The recently designed Tpx3Cam camera based PLIM (Phosphorescence Lifetime IMaging) macro-imager was tested using an array of phosphorescent chemical and biological samples. A series of sensor materials prepared by incorporating the phosphorescent O2-sensitive dye, PtBP, into five polymers with different O2 permeability were imaged along with several commercial and non-commercial sensors based on PtBP and PtOEPK dyes. The PLIM images showed good lifetime contrast between the different materials, and phosphorescence lifetime values obtained were consistent with those measured by alternative methods. A panel of live tissues samples stained with PtBP based nanoparticle probe were also prepared and imaged under resting conditions and upon inhibition of respiration. The macro-imager showed promising results as a tool for PLIM of O2 in chemical and biological samples.
Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.
We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.
Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.
Ultra-fast frame rate CMOS cameras, combined with a photon counting image intensifier, can be used for microsecond resolution wide-field time-correlated single photon counting (TCSPC) microscopy. A sequence of frames is recorded after an excitation pulse, and the number and location of photons in each frame is determined. This process is repeated until enough photons are recorded for a photon arrival time histogram in the pixels of the image. This approach combines low, nanowatt excitation power with single-photon detection sensitivity and arrival timing in many pixels simultaneously, short acquisition times in the order of seconds and allows lifetime mapping with a time resolution of ~1 microsecond. Moreover, we also show that the phosphor decay can be exploited to time the photon arrival well below the exposure time of the camera. This approach yields better time resolution and larger images than direct imaging of photon events. We show that both methods are ideal for lifetime imaging of transition metal compounds in living cells within a few seconds.
In fluorescence microscopy the lateral resolution is limited to about 200 nm because of diffraction. Resolution
improvement by a factor of two can be achieved using structured illumination, where a ine grating is projected
onto the sample, and the final image is reconstructed from a set of images taken at different grating positions.
Further resolution improvement can be achieved by saturating the transitions involved in fluorescence emission.
Recently discovered photoswitchable proteins undergo transitions that are saturable at low illumination intensity.
Combining this concept with structured illumination, theoretically unlimited resolution can be achieved, where
the smallest resolvable distance will be determined by signal-to-noise ratio. This work focuses on the use of
the photoswitchable protein Dronpa with structured illumination to achieve nanometre scale resolution in fixed