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Wide-field time-gated photoluminescence microscopy for fast ultrahigh-sensitivity imaging of photoluminescent probes

Razali WA, Sreenivasan VK, Bradac C, Connor M, Goldys EM, Zvyagin AV

J Biophotonics, 2016 08;9(8):848-58.
PMID: 27264934 DOI: 10.1002/jbio.201600050

Fluorescence microscopy is a fundamental technique for the life sciences, where biocompatible and photostable photoluminescence probes in combination with fast and sensitive imaging systems are continually transforming this field. A wide-field time-gated photoluminescence microscopy system customised for ultrasensitive imaging of unique nanoruby probes with long photoluminescence lifetime is described. The detection sensitivity derived from the long photoluminescence lifetime of the nanoruby makes it possible to discriminate signals from unwanted autofluorescence background and laser backscatter by employing a time-gated image acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete nanorubies dispersed on a coverslip. It enabled recovery of the photoluminescence signal emanating from discrete nanorubies when covered by a layer of an organic fluorescent dye, which were otherwise invisible without the use of spectral filtering approaches. Time-gated imaging also facilitated high sensitivity detection of nanorubies in a biological environment of cultured cells. Finally, we monitor the binding kinetics of nanorubies to a functionalised substrate, which exemplified a real-time assay in biological fluids. 3D-pseudo colour images of nanorubies immersed in a highly fluorescent dye solution. Nanoruby photoluminescence is subdued by that of the dye in continuous excitation/imaging (left), however it can be recovered by time-gated imaging (right). At the bottom is schematic diagram of nanoruby assay in a biological fluid.

Matched MeSH terms: Luminescent Measurements/methods*
Fulltext Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix

Zielinski MS, Vardar E, Vythilingam G, Engelhardt EM, Hubbell JA, Frey P, et al.

Commun Biol, 2019;2:69.
PMID: 30793047 DOI: 10.1038/s42003-019-0313-x

By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.

Matched MeSH terms: Luminescent Measurements/methods
Fulltext Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system

Teow SY, Liew K, Che Mat MF, Marzuki M, Abdul Aziz N, Chu TL, et al.

BMC Biotechnol, 2019 06 14;19(1):34.
PMID: 31200673 DOI: 10.1186/s12896-019-0528-4

BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).
RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.
CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

Matched MeSH terms: Luminescent Measurements/methods
Bioluminescent method for the rapid screening of toxic heayy metals in environmental samples using Photobacterium leiognathi strain AK-MIE

Kassim A, Halmi MIE, Gani SSA, Zaidan UH, Othman R, Mahmud K, et al.

Ecotoxicol Environ Saf, 2020 Jun 15;196:110527.
PMID: 32278138 DOI: 10.1016/j.ecoenv.2020.110527

Assessment of eco-toxicant using bioluminescent bacterial assay is a widely used and globally accepted method. In this work, a new luminescent bacterium was isolated from squid (Loligo duvauceli) and identified as Photobacterium leiognathi strain AK-MIE using 16S rRNA, phylogeny analysis. The predicted optimum conditions by RSM were 2.76% (w/v) NaCl, 2.28% (w/v) peptone, 0.34% (w/v) yeast extract, and pH 6.83 with 541,211.80 RLU of luminescent production whereas the predicted optimum conditions by ANN were 2.21% (w/v) NaCl, 2.27% (w/v) peptone, 0.39% (w/v) yeast extract, and pH 6.94 which produced 541,986.20 RLU. The validation analysis of both RSM and ANN show 0.60% and 0.69% deviation from the predicted results indicating that both models provided good quality predictions with ANN showing a superior data fitting capability for non-linear regression analysis. Toxicity tests show strain AK-MIE was sensitive to mercury (concentration causing 50% inhibition or IC50 of 0.00978 mgL-1), followed by cadmium (IC50 of 0.5288 mgL-1), copper IC50 of (0.8117 mgL-1), silver (IC50 of 1.109 mgL-1), and lead (IC50 of 10.71 mgL-1) which are more sensitive than previously isolated luminescent bacteria, suggesting that strain AK-MIE has the potential to be used in toxicity assessment of heavy metals in the environment. Based on the field trial results, several sediment samples from industrial areas in Bangi, Selangor managed to inhibit the bioluminescence of strain AK-MIE. Validation method carried out using ICP-MS proved the presence of several toxic heavy metal elements.

Matched MeSH terms: Luminescent Measurements/methods*
Effects of diarylpentanoid analogues of curcumin on chemiluminescence and chemotactic activities of phagocytes

Jantan I, Bukhari SN, Lajis NH, Abas F, Wai LK, Jasamai M

J Pharm Pharmacol, 2012 Mar;64(3):404-12.
PMID: 22309272 DOI: 10.1111/j.2042-7158.2011.01423.x

A series of 43 curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on the chemiluminescence and chemotactic activity of phagocytes in vitro.

Matched MeSH terms: Luminescent Measurements/methods