Phosphorescence Lifetime Imaging (PLIM) is similar to Fluorescence Lifetime Imaging (FLIM), only that it images the phosphorescence from the sample and consequently covers time ranges up to milliseconds. Analogous to FLIM, the contrast in a PLIM image is based on the lifetime of individual fluorophores rather than their emission spectra. The phosphorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon.
PLIM, or generally the characterization of phosphorescent compounds has been of great importance in the field of materials science namely chemical sensing for many years , and has renewed its interest over the past decade with the booming development of Organic Light Emitting Diode (OLED) technology. Other typical samples include metal ions complexed with organic ligands, which can be used to, e.g., image oxygen consumption in living cells.
Multi-channel scaling (MCS) or Time-Correlated Single Photon Counting (TCSPC) with multi-stop capabilities is used to determine the phosphorescence lifetime. In both cases, one measures the time between sample excitation by a pulsed laser and the arrival of the emitted photon at the detector. The methods requires a defined “start”, provided by the electronics steering the laser pulse or a photodiode, and a defined “stop” signal, realized by detection with single-photon sensitive detectors (e.g. Single Photon Avalanche Diodes, SPADs or Photomultiplier Tubes, PMTs). The measurement of this time delay is repeated many times to account for the statistical nature of the fluorophores emission. The delay times are sorted into a histogram that plots the occurrence of emission over time after the excitation pulse.
In order to acquire a phosphorescence lifetime image, the photons have to be attributed to the different pixels, which is done by storing the absolute arrival times of the photons additionally to the relative arrival time in respect to the laser pulse. Line and frame marker signals from the scanner of the confocal microscope are additionally recorded in order to sort the time stream of photons into the different pixels.
In contrast to TCSPC based FLIM, PLIM can usually be performed efficiently with galvo scanners for compounds with lifetimes up to a few µs only due to their high scanning speeds that limit the dwell time per pixel. If the dwell time is shorter than the time needed for the phosphorescence to decay completely, photons will be sorted into wrong pixels, leading to distorted images. PLIM is therefore best performed with piezo scanning devices, which enable lower scanning speeds than galvo scanners. As the ideal pixel dwell time is ~50-100 times the luminescence lifetime, measurements on dyes with slow phosphorescence can take a very long time.
Confocal Laser Scanning Microscopes (LSMs) are widely used tools in cell and molecular biology, biochemistry and other related sciences. PicoQuant's LSM Upgrade Kits greatly enhance the capabilities of these microscopes by extension to time-resolved techniques, and thereby providing not only Fluorescence Lifetime Imaging (FLIM) and Phosphorescence Liftetime Imaging (PLIM) but also Fluorescence Correlation Spectroscopy (FCS) and a wealth of other time resolved techniques. The LSM Upgrade Kit combines PicoQuant products to a ready-to-use kit that fits your specific application on a state of the art Laser Scanning Microscope of your choice from Leica, Nikon, Olympus or Zeiss.
The upright time-resolved microscope MicroTime 100 contains the complete optics and electronics for recording F(C)CS or FLCS data as well as fluorescence decays in small volumes by means of Time-Correlated Single Photon Counting (TCSPC). The system is based on a conventional upright microscope body that permist easy access to a wide range of sample shapes and sizes.
The homeostasis of oxygen plays a major role for the maintenance of cellular health and function. Oxygen deprivation, also called hypoxia has been identified as one of the main contributors to tumour aggressiveness. In order to study how hypoxia influences cancer cell behaviour the understanding of its oxygenation is crucial. Here we utilise PLIM to measure oxygen tension within living cells. Intracellular oxygen tension was measured in human neuroblastoma (SK-N-AS) cells based on the quenched phosphorescence detection of Pt-porphyrin based nanoparticles. Cells were incubated with the self-permeable, self-loading nanoparticle NanO2 (Luxcel Biosciences or Ibidi) according to manufacturer’s instructions and imaged using a Zeiss LSM780 with Picoquant Timeharp 260. PLIM measurements were performed at decreasing steps of extracellular oxygen tensions (from 21 % to 0.1 % O2). Cells were left to adjust to the new external oxygen tension for at least 30 mins prior lifetime measurements. Lifetime was found to increase (30, 33.8, 40.9, 53.9 and 60 µs) with decreasing extracellular oxygen 21, 12, 8, 1 and 0.1 %. Calibration was performed with a respiration inhibitor, Antimycin A and at 21 % O2 allowing the conversion of phosphorescence lifetime signal into intracellular O2 concentration [µM].
Oxygen was imaged in male cockroach salivary glands that were incubated with KR341 (a phosphorescent probe on Ru-basis with a lifetime in the µs-range that is quenched by molecular oxygen). The sample was slowly scanned and during the image scan, dopamine was added or removed by washing with Ringer buffer. Stimulation of cellular metabolism by dopamine exposure decreases the oxygen concentration within the salivary glands, which can be measured by an increase in the phosphorescence lifetime of KR341.
Cockroach salivary glands were labeled with KR341 (a phosphorescent probe on Ru-basis with a lifetime in the µs-range, that is quenched by molecular oxygen) and OGB1 (a fluorescent, calcium-sensitive probe with a lifetime in the ns-range). As the lifetimes of the two labels are very different, they can be separated and analyzed independently. The result are separate FLIM and PLIM images, from which the O2 and Ca2+-concentrations can be derived by analyzing local changes in the lifetime.