Quantitative time-resolved fluorescence techniques like FLIM are increasingly employed in fields like phase separation and cellular sensing. PicoQuant`s new microscope Luminosa combines state-of-the-art hardware with cutting edge software, delivering high quality data while simplifying daily operation. The software includes features like context-based workflows, sample-free auto-alignment and laser power calibration which improve reproducibility of experiments. We describe how FLIM is streamlined with Luminosa. Its rapidFLIM hardware records several frames per second with high count rates, which the software handles with a novel dynamic binning format. Combined with GPU-accelerated algorithms, this enables high-speed automated analysis of FLIM images with minimal user interaction. We will also show an outlook about how Luminosa can be used for combining FLIM with super-resolution modalities.

PicoQuant`s new confocal microscope Luminosa combines state-of-the-art hardware with cutting edge software to deliver high quality data while simplifying daily operation. Luminosa's rapidFLIM hardware can record several frames per second, which the software handles with a novel dynamic binning format. In combination with GPU-accelerated algorithms, this enables high-speed automated analysis of FLIM images. We demonstrate the combination of PicoQuant's latest multi-channel TCSPC device and a cooled high-performance 23-pixel SPAD-array developed jointly with Pi Imaging Technologies for time-resolved confocal image scanning microscopy (FLIM-ISM). We also discuss how advanced data processing can be applied to FLIM-ISM for additional performance gains. For certain applications widefield imaging offers unique advantages. Here we show how the combination of a powerful PicoQuant laser and the novel SPAD512S camera from Pi Imaging Technology facilitates time-resolved widefield imaging. We benchmark the camera's performance, demonstrating video rate acquisition speeds with minimal photobleaching.

Single molecule studies and—more specifically—single molecule FRET methodologies have become a standard tool for studying dynamic structural changes in proteins and nucleic acids. These types of measurements can reveal dynamic events on time scales covering several orders of magnitude from ns to several seconds. This allows studying e.g., chain dynamics, binding, folding, allosteric events, oligomerization and aggregation. The power of these methodologies is highlighted by the study of Intrinsically Disordered Proteins (IDPs) whose biological relevance has been increasingly studied over the recent years. The new Luminosa microscope offers context-based software workflows for investigating both diffusing and immobilized molecules. In this poster we illustrate how easily and efficiently these measurements can be performed. For diffusion studies, efficiency vs. stoichiometry histograms are calculated in real time and all necessary correction parameters are automatically determined by employing methodologies benchmarked by the scientific community. We will also show how the variable PSF feature can be used in such measurements to fine-tune the observation window of freely diffusing biomolecules. Immobilized molecules are located automatically and intensity time traces are recorded sequentially with a definable duration. To get large datasets effortlessly, large sample areas can be investigated with tiling and stitching. Long acquisition times are enabled by a hold focus option.

Fluorescence Lifetime Imaging (FLIM) has become more attractive in recent years as it offers increased specificity in many assays as well as the possibility of multiplexing the read out of many markers with a small number of detectors. Here we present how FLIM modalities are implemented in Luminosa, the new single-photon counting confocal microscope by PicoQuant. Thanks to a dynamic binning format and GPU-based algorithms FLIM images of 1024x1024 can be analysed in a few seconds. The FLIM analysis workflow suggests the best fitting model based on statistical arguments and requires minimal user interaction making these modalities become accessible to new users who can then confidently start working with FLIM and incorporate it into their research toolbox combining the strengths of phasor plots with decay fitting.

Detection sensitivity is a key parameter to meet today's demand for handling smallest analyte amounts and short measurement times in the optical evaluation of pharmaceuticals and biotechnology products. The introduction of TCSPC based data acquisition has proven to yield a major sensitivity increase and very high dynamic range - it is the ideal method for measuring weak luminescence. The achievable signal-to-noise ratio of this method is significantly higher than of methods based on analog detection. This allows for accurate quantification of extremely low concentration levels down to femtomolar range. Here we present different kinds of hardware for a state of the art photoluminescence spectrometer for steady-state, anisotropy, and time-resolved measurements. We discuss critical components affecting sensitivity and present a step by step rundown how to find optimal measurement conditions in order to reach ultimate temporal and spectral performance limits. The combination of a double monochromator in subtractive mode for emission selection, new hybrid PMA detector without after pulsing, PicoHarp 300 TCSPC electronics and deconvolution of the fluorescence decay with instrument response function of the spectrometer lead to a temporal resolution of about 6 ps. Without doubt, this allows investigating with high precision fast transfer processes or binding formation by anisotropy measurements in molecule systems or building blocks. The performance of such a spectrometer in terms of time resolution, ability to measure long decays (e.g., phosphorescence) and record time-gated spectra using laser drivers with burst capabilities will be demonstrated. Moreover, the combination of fluorescence spectrometer with confocal microscope systems allows the detection of time-resolved emission spectra with high spatial resolution and all advantages of a microscope (e.g., scanning). The achieved sensitivity allows to also quantify the tiniest changes in luminescence intensities as well as photoluminescence lifetimes.

Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential for imaging dynamic processes in life science. The rapidFLIM approach dramatically reduces acquisition times through a combination of fast beam scanning, hybrid photomultiplier detectors, which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high spatial resolution offered in confocal microscopy. Depending on image size, rapidFLIM enables following dynamic processes like protein interactions, chemical reactions or highly mobile species in live cell imaging with a rate of several frames per second. The separation of overlapping fluorescence emissions in biological samples has been improved in the last years by using spectral confocal microscopy in combination with linear unmixing. However, the separation of multiple labels in biological samples remains challenging, especially when strong tissue autofluorescence (AF) overshadows specifically labeled structures. Combining the spectral approach with fluorescence lifetime measurements based on a simultaneous acquisition of both spectral and lifetime parameters could significantly improve the separation quality between multiple labels and tissue AF. We demonstrate this approach in highly autofluorescent human lung tissue, where the fluorescence signals from specific stainings are sometimes weaker than tissue AF. We use dual color Pulsed Interlevaed Excitation (PIE) in conjunction with a spectral FLIM (sFLIM) detection system featuring eight separate TCSPC timing channels and analyze the data by applying a unique pattern matching technique.

Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential to cementing its importance as a tool in the Life Sciences. This technique is already well established, but imaging dynamic processes requires shorter acquisition times. Our novel rapidFLIM approach dramatically reduces the acquisition time through a combination of fast scanning, hybrid photomultiplier detectors which are capable of handling very high count rates, and TCSPC modules with ultra short dead times. With the new FLIMbee fast scanning add-on for the MicroTime 200, this technique can be used with our microscopy platform as well as being offered as an upgrade kit for conventional Laser Scanning Microscopes (LSMs). With this hardware combination, excellent photon statistics can be achieved in significantly shorter time spans, allowing fast processes to be measured with the high resolution achieveable in confocal microscopy. Depending on the image size, rapidFLIM allows imaging at a rate of several frames per second, enabling dynamic processes, such as protein interactions, FRET dynamics, or chemical reactions to be imaged in a time-resolved manner. With these high frame rates, FLIM can also be used on highly mobile species such as cell organelles and for other live cell imaging applications. Recently, we have further pushed the limits of this method by systematically reducing the effects of decay distortions at very high count rates, allowing quantitative data analysis to be performed even at count rates above 10 Mcps. This technique has been applied to quantitatively analyze FRET measurements using fluorescent proteins.

Using Time-Correlated Single Photon Counting (TCSPC) for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation this limitation can be lifted significantly but some artefacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remains an issue to be addressed. We propose here a data analysis method correcting for this type of artefact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction show a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescense lifetime imaging (FLIM) at very high frame rates.

Many applications in biology, medicine, and material science have a demand for a time and spatially resolved detection of luminescence, particularly in the near infrared spectral region. For example, observation of singlet oxygen phosphorescence is important in understanding cell-cell interactions as well as for research involving photo-dynamic therapy. Furthermore, investigating the temporal behavior of semiconductor luminescence has become an important quality indicator for the quality of wafer material, especially many kinds of solar cell research and production.

Time-resolved fluorescence spectroscopy is a spectroscopist's most valuable tool for the investigation of excited state dynamics in molecules, complexes, or semi-conductors. In recent years, the study of luminescence properties has gained in popularity in many scientific fields, including Chemistry, Biology, Physics, as well as in Life, Material or Environmental Sciences. The investigations to be carried out in each of these fields impose different requirements. On one side, monitoring dynamic processes in the excited state necessitates high time resolution that can be achieved by fast pulsed lasers and detectors along with appropriate time-correlated single photon counting (TCSPC) units and small monochromators. On the other hand, high spectral resolution is desirable for fluorophore characterization, requiring detectors with high quantum efficiencies, flash lamps for phosphorescence measurements and large monochromators. Up to now, spectrometers have been usually targeted towards either one of these two specifications. Spectrometers equipped with hybrid detectors, versatile TCSPC cards with optional longer time ranges, and pulsed lasers capable of working in a burst mode can offer an combined solution, covering most of the demands of either high time or spectral resolution. We will demonstrate the performance of such a spectrometer in terms of its time resolution, the ability to measure long decays and record time-gated spectra using laser drivers with burst capabilities. This type of instrument is of great value for analytical facilities in research centers, as it offers a wide range of possible spectroscopic applications in a single, easy to use instrument.

Stimulated Emission Depletion (STED) microscopy has evolved into an established imaging method offering super-resolution well beyond 50 nm. Whereas STED is now available in many laboratories, it is still in the focus of research to push the boundaries of its capabilities and applications. Time-resolved STED microscopy using time correlated single photon counting (TCSPC), is advantageous for many applications and promises further development for increased resolution and less photo-damage. Here, we show the application of established methods (e.g. gSTED) as well as emerging applications of time-resolved STED. We employ pulsed interleaved excitation (PIE), where the STED laser is pulsed at half the frequency of the excitation laser, such that STED and confocal data is taken practically at the same time. By using this approach, single molecule STED experiments can be carried out while the confocal control-experiment is performed simultaneously, allowing to account for measurement artifacts due to the high power of the STED laser. We will show examples from single molecule imaging, where blinking and bleaching are monitored using the confocal data. Furthermore, we will present STED-FCS data, where the confocal data allows insight into changes of the sample due to the STED laser. Since the control experiment for the influence of the STED laser is performed at the same time as the STED measurement, experimental parameters can be adjusted online to give highest resolution while ascertaining that the relevant information drawn from the experiment is not affected. Furthermore, we will present how electronically delaying the STED laser with respect to the excitation laser can increase resolution with no increase in photo-bleaching. By setting the arrival of the STED laser with an accuracy of about 20 ps, experimental conditions for fluorophores with different fluorescence lifetimes can be adjusted.

Biology, medicine, and material science, especially many kinds of solar cell research and production demand for time- and spatially resolved detection in the near infrared spectral region. Observation of singlet oxygen phosphorescence is, for example, important for understanding cell-cell interactions and in the field of photo-dynamic therapy. Whereas the temporal behavior of the luminescence from semiconductors is an agreed indicator for the quality of wafer material. Single photon counting based data acquisition has proven to yield the best sensitivity and a very high dynamic range - it is therefore the ideal method for measuring weak luminescence in the near infrared spectral region. Based on this principle, we present the methodology for state-of-the-art luminescence measurements. Our hardware is designed to detect spectrally- and spatially-resolved weak emission in steady-state and time-resolved manner. Depending on the samples size and aggregation state, large excitation volume spectrometer or diffraction limited small excitation spot microscope systems are employed. A combination of both types of set-ups allows us to record even spectrally-resolved images. The high sensitivity of these systems was proven by detecting singlet oxygen phosphorescence from aqueous samples of photo-sensitizers. With the reached detection sensitivity level it is feasible to record even singlet oxygen phosphorescence images.

Detection sensitivity from the ultraviolet to the near infrared spectral region is a key parameter to meet today's demand for handling smallest analyte amounts and short measurement times in the optical evaluation of miscellaneous samples. The introduction of single photon counting based data acquisition has proven to yield a major sensitivity increase and very high dynamic range - it is the ideal method for measuring weak luminescence. We present the hardware and handling optimization of a state of the art spectrometer for steady-state and time-resolved fluorescence measurements. The high sensitivity of the spectrometer was shown by measurements of popular fluorescent dyes as well as the Raman spectrum of water under well defined and reproducible conditions. The achieved sensitivity allows us to quantify singlet oxygen generation and to characterize the singlet oxygen phosphorescence decay, a prerequisite when studying photosensitisers like porphyrins and phthalocyanines used for example in photodynamic therapy (PDT). Moreover, with the help of an integrating sphere fluorescence quantum yields of low fluorescent samples like Ru(bpy)3 in water can be determined very precisely. The fibre connection of the spectrometer to a time-resolved fluorescence microscope (MicroTime100/200) was also realized. The combination of the advantages of both setups makes it e.g. possible to perform 2D-lifetime imaging with a freely tunable detection window for low luminescent samples even far into the near infrared region. The measurements with such a combination give not only the spectral and lifetime information of a luminescent sample but also the spatial information which is especially important for hetergeneous samples.

Single photon counting based data acquisition has proven to yield a major sensitivity increase in the optical evaluation of pharmaceuticals and biotechnology products. We will show for the first time that a state of the art time-correlated single photon counting (TCSPC) based fluorescence lifetime spectrometer is able to quantify singlet oxygen generation and to characterize the singlet oxygen phosphorescence decay. This makes TCSPC based fluorescence lifetime spectrometers a valuable tool for studying photosensitizers widely used for example in photodynamic therapy (PDT). The detection of the faint singlet oxygen phosphorescence signal has been made possible by using a special burst mode for the pulsed laser excitation and a new generation of TCSPC electronics with a significantly reduced dead-time which enables efficient multi-stop photon detection. Thanks to a recently developed integrating sphere add-on we are also able to measure fluorescence quantum yields with the same instrument. Leveraging the possibility to measure fluorescence lifetime in conjunction with quantum yield, we performed a systematic investigation of the relation between reduced fluorescence emission and different contributions of dynamic and static quenching processes. Furthermore, since quenching normally does not affect the radiative rate constant, this combined set-up allows to verify the accuracy of the extracted lifetime. Especially for very short lifetimes in the range of the instrument response function the presented method allows to assess whether the proper fluorescence lifetime was extracted.