News from Journal of Biophotonics

Plate reader for drug discovery

2013-05-07T00:00:00+02:00

A new automated fluorescence lifetime imaging plate reader was used to study aggregation of HIV-1 Gag proteins, the molecular machine responsible for orchestrating the assembly of nascent HIV-1 virions at the cell membrane.

London (UK) The trend towards automated high content assays, particularly for systems biology and drug discovery, has stimulated the development of automated fluorescence microscopy systems to image sample arrays. Todays commercially available high content analysis (HCA) instruments predominantly employ fluorescence intensity imaging to map the relative distribution and colocalization of labelled proteins with subcellular resolution. Although commonplace in biology laboratories, Förster resonant energy transfer (FRET) measurements have not been widely taken up for drug discovery. Conventional (manual) FRET microscopy is too time-consuming and labour-intensive for high throughput applications. One of the most widely used approaches to map FRET is fluorescence lifetime imaging (FLIM). However, a lack of available FLIM instrumentation for automated multiwell plate readouts is constricting its use in drug discovery.
Recently, a team led by Paul M. W. French from Imperial College London developed a system for rapid FLIM and FRET for HCA which has been implemented in the time domain using a gated optical image intensifier to provide wide-field time-gated imaging and incorporated a quasi-wide-field Nipkow (spinning disc) microscope unit to provide optical sectioning. The British scientists now present an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples. They illustrate its potential to assay protein-protein interactions through application to Gag protein aggregation during the HIV life cycle.
HIV-1 Gag proteins are the molecular machine responsible for orchestrating the assembly of nascent HIV-1 virions at the cell membrane. Upon oligomerisation of two or more Gag proteins, a sequestered myristic acid moiety present in Gag monomers is exposed to the solvent. This myristic switch mechanism drives membrane binding providing an opportunity to interfere with virion formation. Labeling Gag proteins with fluorescent protein tags enables their distribution to be visualized and formation of viral like particles can be assayed using FLIM-FRET.
For their improved FLIM FRET assay of Gag protein aggregation, the researchers chose HeLa cells as a superior biological host. With help of the new automated FLIM plate reader it was possible to discriminate different biomolecular processes as the HIV-1 Gag protein interactions were manipulated through the use of myristic acid negative mutants that are unable to bind the plasma membrane. Furthermore the scientists applied an inhibitor of myristoylation to permit, for the first time, a quantitative evaluation of FLIM FRET assay performance using dose response curves and calculation of Z factors. They used this metric to compare the assay performance of hetero-FRET and homo-FRET readouts of the Gag protein interactions. This study is the first FLIM assay of protein aggregation based on homo-FRET (donor and acceptor molecules are the same).
The researchers are planning to utilise this new capability to screen for other novel inhibitors of HIV formation with a view to developing therapies for HIV and other viral infections. (Text contributed by K. Maedefessel-Herrmann)

Alibhai, D., et al; J. Biophotonics 6(5), 398-408 (2013); DOI 10.1002/jbio.201200185
http://onlinelibrary.wiley.com/doi/10.1002/jbio.201200185/abstract

A fruitful combination

2013-04-08T00:00:00+02:00

Combining two methods into a single platform opens up new possibilities for non-invasive 3D skin imaging: optical coherence tomography to locate abnormal skin regions followed by multiphoton tomography to examine sub-cellular features of a specific abnormal region.

Vienna (Austria) The development of optical non-invasive in vivo 3D imaging techniques suitable for clinical dermatological applications has been an area of intense research since the 1990s. Now, two methods as a twin pack might pave the way to significant improvements in the field of dermatology by exploiting the benefits of each modality: Multiphoton tomography (MPT) and optical coherence tomography (OCT) which are both imaging modalities capable of performing in vivo optical biopsy and have been successfully used for clinical as well as research applications in dermatology.
MPT is based on non-linear processes such as two photon excited fluorescence (TPEF) from endogenous fluorophores and second harmonic generation (SHG) from non-centrosymmetric molecules. OCT is an imaging technique capable of reconstructing 3D internal structure of a target tissue by measuring the amplitude and time delay of backscattered light. As these two techniques are complimentary in terms of imaging performance such as resolution, penetration depth and contrast, a combination of MPT and OCT could be fruitful as a diagnostic tool.
Wolfgang Drexler and his colleagues from Medical University Vienna, Austria, and JenLab GmbH, Jena, Germany investigated the diagnostic potential of a multimodal MPT/OCT approach in various dermatological applications. In a preliminary clinical trial, they sequentially used state-of-the-art MPT and OCT systems to acquire 3D images of normal skin, nevi, scars and pathologic skin lesions in vivo. OCT was used for visualizing and pre-screening micron-scale morphology of a relatively large tissue volume. MPT was subsequently employed to reveal sub-cellular details of a zoomed-in tissue volume. The aim of this study was to visualize the 3D morphology of different skin layers and to identify characteristic features of various skin pathologies.
MPT proved to be capable of revealing sub-cellular details of different skin layers up to a depth of 200 mm, without need for any extrinsic contrast agents. Meanwhile, OCT could be used to reveal the layered architectural arrangement of skin tissue over a volume of about 10 X 10 X 1.5 mm3. The study demonstrated the clinical diagnostic potential of MPT/OCT for pre-screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub-cellular level information of the respective regions. With the help of metabolic information extracted using MPT, it should even be possible to perform non-invasive in vivo functional imaging of the target tissue. (Text contributed by K. Maedefessel-Herrmann)

A. Alex. et al.; J. Biophotonics 6, 352-362 (2013); DOI 10.1002/jbio.201200085
http://onlinelibrary.wiley.com/doi/10.1002/jbio.201200085/abstract

Go with the flow: A review of methods and advancements in blood flow imaging

2013-03-27T00:00:00+01:00

Obstructions and alterations of the blood flow are associated with many diseases. Sophisticated modern methods of blood flow imaging help determining the presence or extent of such a disease.

Limerick (Ireland) The flow of our blood is essential for our health and live. A view at our blood flow in general or in special parts or tissues of the body can help diagnosing a wide range of health problems. Historically, the first means of measuring flow and perfusion were performed through invasive procedures, for example by direct measurements of capillary blood pressure, transcutaneous oxygen measurements, and radionuclide techniques. Nowadays, a whole wealth of modern methods is available or in development, many of them being non-invasive. Recently high resolution label-free imaging of the microcirculation at clinically relevant depths and has become available in the research domain.
In a review article, Susan M. Daly from the Biophotonics Research Facility at the University of Limerick and Martin J. Leahy from the Tissue Optics and Microcirculation Imaging Group at the National University of Ireland, Galway, Ireland present a comprehensive overview on current imaging techniques, state-of-the-art advancements and applications, as well as general perspectives on the prospects for these modalities in the clinical realm. Their modus operandi as well as associated advantages and limitations are outlined.
The authors present a broad variety of very different methods, including, among others, Indocyanine fluorescence, thermography, Laser Doppler Flowmetry, side-stream dark field illumination imaging, ultrasound imaging, Magnetic Resonance Imaging, positron emission tomography, optical coherence tomography, particle tracking velocimetry, and correlation spectroscopy.
Daly and Leahy conclude: Although the techniques discussed may differ inherently in their construction, operation, resolution, etc., they are yet intrinsically linked. The prevalence of complementary modalities is rife at present, utilising supplementary information to yield multifaceted information with affirming, real-world applications.
Text contributed by K. Maedefessel-Herrmann

Daly, S.M.; Leahy, M.J.; J. Biophotonics 6, 217-255 (2013); http://onlinelibrary.wiley.com/doi/10.1002/jbio.201200071/abstract

A closer look at collagen

2013-02-25T00:00:00+01:00

From molecular structure to tissue architecture: second-harmonic generation (SHG) microscopy is an ideal tool for probing collagen organization. For example, it can be used to study skin tumors and skin ageing.

Sesto Fiorentino (Italy) Second-harmonic generation (SHG) is a nonlinear second order optical process occurring in noncentrosymmetric systems with a large hyperpolarizability. This condition is satisfied at the molecular level by the presence of electron-donor and electronacceptor moieties connected by a π-conjugated system and by a structural organization of the molecular emitters at the focal volume scale. Both conditions are satisfied in anisotropic biological molecules such as collagen, making SHG microscopy an ideal tool for imaging collagen and probing its hierarchical organization from molecular scale up to tissue architectural level. SHG combines the advantages of a non-linear microscopy approach with a coherent modality able to probe molecular organization.
In a review article, Riccardo Cicchi from European Laboratory for Non-linear Spectroscopy (LENS), in Sesto Fiorentino, Italy, and his co-authors from Italy and Jena, Germany, sum up the physical concepts of SHG from collagen. They explain how this optical process allows probing structures ranging from molecular sizes to tissue architecture, through image pattern analysis and scoring methods. Starting from the description of the most relevant approaches employing SHG polarization anisotropy and forward backward SHG detection, the scientists then focus on the most relevant methods for imaging and characterizing collagen organization in tissues through image pattern analysis methods. They outline the advantages and limitations of the methods applied to tissue imaging as well as potential clinical applications.
SHG has already been used for imaging collagen rich tissues such as cornea, tendon, and arteries. In particular, SHG microscopy has been mainly used for selectively investigating collagen fibres orientation and their structural changes in human dermis, keloid, fibrosis, thermally-treated samples, and also in tumor microenvironments.
The Second-harmonic to Autofluorescence Ageing Index of Dermis (SAAID) is a simple scoring method to be used when photon excited fluorescence microscopy (TPEF) and SHG microscopy are used simultaneously for imaging connective tissue. Since collagen and elastic fibres can be respectively imaged by SHG and TPEF microscopy, this method can be used not only to determine ageing but also other altered physiological conditions of skin dermis and, more in general, of connective tissue. It can be used to study skin tumors. (Text contributed by K. Maedefessel-Herrmann)
R. Cicchi et al., J. Biophotonics 6(2), 129-142 (2013); DOI 10.1002/jbio.201200092