Multiphoton fluorescence lifetime imaging of NADH reveals spatio-temporal patterns in cell metabolism during collective migration

2021 ◽  
Author(s):  
Jacopo Ferruzzi ◽  
Stephen J. DeCamp ◽  
Victor M. K. Tsuda ◽  
Muhammad H. Zaman ◽  
Jeffrey J. Fredberg ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Cell Metabolism ◽  
Temporal Patterns ◽  
Fluorescence Lifetime Imaging ◽  
Collective Migration ◽  
Lifetime Imaging ◽  
Spatio Temporal

scholarly journals Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

2021 ◽  
Vol 22 (11) ◽  
pp. 5952
Author(s):  
Sviatlana Kalinina ◽  
Christian Freymueller ◽  
Nilanjon Naskar ◽  
Bjoern von Einem ◽  
Kirsten Reess ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Imaging Techniques ◽  
Cell Metabolism ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Flavin Mononucleotide ◽  
Exponential Fitting ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Significant Difference ◽  
Redox Coenzymes

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.

scholarly journals Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

2020 ◽  
Author(s):  
V. Zickus ◽  
M.-L. Wu ◽  
K. Morimoto ◽  
V. Kapitany ◽  
A. Fatima ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
High Speed ◽  
Single Photon ◽  
Fluorescent Proteins ◽  
Cell Metabolism ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Protein Activity ◽  
Lifetime Imaging

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 Megapixel resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080 – human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 megapixels.

scholarly journals Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Vytautas Zickus ◽  
Ming-Lo Wu ◽  
Kazuhiro Morimoto ◽  
Valentin Kapitany ◽  
Areeba Fatima ◽  
...  
Keyword(s):  
Neural Network ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Fluorescent Proteins ◽  
Cell Metabolism ◽  
Wide Field ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Estimation ◽  
Protein Activity ◽  
Lifetime Imaging

AbstractFluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080—human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

2021 ◽  
Author(s):  
Lorenzo Scipioni ◽  
Alessandro Rossetta ◽  
Giulia Tedeschi ◽  
Enrico Gratton
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
New Paradigm ◽  
Lifetime Imaging
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