Rumelo Amor

Antitumor activity of the tea polyphenol epigallocatechin-3-gallate encapsulated in targeted vesicles after intravenous administration

AIM The therapeutic potential of epigallocatechin-3-gallate (EGCG), a green tea polyphenol with anticancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study was to determine whether a tumor-targeted vesicular formulation of EGCG would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. MATERIALS & METHODS Transferrin-bearing vesicles encapsulating EGCG were administered intravenously to mice bearing subcutaneous A431 and B16-F10 tumors. RESULTS The intravenous administration of EGCG encapsulated in transferrin-bearing vesicles resulted in tumor suppression in 40% of A431 and B16-F10 tumors. Animal survival was improved by more than 20 days compared with controls. CONCLUSION Encapsulation of EGCG in transferrin-bearing vesicles is a promising therapeutic strategy.

A promising new wavelength region for three-photon fluorescence microscopy of live cells

We report three‐photon laser scanning microscopy (3PLSM) using a bi‐directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto‐fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two‐photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout

Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos. DOI: http://dx.doi.org/10.7554/eLife.18659.001

Exploration of the two-photon excitation spectrum of fluorescent dyes at wavelengths below the range of the Ti:Sapphire laser.

We have studied the wavelength dependence of the two‐photon excitation efficiency for a number of common UV excitable fluorescent dyes; the nuclear stains DAPI, Hoechst and SYTOX Green, chitin‐ and cellulose‐staining dye Calcofluor White and Alexa Fluor 350, in the visible and near‐infrared wavelength range (540–800 nm). For several of the dyes, we observe a substantial increase in the fluorescence emission intensity for shorter excitation wavelengths than the 680 nm which is the shortest wavelength usually available for two‐photon microscopy. We also find that although the rate of photo‐bleaching increases at shorter wavelengths, it is still possible to acquire many images with higher fluorescence intensity. This is particularly useful for applications where the aim is to image the structure, rather than monitoring changes in emission intensity over extended periods of time. We measure the excitation spectrum when the dyes are used to stain biological specimens to get a more accurate representation of the spectrum of the dye in a cell environment as compared to solution‐based measurements.

Tumor regression after intravenous administration of targeted vesicles entrapping the vitamin E α-tocotrienol

Abstract The therapeutic potential of tocotrienol, a member of the vitamin E family of compounds with potent in vitro anti‐cancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study is to determine whether a novel tumor‐targeted vesicular formulation of tocotrienol would suppress the growth of A431 epidermoid carcinoma and B16‐F10 melanoma in vitro and in vivo. In this work, we demonstrated that novel transferrin‐bearing multilamellar vesicles entrapping &agr;‐T3 resulted in a dramatically improved (by at least 52‐fold) therapeutic efficacy in vitro on A431 cell line, compared to the free drug. In addition, the intravenous administration of tocotrienol entrapped in transferrin‐bearing vesicles resulted in tumor suppression for 30% of A431 and 60% of B16‐F10 tumors, without visible toxicity. Mouse survival was enhanced by >13 days compared to controls administered with the drug solution only. This tumor‐targeted, tocotrienol‐based nanomedicine therefore significantly improved the therapeutic response in cancer treatment. Graphical abstract Tumor growth studies in a mouse A431 xenograft model after intravenous administration of transferrin‐bearing vesicles encapsulating &agr;‐T3 (10 &mgr;g/injection) (green) (controls: control vesicles entrapping &agr;‐T3 (orange), &agr;‐T3 solution (red), untreated tumors (black)) (n = 10). Figure. No Caption available.

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ≈90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

A compact instrument for adjusting laser beams to be accurately coincident and coaxial and its use in biomedical imaging using wave-mixed laser sources

Biomedical imaging applications that involve nonlinear optical processes such as sum-frequency generation (SFG) and four-wave mixing require that the pulses are synchronized in time and the beams are coaxial to better than 400 μrad. For this reason, folding mirrors are normally used to extend the beam path over a few meters so that detectors can be put into the beams to check their overlap at the start of a long path and also at the end of it. We have made a portable instrument with a footprint of only 22 cm × 11 cm × 16 cm that uses a short focal length lens and a telephoto combination for viewing the near-field and far-field simultaneously. Our instrument is simple to build and use, and we show its application in coherent anti-Stokes Raman scattering microscopy and SFG-based two-photon fluorescence microscopy.

Increased signals from short-wavelength-excited fluorescent molecules using sub-Ti:Sapphire wavelengths

We report the use of an all‐solid‐state ultrashort pulsed source specifically for two‐photon microscopy at wavelengths shorter than those of the conventional Ti:Sapphire laser. Our approach involves sum–frequency mixing of the output from an optical parametric oscillator (λ= 1400–1640 nm) synchronously pumped by a Yb‐doped fibre laser (λ= 1064 nm), with the residual pump radiation. This generated an fs‐pulsed output tunable in the red spectral region (λ= 620–636 nm, ∼150 mW, 405 fs, 80 MHz, M2∼ 1.3). We demonstrate the performance of our ultrashort pulsed system using fluorescently labelled and autofluorescent tissue, and compare with conventional Ti:Sapphire excitation. We observe a more than 3‐fold increase in fluorescence signal intensity using our visible laser source in comparison with the Ti:Sapphire laser for two‐photon excitation at equal illumination peak powers of 1.16 kW or less.