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 that the relative intensities in each plane of excitation depend on the Stokes shift of the fluorochrome. 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.Comment: 15 pages, 4 figures; changed the discussion of narrow-band detected fringes (Fig. 3) to describe the phenomenon as a moire pattern between the excitation and emission standing-wave fields, rather than a beats pattern; added DiI(5)-labelled red blood cell in Fig. 4 to show that standing-wave fringes are present even when the dye excitation wavelength is outside the haemoglobin absorption ban

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

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

The therapeutic potential of epigallocatechin gallate, a green tea polyphenol with 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 tumor-targeted vesicular formulation of epigallocatechin gallate would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. Transferrin-bearing vesicles encapsulating epigallocatechin gallate were intravenously administered to mice bearing subcutaneous A431 and B16-F10 tumors. The intravenous administration of epigallocatechin gallate encapsulated in transferrin-bearing vesicles resulted in tumor suppression for 40% of A431 and B16-F10 tumors. Animal survival was improved by more than 20 days compared to controls. Encapsulation of epigallocatechin gallate in transferrin-bearing vesicles is a promising therapeutic strategy

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

Whole-brain imaging of freely-moving zebrafish

One of the holy grails of neuroscience is to record the activity of every neuron in the brain while an animal moves freely and performs complex behavioral tasks. While important steps forward have been taken recently in large-scale neural recording in rodent models, single neuron resolution across the entire mammalian brain remains elusive. In contrast the larval zebrafish offers great promise in this regard. Zebrafish are a vertebrate model with substantial homology to the mammalian brain, but their transparency allows whole-brain recordings of genetically-encoded fluorescent indicators at single-neuron resolution using optical microscopy techniques. Furthermore zebrafish begin to show a complex repertoire of natural behavior from an early age, including hunting small, fast-moving prey using visual cues. Until recently work to address the neural bases of these behaviors mostly relied on assays where the fish was immobilized under the microscope objective, and stimuli such as prey were presented virtually. However significant progress has recently been made in developing brain imaging techniques for zebrafish which are not immobilized. Here we discuss recent advances, focusing particularly on techniques based on light-field microscopy. We also draw attention to several important outstanding issues which remain to be addressed to increase the ecological validity of the results obtained

Nonlinear and interference techniques for biomedical imaging

Optical microscopy has long been an established tool in the biomedical sciences, being the preferred choice in the study of single cells and tissue sections. The realisation of the confocal laser scanning microscope in the 1980s led to major advances in the way optical microscopy is implemented, paving the way for the use of interference techniques such as 4Pi microscopy to increase the optical resolution, and for nonlinear microscopy techniques such as two-photon microscopy, which allows deeper penetration and the imaging of live specimens as a consequence of reduced photo-bleaching, and coherent anti-Stokes Raman scattering (CARS) microscopy, which produces high-contrast images without the need for fluorescent staining. In this work, I discuss advances in nonlinear and interference techniques available for biomedical imaging. I present a simultaneous near-field and far-field viewer for use in aligning the input beams in a CARS microscope and in a sum-frequency-generation- based two-photon microscope. I show 3D optical sectioning of whole mouse embryos using the Mesolens, a giant microscope objective capable of subcellular resolution in a 5 mm field of view, and present theoretical calculations on its use for two-photon microscopy. I present fast recording of synaptic events in neurones, with reduced photo-bleaching, using widefield two-photon microscopy. Finally, I show multiple super-resolved sections are obtained using a laser scanning standing wave microscope, generating precise contour maps of the surface membrane of red blood cells and revealing 3D information from a single image.Optical microscopy has long been an established tool in the biomedical sciences, being the preferred choice in the study of single cells and tissue sections. The realisation of the confocal laser scanning microscope in the 1980s led to major advances in the way optical microscopy is implemented, paving the way for the use of interference techniques such as 4Pi microscopy to increase the optical resolution, and for nonlinear microscopy techniques such as two-photon microscopy, which allows deeper penetration and the imaging of live specimens as a consequence of reduced photo-bleaching, and coherent anti-Stokes Raman scattering (CARS) microscopy, which produces high-contrast images without the need for fluorescent staining. In this work, I discuss advances in nonlinear and interference techniques available for biomedical imaging. I present a simultaneous near-field and far-field viewer for use in aligning the input beams in a CARS microscope and in a sum-frequency-generation- based two-photon microscope. I show 3D optical sectioning of whole mouse embryos using the Mesolens, a giant microscope objective capable of subcellular resolution in a 5 mm field of view, and present theoretical calculations on its use for two-photon microscopy. I present fast recording of synaptic events in neurones, with reduced photo-bleaching, using widefield two-photon microscopy. Finally, I show multiple super-resolved sections are obtained using a laser scanning standing wave microscope, generating precise contour maps of the surface membrane of red blood cells and revealing 3D information from a single image

A new giant lens for confocal mesoscopy

Optical lenses reached the limit of resolution set by the wavelength of light more than a century ago. However, no attempt was made to achieve the maximum resolution in the case of low-magnification lenses, probably because the visual image would then have contained detail too fine to be perceived by the human eye. Currently available lenses of less than 10x magnification are of simple construction and their numerical apertures are 0.2 or less, as compared with 1.3 or more in high-power lenses. They are adequate for the eye or a standard camera, but thin confocal optical sections are not possible because of the low numerical aperture. We have developed a novel lens system called the Mesolens, which, with a magnification of 4x and an N.A. of 0.5, combines high spatial resolution with an wide field of view. When compared with a standard 4x objective, its lateral resolution is 2.5 to 5 times better and its depth resolution is 10 times better. This lens provides, for the first time, good optical sectioning of specimens as large as entire 10 day mouse embryos (5mm long) with subcellular detail in every developing organ. The lens is difficult to make because of the need for a higher degree of aberration control than in any standard camera lens and it is too large (optical train 50cm x 7 cm) to fit on any standard microscope [1]. Figure 1: A single confocal optical section of 11-day mouse embryo taken using the Mesolens. This specimen was stained for Golgi bodies and clearly shows in a single image (with no stitching and tiling) the whole organism, which is 5mm in diameter, with sub-micron spatial resolution. The full size image shows the microanatomy of the whole embryo, while in the enlarged portion at the bottom left, bright dots corresponding to the Golgi bodies of individual cells can be seen in the developing heart. We will show recent data and discuss the Mesolens and its applications, including unexpected ones such as the detection of bioluminescent labels in individual cells at near-video rates, which is made possible by the high capture efficiency of the lens. [1] “New lens offers a brighter outlook”, Science 335 1562-3 (2012)

Standing-wave excitation of fluorescence in a laser-scanning microscope allows precise contour mapping of the red blood cell membrane

We demonstrate fluorescence excitation at multiple planes in a laser-scanning microscope by using the standing wave from a mirror placed close to the specimen. We have observed precise modulation of the standing waves close to a mirror, with a frequency proportional to the Stokes shift, corresponding to a moiré pattern between the excitation and emission standing-wave fields. We use standing-wave excitation to plot the exact contour maps of the red blood cell membrane, with an axial resolution of ≈90 nm. The method may prove useful in the study of diseases which involve the surface membrane of red blood cells

Efficient excitation of blue emitting dyes by two-photon microscopy at visible wavelengths

An important advantage of multi-photon microscopy over more traditional single-photon imaging techniques is the elimination of high-energy laser sources that can cause significant photo-damage to delicate biological specimens. This is particularly acute when short wavelength excitation is required. A number of papers (including [1,2]) have investigated the optimum two-photon excitation wavelengths for short-wavelength excitable molecules and have reported that for several fluorochromes the two-photon cross-section increases rapidly at shorter wavelengths. However, most papers thus far have used a Ti:Sapphire laser, which has a wavelength tuning range of 680-1080nm, and therefore few (and mainly cuvette based) data of two-photon excitation efficiency is available for excitation wavelengths shorter than 680nm. Using a new wavelength tunable optical parametric oscillator and frequency mixing system (Coherent Chameleon OPOVis), we have studied the wavelength dependence of two-photon excitation efficiency of a number of common UV excitable dyes, such as the nuclear stain DAPI, starch staining dye Calcofluor White and Alexa 350, in the wavelength range 530nm to 770nm. We will report details of the optical system and biomedical specimens used to make our measurements, together with a summary of our results. One key finding is that DAPI, a very commonly used nuclear marker, when excited at 590nm, results in a 7 times increase in the fluorescence signal output when compared to excitation at 680nm with the Ti:Sapphire laser. 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