Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

Görlitz, Frederik; Corcoran, David S; Castano, Edwin A. Garcia; Leitinger, Birgit; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.

doi:10.3390/photonics4030040

Cited by 15 publications

(12 citation statements)

References 35 publications

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“…FLIM has been used to study the chromatin compaction state during cell cycle progression in fixed samples where heterogeneous DNA distribution was seen (Murata et al 2000;Murata et al 2001;Görlitz et al 2017). FLIM on live interphase nuclei has shown chromatin decompaction upon chemical or radiation stimulus (Abdollahi et al 2018;Lou et al 2019;Pelicci et al 2019;Sherrard et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

et al. 2021

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The organization of chromatin into higher-order structures and its condensation process represent one of the key challenges in structural biology. This is important for elucidating several disease states. To address this long-standing problem, development of advanced imaging methods has played an essential role in providing understanding into mitotic chromosome structure and compaction. Amongst these are two fast evolving fluorescence imaging technologies, specifically fluorescence lifetime imaging (FLIM) and super-resolution microscopy (SRM). FLIM in particular has been lacking in the application of chromosome research while SRM has been successfully applied although not widely. Both these techniques are capable of providing fluorescence imaging with nanometer information. SRM or “nanoscopy” is capable of generating images of DNA with less than 50 nm resolution while FLIM when coupled with energy transfer may provide less than 20 nm information. Here, we discuss the advantages and limitations of both methods followed by their contribution to mitotic chromosome studies. Furthermore, we highlight the future prospects of how advancements in new technologies can contribute in the field of chromosome science.

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Section: Introductionmentioning

confidence: 99%

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

et al. 2021

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“…Double-exponential decay pro¯les can be determined by using additional time windows. 150 The method is fast and e±cient for gated images intensi¯ers, 95,96,105,109 gated photon-counting systems, [84][85][86][87][88][89][90] and directly gated CCD sensors. 100 Liu et al 151 have optimized data analysis in timegated FLIM technology by combining the 4th-order partial di®erential equations of each pixel intensity with RLD to correct for the existing noise of the algorithm in the calculation process, thus enabling time-gated FLIM to quickly acquire lifetime images with high lifetime accuracy.…”

Section: Rapid Lifetime Determination Methodsmentioning

confidence: 99%

“…Nipkow-disk, [107][108][109] light sheet systems, [110][111][112][113][114] and total internal re°ection (TIR) illumination 115 can inherently provide depth resolution. For the Nipkow systems described by Grant et al 107 and G€ orlitz et al 109 the acquisition times for 256 Â 256 pixels range from 1 s for low-quality images recorded in two time gates and 30 s for high-quality images sequentially recorded in eight time gates. A comparison of FLIM images recorded by a gated image intensi¯er with and without optical sectioning by a Nipkow disk is shown in Ref.…”

Section: Nipkow-disk Systemsmentioning

confidence: 99%

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Liu

Lin

Becker

et al. 2019

J. Innov. Opt. Health Sci.

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Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

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“…While optical sectioning techniques such as confocal FLIM may be prohibitively slow for clinical imaging, structured illumination microscopy (SIM) has been used and combined with FLIM to accurately recover fluorescence lifetimes at reasonable acquisition rates 26 . More recently, SIM and widefield FLIM were combined and used to acquire super-resolved images of cell morphology and map the corresponding Förster resonant energy transfer (FRET) readouts to the cellular nanostructures 29 .…”

Section: Introductionmentioning

confidence: 99%

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy

Hinsdale

Malik

Cheng

et al. 2021

Sci Rep

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We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

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Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

Cited by 15 publications

References 35 publications

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy

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