Journal of Biophotonics

Cover image for Vol. 9 Issue 8

Editor: Jürgen Popp

Impact Factor: 3.818

ISI Journal Citation Reports © Ranking: 2015: 10/90 (Optics); 16/77 (BIOCHEMICAL RESEARCH METHODS); 16/72 (Biophysics)

Online ISSN: 1864-0648

Associated Title(s): Laser & Photonics Reviews

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July 21, 2016

easySTORM: a robust, lower-cost approach to localization and TIRF microscopy

easySTORM: a robust, lower-cost approach to localization and TIRF microscopyA broad range of new microscopy methodologies have recently been introduced collectively referred to as “super-resolved microscopy (SRM)”. They offer resolution measured in tens of nanometers or less – overcoming the diffraction limit and thereby opening up a wealth of new possibilities for biological studies. There is an increasing emphasis now on making SRM techniques and instrumentation more accessible. Their implementation with commercially available instrumentation typically requires complete new microscope systems to be purchased, which are relatively expensive. While techniques such as stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) should be integrated into tailored microscopes, localization microscopy techniques can be implemented on existing wide-field or total internal reflection fluorescence (TIRF) microscopes.
Researchers from the Imperial College London now adapted an existing wide-field epifluorescence microscope for localization microscopy. They developed a straightforward and relatively low-cost approach to implement stochastic optical reconstruction microscopy (STORM) and TIRF – a usually significantly complex and costly affair.
For their new approach, which they called “easySTORM”, the scientists took advantage of multimode optical fibers and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non-TIRF imaging, this enables TIRF and STORM imaging of cells labeled with appropriate dyes or expressing suitable fluorescent proteins.
The costs of the components needed for the new setup amounted to less than about ₤ 20,000. The scientists believe this should enable manufacturers to reduce costs and research groups to upgrade existing instruments such that these techniques can be routinely implemented for biological studies.
(Text contributed by K. Maedefessel-Herrmann)

Reference: Kwasi Kwakwa, Alexander Savell, Timothy Davies, Ian Munro, Simona Parrinello, Marco A. Purbhoo, Chris Dunsby, Mark A.A. Neil and Paul M.W. French, easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy, J. Biophotonics (2016)DOI http://dx.doi.org/10.1002/jbio.201500324

May 09, 2016

Better healing out of the blue

Better healing out of the blue

Irradiation with a blue-LED haemostatic device improved the healing process in superficial skin wounds without adverse side effects.

Sesto Fiorentino (Florence, Italy) – Since its invention in the early ’90s, blue-LED technology has found several applications in the biochemical and biomedical field. One is that irradiation with blue light can stop the bleeding of wounds. The absorption of haemoglobin at narrow peaks in the blue range (410 nm and 430 nm for oxygenated and non-oxygenated haemoglobin respectively), showing an absorption coefficient much higher than skin chromophores. The effect can be used to ensure a local temperature increase that is able to induce hemostasis through a photo-thermo-coagulation process.
Does blue light treatment also affect the healing process of superficial abrasions? Researchers from the National Research Council and the University of Florence in Sesto Fiorentino and Florence (Italy) now addressed this question. They used a blue-LED photo-haemostatic device which had been developd as a part of a project called LighTPatcH within the Biophotonics plus call 2012 initiative. The compact, easy-to-handle optical fiber-coupled device emits the two wavelengths matching the main absorption peaks of haemoglobin in the blue region.
In the study, mechanical abrasions were induced on the back of 10 Sprague Dawley rats. Half of them were treated with the blue-LED device, while the others were left to naturally recover. The induced photothermal effect was monitored during treatment by the use of a thermocamera. Visual observations, non-linear microscopic imaging, histological and immunofluorescence analyses were used to study the healing process 8 days after the treatment, and LED treated and untreated wounds were compared.
The Italian scientists found a faster healing process and a better-recovered skin morphology in treated abrasions with respect to the control wounds. The wounds irradiated with blue light showed a reduced inflammatory response, a higher collagen content as well as a better recovered morphology and organization of dermal collagen. No adverse reactions neither thermal damages in both abraded areas and surrounding tissue caused by the blue irradiation were observed. The results support the hypothesis that the selective photo-thermal effect used for inducing haemostasis in superficial skin wounds is associated to a faster healing process.
(Text contributed by K. Maedefessel-Herrmann)

Reference: Riccardo Cicchi, Francesca Rossi, Domenico Alfieri, Stefano Bacci, Francesca Tatini, Gaetano De Siena, Gaia Paroli, Roberto Pini, and Francesco S. Pavone, Observation of an improved healing process in superficial skin wounds after irradiation with a blue-LED haemostatic device, J. Biophotonics (2016)
DOI: http://dx.doi.org/10.1002/jbio.201500191

May 03, 2016

Watching apoptosis in 3D

Watching apoptosis in 3D

A promising approach for watching cell signaling processes in their physiological context: Scientists visualize apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

London (UK) – For the study of systemic effects of disease, for the development of potential therapies, and for a deeper understanding of cellular signaling responses, microscopy of cells in culture is not sufficient. Imaging functional cell behavior in the context of whole live organisms would be of great interest in this context. Zebrafish are ideal organisms for such whole-body imaging techniques: they are relatively transparent to optical radiation, they are genetically tractable, and transparent mutants as well as fluorophore-tagged lines are readily available. 3-D imaging technology that is suitable for zebrafish, however, is still under development.
Scientists from the Imperial College London and the University College London (UK) now present the application of optical projection tomography (OPT) to map the activity of a Förster resonant energy transfer (FRET) biosensor throughout a whole intact zebrafish using fluorescence lifetime imaging (FLIM). FRET essentially reads out the colocalization (≲10 nm) of two or more fluorophores via the strength of the resonant energy transfer that occurs between them. OPT is analogous to x-ray CT, but uses visible optical radiation. It can be implemented to image with transmitted light and with fluorescence, and so can utilize a vast array of fluorescent markers and biosensors.
The method, termed as FLIM OPT, provides a powerful means to visualize cell signaling processes in their physiological context. Now, the performance of FLIM OPT was demonstrated in live transgenic zebrafish larvae using a genetically expressed FRET biosensor for Caspase 3. The researchers had generated a novel transgenic zebrafish line expressing a Caspase 3 FRET biosensor under the control of a ubiquitous promoter in a non-pigmented zebrafish. As soon as Caspase 3 is activated, it starts cleaving the biosensor, the fluorophores are separated, and the energy transfer between them decreases. Caspase 3 is a member of the cysteine dependent aspartate proteases, a family of proteases that cleave specific peptide sequences after an aspartate residue. It is a key enzyme in apoptosis, or programmed cell death, which is essential during development and homeostatic tissue turnover as well as during disease. If apoptosis becomes dysregulated it can lead to cancer, autoimmune disorders and neurodegenerative diseases.
Apoptosis was triggered in live zebrafish larvae by gamma irradiation at 24 hours post fertilization. Changes in Caspase 3 activation were monitored over time. The scientists observed significant apoptosis at 3.5 hours post irradiation, predominantly in the head region. Only 150 s were required to acquire the 3-D FLIM dataset, compared to 300 s required to image a single optical section using laser scanning confocal microscopy with time-correlated single photon counting. This should impose significantly less stress on the organism. The low light dose associated with wide-field imaging enables extended time-lapse studies to be undertaken with the potential to recover the zebrafish after imaging for further longitudinal studies.
The researchers believe that the presented approach could be useful in drug discovery, as zebrafish are an ideal model for drug screening. FLIM OPT could provide a way to visualize whole body responses to potential therapies in space and time. (Text contributed by K. Maedefessel-Herrmann)

Original publication: [Natalie Andrews, Marie-Christine Ramel, Sunil Kumar, Yuriy Alexandrov, Douglas J. Kelly, Sean C. Warren, Louise Kerry, Nicola Lockwood, Antonina Frolov, Paul Frankel, Laurence Bugeon, James McGinty, Margaret J. Dallman, Paul M. W. French, Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time, J. Biophotonics 9(4), 414-424 (2016)]

July 21, 2016
easySTORM: a robust, lower-cost approach to localization and TIRF microscopy

May 09, 2016
Better healing out of the blue

May 03, 2016
Watching apoptosis in 3D

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