Fluorescence Microscopy

Diaspro, Alberto; Bianchini, Paolo; Zanacchi, Francesca Cella; Lanzanò, Luca; Vicidomini, Giuseppe; Oneto, Michele; Pesce, Luca; Cainero, Isotta

doi:10.1007/978-3-030-00069-1_21

Cited by 10 publications

(8 citation statements)

References 313 publications

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“…Comprehensive reviews of CLSM are available elsewhere. [150][151][152] Multiphoton FLIM uses MPE, generally two-photon (2P) or three-photon (3P) excitation, which relies on high photon density to achieve nonlinear excitation of fluorescence. This high density is achieved by lower energy, higher wavelength photons in the near-infrared region.…”

Section: Confocal and Multiphoton Microscopesmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. Approach: This review covers FLIM principles and theory, including advantages over intensitybased fluorescence measurements. Fundamentals of FLIM instrumentation in time-and frequencydomains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell-and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

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Section: Confocal and Multiphoton Microscopesmentioning

confidence: 99%

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

et al. 2020

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“…The difference could be explained by the overestimation of the number of bacteria captured on the surface by fluorescence microscopy. According to Diaspro et al, fluorescent proteins generally exhibit multiple localized events from each single fluorophore, which could lead to an overestimation of the real molecule number [ 47 ]. These results are also consistent with Lisle et al who demonstrated that fluorescent microscopy increasingly overestimated (15.0 to 99.3%) the true bacterial abundance [ 48 ].…”

Section: Resultsmentioning

confidence: 99%

Regenerable ZnO/GaAs Bulk Acoustic Wave Biosensor for Detection of Escherichia coli in “Complex” Biological Medium

et al. 2021

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A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of Escherichia coli in liquid media. The geometry of the biosensor consists of a GaAs membrane coated with a thin film of piezoelectric ZnO on its top surface. A pair of electrodes deposited on the ZnO film allows the generation of BAWs by lateral field excitation. The back surface of the membrane is functionalized with alkanethiol self-assembled monolayers and antibodies against E. coli. The antibody immobilization was investigated as a function of the concentration of antibody suspensions, their pH and incubation time, designed to optimize the immunocapture of bacteria. The performance of the biosensor was evaluated by detection tests in different environments for bacterial suspensions ranging between 103 and 108 CFU/mL. A linear dependence between the frequency response and the logarithm of E. coli concentration was observed for suspensions ranging between 103 and 107 CFU/mL, with the limit of detection of the biosensor estimated at 103 CFU/mL. The 5-fold regeneration and excellent selectivity towards E. coli detected at 104 CFU/mL in a suspension tinted with Bacillus subtilis at 106 CFU/mL illustrate the biosensor potential for the attractive operation in complex biological media.

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“…) that can be brought down to *200 nm at visible wavelengths with the use of high numerical aperture (NA) microscope objectives [1,2].…”

Section: Namentioning

confidence: 99%

“…A kernel function was designed with SIMD (Single Instruction, Multiple Data) approach to perform this calculation made in MATLAB with the Parallel CUDA kernel function. In multidimensional data, initial block and thread sizes (1,2). In this structure, there are a total of 192 threads that operate simultaneously with the Matrix items.…”

Section: Gpu Accelerationmentioning

confidence: 99%

An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction

Aydin

Uysallı²,

Özgönül³

et al. 2022

PLoS ONE

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When combined with computational approaches, fluorescence imaging becomes one of the most powerful tools in biomedical research. It is possible to achieve resolution figures beyond the diffraction limit, and improve the performance and flexibility of high-resolution imaging systems with techniques such as structured illumination microscopy (SIM) reconstruction. In this study, the hardware and software implementation of an LED-based super-resolution imaging system using SIM employing GPU accelerated parallel image reconstruction is presented. The sample is illuminated with two-dimensional sinusoidal patterns with various orientations and lateral phase shifts generated using a digital micromirror device (DMD). SIM reconstruction is carried out in frequency space using parallel CUDA kernel functions. Furthermore, a general purpose toolbox for the parallel image reconstruction algorithm and an infrastructure that allows all users to perform parallel operations on images without developing any CUDA kernel code is presented. The developed image reconstruction algorithm was run separately on a CPU and a GPU. Two different SIM reconstruction algorithms have been developed for the CPU as mono-thread CPU algorithm and multi-thread OpenMP CPU algorithm. SIM reconstruction of 1024 × 1024 px images was achieved in 1.49 s using GPU computation, indicating an enhancement by ∼28 and ∼20 in computation time when compared with mono-thread CPU computation and multi-thread OpenMP CPU computation, respectively.

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Fluorescence Microscopy

Cited by 10 publications

References 313 publications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

Regenerable ZnO/GaAs Bulk Acoustic Wave Biosensor for Detection of Escherichia coli in “Complex” Biological Medium

An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction

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