Real-time fluorescence lifetime imaging system with a 32 x 32 0.13microm CMOS low dark-count single-photon avalanche diode array.

A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13microm CMOS single-photon avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called 'Integration for Extraction Method' (IEM) [J. Opt. Soc. Am. A 25, 1190 (2008)]. Here, IEM is modified for a wider resolvability range and implemented on the FPGA of the new SPAD array imager. We experimentally demonstrate that the dynamic range and accuracy of calculated lifetimes of this new camera is suitable for widefield FLIM applications by imaging a variety of test samples, including various standard fluorophores covering a lifetime range from 1.6ns to 16ns, microfluidic mixing of fluorophore solutions, and living fungal spores of Neurospora Crassa. The calculated lifetimes are in a good agreement with literature values. Real-time fluorescence lifetime imaging is also achieved, by performing parallel 32 x 16 lifetime calculations, realizing a compact and low-cost FLIM camera and promising for bigger detector arrays.

Hardware implementation and calibration of background noise for an integration-based fluorescence lifetime sensing algorithm.

A new integration based fluorescence lifetime imaging microscopy (FLIM) called IEM has been proposed to implement lifetime extraction [J. Opt. Soc. Am. A25, 1190 (2008)]. A real-time hardware implementation of the IEM FLIM algorithm suitable for single photon avalanche diode arrays in nanometer-scale CMOS technology is now proposed. The problems of reduced pixel readout bandwidth and background noise are studied and a calibration method suitable for FPGA implementation is introduced. In particular, the relationship between signal-to-noise ratio and background noise is considered based on statistics theory and compared with a rapid lifetime determination method and maximum-likelihood estimator with-without background correction. The results are also compared with Monte Carlo simulations giving good agreement. The performance of the proposed methods has been tested on monoexponential decay experimental data. The high flexibility, wide range, and hardware friendliness make IEM the best candidate for system-on-chip integration to our knowledge.

Absolute assessment of adsorption-based porous solid characterization methods: comparison methods.

The design and use of microporous solids depends on having access to characteristics such as the pore volume and surface area. Comparison methods such as the alpha(s) method are one of the most widely used means of determining these parameters. An assessment of this group of methods was undertaken by comparing estimates obtained from them using adsorption isotherms generated by grand canonical Monte Carlo simulation on a selection of model nanoporous solids with exactly known surface areas and pore volumes. Conclusions are drawn from this absolute assessment in regard to the validity of the alpha(s) method for determining the micropore volume, the mesopore surface area, and the separation of pore groups based on the concept of primary and cooperative filling, the subtracting pore effect (SPE) method, and the required character of the reference surface.

Molecular simulation evidence for solidlike adsorbate in complex carbonaceous micropore structures.

Adsorption of a model nitrogen vapor on a range of complex nanoporous carbon structures is simulated by grand canonical Monte Carlo simulation for a single subcritical temperature above the bulk freezing point. Adsorption and desorption isotherms, heats of adsorption, and three-dimensional singlet distribution functions (SDFs) were generated. Inspection of the SDFs reveals significant levels of solidlike adsorbate at saturation even in the most complex of the microporous solids considered. This strongly suggests that solidlike adsorbate will also occur for simple subcritical vapors adsorbed on real noncrystalline solids such as microporous carbons at temperatures above the bulk freezing point, supporting indirect experimental observations. The presence of significant levels of solidlike adsorbate has implications for characterization of microporous solids where adsorbate density is used (e.g., determination of pore volume from loading). Detailed consideration of the SDF at different loadings for a model microporous solid indicates solidlike adsorbate forms at distributed points throughout the pore space at pressures dependent on the nature of the local porosity. The nature of the local porosity also dictates the freezing mechanism. A local freezing/ melting/refreezing process is also observed. Introduction of mesoporosity into the model causes hysteresis between the adsorption and desorption isotherms. Adsorption in the hysteresis loop occurs by a series of local condensation events. It appears as if the presence of adjacent microporosity and/or adsorbate within it affects the pressure at which these events occur. Reversal of the condensation during desorption occurs throughout the mesoporosity at a single pressure; this pressure is unaffected by the presence of adjacent microporosity or the adsorbate within it. It is also shown that the empirical concept of "pore size" is not consistent for describing adsorption in the complex solids considered here. A new concept is, therefore, proposed that seeks to account for the factors that affect local adsorption energy: local geometry, microtexture, surface atom density, and surface chemistry.