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Autofluorescence Spectroscopy and Imaging II

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Photochemistry".

Deadline for manuscript submissions: closed (31 May 2022) | Viewed by 23541

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Dr. Anna Cleta Croce

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Institute of Molecular Genetics, National Research Council (IGM-CNR), c/o Department of Biology and Biotechnology, University of Pavia, Pavia, Italy
Interests: photobiology; UV-visible autofluorescence analysis; endogenous fluorophores; label-free and real-time diagnosis; optical biopsy
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Dear Colleagues,

Autofluorescence defines the ability of biological substrates to give rise to fluorescence emission when excited with light at a suitable wavelength, in the absence of fixation or labelling with exogenous dyes. Various kinds of natural biomolecules may act as endogenous fluorophores and contribute to different extents to the overall autofluorescence signal of a biological substrate. The ubiquitous presence in living organisms of endogenous fluorophores and their possible changes, dependent on their close involvement in cell metabolic and catabolic pathways or participation to tissue architecture under normal or diseased conditions, is at the basis of unceasing and countless studies and of technological advances for label-free, real-time analytical and diagnostic applications. In biomedicine, autofluorescence-based diagnostic procedures rely on endogenous fluorophores typical of animal cells and tissues, such as collagen and elastin, relatable to tissue structure and its alteration, lipofuscins and porphyrins or glycation end products of lipids and proteins, relatable to oxidative stress and metabolic diseases, NAD(P)H and flavins, relatable to cell energy metabolism, and reductive biosynthesis. In the vegetable kingdom, chlorophylls and carotenoids involved in light harvesting and the consequent chemical energy production and in antioxidant protection are increasingly considered as biomarkers for applications ranging from the remote surveillance of plant pathologies and environment pollution, to the monitoring of biomass production. In addition to these common topics, many additional endogenous fluorophores can act as valuable biomarkers, for example, lignin, relevant to wood quality, or flavonoids, valuable for plant physiological activities or for their antioxidant role as food components or additives.

In this context, the unceasing attention to the multiple aspects of autofluorescence is attested to by the various contributions already collected in the first edition of the Special Issue, in parallel with other works recently published in Molecules. Therefore, the second edition of the Special Issue on autofluorescence is expected to attract new contributions on the various aspects of autofluorescence and its related technological advances and to further promote knowledge and development of wide ranging in situ, label-free, and real-time analytical and diagnostic procedures.

You may choose our Joint Special Issue in Photochem.

Dr. Anna Cleta Croce
Guest Editor

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Keywords

autofluorescence
NA(P)DH
flavins
lipofuscins
proteins
collagen
porphyrins
bile pigments
carotenoids and retinoids
chlorophyll
cultured cells
animal tissues and organs
biological fluids
energy/lipid metabolism
mitochondria
oxidative stress
plants
food
environment
optical redox
spectroscopy
imaging
time resolved analysis
multiphoton excitation

Related Special Issue

Autofluorescence Spectroscopy and Imaging II in Photochem (5 articles)

Published Papers (9 papers)

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Research

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11 pages, 2157 KiB

Open AccessArticle

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging

by Kana Kobayashi-Taguchi, Takashi Saitou, Yoshiaki Kamei, Akari Murakami, Kanako Nishiyama, Reina Aoki, Erina Kusakabe, Haruna Noda, Michiko Yamashita, Riko Kitazawa, Takeshi Imamura and Yasutsugu Takada

Molecules 2022, 27(10), 3340; https://doi.org/10.3390/molecules27103340 - 23 May 2022

Cited by 1 | Viewed by 2383

Abstract

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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13 pages, 17999 KiB

Open AccessArticle

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging

by Alba Alfonso-Garcia, Stephanie A. Cevallos, Jee-Yon Lee, Cai Li, Julien Bec, Andreas J. Bäumler and Laura Marcu

Molecules 2022, 27(4), 1317; https://doi.org/10.3390/molecules27041317 - 15 Feb 2022

Cited by 4 | Viewed by 2064

Abstract

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50–80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between

β

-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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16 pages, 8637 KiB

Open AccessArticle

The Bright Side of the Tiger: Autofluorescence Patterns in Aedes albopictus (Diptera, Culicidae) Male and Female Mosquitoes

by Anna C. Croce and Francesca Scolari

Molecules 2022, 27(3), 713; https://doi.org/10.3390/molecules27030713 - 21 Jan 2022

Cited by 4 | Viewed by 2298

Abstract

Light-based events in insects deserve increasing attention for various reasons. Besides their roles in inter- and intra-specific visual communication, with biological, ecological and taxonomical implications, optical properties are also promising tools for the monitoring of insect pests and disease vectors. Among these is the Asian tiger mosquito, Aedes albopictus, a global arbovirus vector. Here we have focused on the autofluorescence characterization of Ae. albopictus adults using a combined imaging and spectrofluorometric approach. Imaging has evidenced that autofluorescence rises from specific body compartments, such as the head appendages, and the abdominal and leg scales. Spectrofluorometry has demonstrated that emission consists of a main band in the 410–600 nm region. The changes in the maximum peak position, between 430 nm and 500 nm, and in the spectral width, dependent on the target structure, indicate the presence, at variable degrees, of different fluorophores, likely resilin, chitin and melanins. The aim of this work has been to provide initial evidence on the so far largely unexplored autofluorescence of Ae. albopictus, to furnish new perspectives for the set-up of species- and sex-specific investigation of biological functions as well as of strategies for in-flight direct detection and surveillance of mosquito vectors. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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18 pages, 1788 KiB

Open AccessFeature PaperArticle

Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions

by Martin Heidelman, Bibek Dhakal, Millicent Gikunda, Kalinga Pavan Thushara Silva, Laxmi Risal, Andrew I. Rodriguez, Fumiyoshi Abe and Paul Urayama

Molecules 2021, 26(16), 5020; https://doi.org/10.3390/molecules26165020 - 19 Aug 2021

Cited by 2 | Viewed by 2300

Abstract

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker’s yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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9 pages, 1880 KiB

Open AccessCommunication

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells

by Alexandr Zalessky, Yuriy Fedotov, Elizaveta Yashkina, Viktor Nadtochenko and Andreyan N. Osipov

Molecules 2021, 26(13), 4027; https://doi.org/10.3390/molecules26134027 - 01 Jul 2021

Cited by 4 | Viewed by 2125

Abstract

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 10¹¹ W∙cm⁻² peak power density. An increase in the pulse power density to 2 × 10¹¹ W∙cm⁻² led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 10¹¹ W∙cm⁻² led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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12 pages, 4016 KiB

Open AccessArticle

Analysis of Pathogenic Bacterial and Yeast Biofilms Using the Combination of Synchrotron ATR-FTIR Microspectroscopy and Chemometric Approaches

by Samuel Cheeseman, Z. L. Shaw, Jitraporn Vongsvivut, Russell J. Crawford, Madeleine F. Dupont, Kylie J. Boyce, Sheeana Gangadoo, Saffron J. Bryant, Gary Bryant, Daniel Cozzolino, James Chapman, Aaron Elbourne and Vi Khanh Truong

Molecules 2021, 26(13), 3890; https://doi.org/10.3390/molecules26133890 - 25 Jun 2021

Cited by 28 | Viewed by 2882

Abstract

Biofilms are assemblages of microbial cells, extracellular polymeric substances (EPS), and other components extracted from the environment in which they develop. Within biofilms, the spatial distribution of these components can vary. Here we present a fundamental characterization study to show differences between biofilms formed by Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), Gram-negative Pseudomonas aeruginosa, and the yeast-type Candida albicans using synchrotron macro attenuated total reflectance-Fourier transform infrared (ATR-FTIR) microspectroscopy. We were able to characterise the pathogenic biofilms’ heterogeneous distribution, which is challenging to do using traditional techniques. Multivariate analyses revealed that the polysaccharides area (1200–950 cm⁻¹) accounted for the most significant variance between biofilm samples, and other spectral regions corresponding to amides, lipids, and polysaccharides all contributed to sample variation. In general, this study will advance our understanding of microbial biofilms and serve as a model for future research on how to use synchrotron source ATR-FTIR microspectroscopy to analyse their variations and spatial arrangements. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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10 pages, 1990 KiB

Open AccessArticle

Identification of Binding Regions of Bilirubin in the Ligand-Binding Pocket of the Peroxisome Proliferator-Activated Receptor-A (PPARalpha)

by Darren M. Gordon, Stephen H. Hong, Zachary A. Kipp and Terry D. Hinds, Jr.

Molecules 2021, 26(10), 2975; https://doi.org/10.3390/molecules26102975 - 17 May 2021

Cited by 25 | Viewed by 2931

Abstract

Recent work has shown that bilirubin has a hormonal function by binding to the peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor that drives the transcription of genes to control adiposity. Our previous in silico work predicted three potential amino acids that bilirubin may interact with by hydrogen bonding in the PPARα ligand-binding domain (LBD), which could be responsible for the ligand-induced function. To further reveal the amino acids that bilirubin interacts with in the PPARα LBD, we harnessed bilirubin’s known fluorescent properties when bound to proteins such as albumin. Our work here revealed that bilirubin interacts with threonine 283 (T283) and alanine 333 (A333) for ligand binding. Mutational analysis of T283 and A333 showed significantly reduced bilirubin binding, reductions of 11.4% and 17.0%, respectively. Fenofibrate competitive binding studies for the PPARα LBD showed that bilirubin and fenofibrate possibly interact with different amino acid residues. Furthermore, bilirubin showed no interaction with PPARγ. This is the first study to reveal the amino acids responsible for bilirubin binding in the ligand-binding pocket of PPARα. Our work offers new insight into the mechanistic actions of a well-known molecule, bilirubin, and new fronts into its mechanisms. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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14 pages, 5601 KiB

Open AccessArticle

A New Coumarin-Acridone Compound as a Fluorescence Probe for Fe³⁺ and Its Application in Living Cells and Zebrafish

by Jiayong Huang, Zhenshuo Yan, Peiling Qiu, Yufeng Mo, Qizhen Cao, Qiuhong Li, Lini Huo and Lichun Zhao

Molecules 2021, 26(8), 2115; https://doi.org/10.3390/molecules26082115 - 07 Apr 2021

Cited by 7 | Viewed by 2301

Abstract

A new coumarin-acridone fluorescent probe S was designed and synthesized, and the structure was confirmed with ¹H/¹³C NMR spectrometry, single-crystal X-ray diffraction, and high-resolution mass spectrometry. This probe has high sensitivity and selectivity for Fe³⁺ over other testing metal ions at 420 or 436 nm in acetonitrile–MOPS (3-Morpholinopropanesulfonic Acid) buffer solution (20.0 μM, pH = 6.9, 8:2 (v/v)). Under physiological conditions, the probe displayed satisfying time stability with a detection limit of 1.77 µM. In addition, probe S was successfully used to detect intracellular iron changes through a fluorescence-off mode, and the imaging results of cells and zebrafish confirmed their low cytotoxicity and satisfactory cell membrane permeability, as well as their potential biological applications. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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Review

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27 pages, 8226 KiB

Open AccessReview

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior

by Anna C. Croce and Francesca Scolari

Molecules 2022, 27(14), 4458; https://doi.org/10.3390/molecules27144458 - 12 Jul 2022

Cited by 5 | Viewed by 3167

Abstract

Light-based phenomena in insects have long attracted researchers’ attention. Surface color distribution patterns are commonly used for taxonomical purposes, while optically-active structures from Coleoptera cuticle or Lepidoptera wings have inspired technological applications, such as biosensors and energy accumulation devices. In Diptera, besides optically-based phenomena, biomolecules able to fluoresce can act as markers of bio-metabolic, structural and behavioral features. Resilin or chitinous compounds, with their respective blue or green-to-red autofluorescence (AF), are commonly related to biomechanical and structural properties, helpful to clarify the mechanisms underlying substrate adhesion of ectoparasites’ leg appendages, or the antennal abilities in tuning sound detection. Metarhodopsin, a red fluorescing photoproduct of rhodopsin, allows to investigate visual mechanisms, whereas NAD(P)H and flavins, commonly relatable to energy metabolism, favor the investigation of sperm vitality. Lipofuscins are AF biomarkers of aging, as well as pteridines, which, similarly to kynurenines, are also exploited in metabolic investigations. Beside the knowledge available in Drosophila melanogaster, a widely used model to study also human disorder and disease mechanisms, here we review optically-based studies in other dipteran species, including mosquitoes and fruit flies, discussing future perspectives for targeted studies with various practical applications, including pest and vector control. Full article

(This article belongs to the Special Issue Autofluorescence Spectroscopy and Imaging II)

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