygen is generated causing cytotoxicity. However, with low light there is no problem. Compared to other probes, the NanO2 and MM2 have higher photo stability and brightness, and also lower photo toxicity. They localize in the cytosol, but not in the nucleus or other vital organelles, and this limits their cytotoxicity. It is recommended to limit scanning to 10 scans per object with a PLIM. Image acquisition parameters should be carefully optimized.
Is it possible to use the extracellular (CPOx) and intracellular (NanO2) probes together?
Yes, this is possible. You would need to use the CPOx-beads, orange for these kinds of experiments. NanO2 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 is the selectivity of the O2 probes?
In our extensive experience with MM2 and NanO2, we have not observed any interference with drugs, cellular components, or other (fluorescence) probes. With respect to O2 sensitivity, NanO2 and MM2 probes cover the whole physiological range from 0 - 200 µm.
How do you get the probes into a spheroid?
The best way using NanO2 / MM2 is to add the probe to the medium during the formation / growth of the spheroid. In this case, you will get a uniform staining of the spheroid. Pre-formed spheroids can only be stained to a depth of 50-100 µm. 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.
How long can these intracellular probes stay inside the spheroid? Will they harm the cells after a certain amount of time?
Experiments were performed for up to 3 or 4 days without any harm to the spheroids. The signal was quite stable in this time frame.
Do you need a certain density of beads in a small area to get a good reading? Do the beads affect the growing cells?
Nanoparticle probes, like NanO2 or MM2, give a uniform staining inside the cell. 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.
What is the difference between NanO2 and MM2, and which one is better for lifetime imaging?
NanO2 is used to measure phosphorescence lifetime (i.e., you can use a PLIM microscope or the OPAL System). MM2 allows ratiometric intensity measurements and two-photon excitation. This yields semi-quantitative results on many standard fluorescence microscopes (e.g., wide-field, confocal, or two-photon).
Are the NanO2 and MM2 probes specific to oxygen, or will other dissolved gasses also affect the signals (e.g., dissolved superoxide, nitric oxide, or carbon monoxide)?
The NanO2 and MM2 probes are specific to O2 (molecular oxygen). So far, no cross-sensitivity to ROS or other species has been observed. Both nanoparticle sensors are shielded with a polymer matrix, so that only small gaseous O2 molecules can interact with the sensitive dye.
How do I determine the absolute O2 concentration from the ratio of the MM2 reference signal to the O2-sensitive signal?
To get an absolute O2 concentration from the ratio between reference signal and the O2-sensitive signal, you need to perform a calibration at 0% and 21% O2 first. After you got the ratio from these references, you can calculate the O2 concentration of your spheroid via a calibration curve.
To generate a 0% oxygen atmosphere, we recommend using MM2-positive cells and add sodium sulfide, which is an oxygen scavenger agent. To obtain a 21% oxygen atmosphere please do the following: incubate the cells in 2D (since it is easier than in 3D) with MM2 and remove the medium afterwards. Perform the measurement after the cells dried out/died (because living cells consume O2 and you would not get 21% total in the end if they are still alive).
Since temperature has a great impact on oxygen concentration, make sure that you perform the calibration measurements at the same temperature as your probe. To be even more accurate, you might also need to subtract the background fluorescence from all values, including those of your probe.
What does the ratio of the MM2 reference signal to the O2-sensitive signal mean?
A ratio has to be measured in each plane and in all the regions you want to analyze. You cannot use the reference signal of only one region and compare it to all others. The reference signal is never constant since you cannot guarantee that the same amount of MM2 reagent is present in the inside of your spheroid compared to the outer cell layer. However, we would not recommend using single pixels for this. Rather stick to an area or generate a mean value of several pixels.
Does the MM2 binding affinity to cellular organelles (e.g., DNA) affect the lifetime of the MM2, and will it affect the intracellular O2 measurements?
The process of staining is complex and cell-specific. Most cells are stained after endocytosis, but other uptake mechanisms exist. Usually, the probe remains in the cytosol and does not enter the mitochondria or the nucleus. Therefore, you cannot use the term "binding affinity". The phosphorescent dye within this nanoparticle is shielded by a polymer matrix, which means that the lifetime calibration is stable with different cells and measurement set-ups. MM2 gives reproducible results, however the calibration is affected by temperature.