Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems.
Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.
The characterization of a 10x10-SPAD detector module fabricated in a 0.35μm High Voltage CMOS technology is presented. The detector is designed to find application in Fluorescence Lifetime spectroscopy and is capable of performing lifetime measurement by using the time-gated technique. The characterization explores the dark count distribution, the detector dynamic range and the gating performances. More than 70% of single SPADs have a dark count rate lower than 1 kHz at the excess bias voltage of 1.8V and do not exceed 2 kHz at the bias voltage of 4.8V. Exploiting the intrinsic features of the detector a direct measurement of the optical cross talk between neighboring SPAD in the matrix was performed. The cross talk was found to be in the range from 2% to 3% for lateral neighbors and between 0.3% and 0.5% for diagonal neighbors. A dynamic range exceeding 120 dB was observed with a maximum count rate before saturation of 500 MHz. Time gating resolution was found to be less than 1 ns. A fluorescent lifetime measurement of ZnS-ZnSe quantum-dot reference slides was performed and the non-uniformity of the calculated lifetime value was lower than 1% across the matrix. The application of the detector in the construction of a lifetime acquisition system is analyzed estimating the detector data throughput. External resources needed to build the acquisition system are estimated and the FPGA-based acquisition system used during characterizations is described.
Fluorescence lifetime measurement is used in biological research to enhance the contrast of fluorescence images. The outstanding sensitivity that can be achieved with this method is obtained at the expense of a high data throughput. A substantial data reduction can be achieved using the time-gated technique, which consists in counting the number of photons occurring inside different time windows. Thanks to the recent developments in the realization of Single Photon Avalanche Diodes (SPAD) in standard CMOS technologies, this technique can be monolithically implemented on-chip. In this work, three different detectors fabricated within a 0.35 m High Voltage CMOS technology will be described, focusing onto their use in lifetime imaging. The sensors have been designed for different optical setups and for different applications, ranging from Fluorescence Lifetime Imaging Microscopy to miniaturized Labon-Chip. The advantages and limitation of the proposed sensors will be pointed out and a case study of a specific application will be presented.
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