Advantages and Limitations of Fluorescence Lifetime Measurements Using Single-Photon Avalanche Diode (SPAD) Array Detector: A Comprehensive Theoretical and Experimental Study

Fast fluorescence lifetime (FL) determination is a major factor for studying dynamic processes. To achieve a required precision and accuracy a certain number of photon counts must be detected. FL methods based on single-photon counting have strongly limited count rates because of the detector’s pile-up issue and are suffering from long measurement times in the order of tens of seconds. Here, we present an experimental and Monte Carlo simulation-based study of how this limitation can be overcome using array detectors based on single-photon avalanche diodes (SPADs). We investigated the maximum count rate per pixel to determine FL with a certain precision and accuracy before pile-up occurs. Based on that, we derived an analytical expression to calculate the total measurement time which is proportional to the FL and inversely proportional to the number of pixels. However, a higher number of pixels drastically increases data rate. This can be counteracted by lowering the time resolution. We found that even with a time resolution of four times the FL, an accuracy of 10% can be achieved. Taken all together, FLs between 10 ns and 3 ns can be determined with a 300-pixel SPAD array detector with a measurement time and data rate less than 1 µs and 700 Mbit/s, respectively. This shows the enormous potential of SPAD array detector for high-speed applications requiring continuous data read out

Macro-SICM : A Scanning Ion Conductance Microscope for Large-Range Imaging

The scanning ion conductance microscope (SICM) is a versatile, high-resolution imaging technique that uses an electrolyte-filled nanopipet as a probe. Its noncontact imaging principle makes the SICM uniquely suited for the investigation of soft and delicate surface structures in a liquid environment. The SICM has found an ever-increasing number of applications in chemistry, physics, and biology. However, a drawback of conventional SICMs is their relatively small scan range (typically 100 μm × 100 μm in the lateral and 10 μm in the vertical direction). We have developed a Macro-SICM with an exceedingly large scan range of 25 mm × 25 mm in the lateral and 0.25 mm in the vertical direction. We demonstrate the high versatility of the Macro-SICM by imaging at different length scales: from centimeters (fingerprint, coin) to millimeters (bovine tongue tissue, insect wing) to micrometers (cellular extensions). We applied the Macro-SICM to the study of collective cell migration in epithelial wound healing

Single-photon avalanche diode (SPAD) array detector for high-throughput fluorescence lifetime flow cytometry

Time-domain measurement of the fluorescence lifetime in flow cytometry offers a complementary method to traditional flow cytometry, especially for the analysis of cells and biomolecules. Short measurement times due to high throughputs (cells/s) make a suited detector mandatory for a sufficient detection of the fluorescence signal and therefore the application of this method. We summarize the requirements for a suited detector for time resolved flow cytometry (TRFC) and present a new developed SPAD array detector with 100 pixels and 340 ns time between measurement windows, allowing a high-throughput of up to 60,000 cells/s. The functionality of the detector is shown with the measurement of a 22.2 ns laser pulse, making it applicable for an improved TRFC

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force–distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50–60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells