We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.
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"No prechirping is required and the laser beam can be coupled straight into the fiber. Photonic crystal fibers have been successfully used in a number of twophoton fiberscope designs (Flusberg et al. 2005b; Engelbrecht et al. 2008; Piyawattanametha et al. 2009). A disadvantage is, however, their susceptibility to contamination of the fiber tip, probably attributable to the intricate photonic crystal structure, which poses a particular problem when the fiber end is integrated into a fiber scanner device and thus cannot be easily recleaved. "
[Show abstract][Hide abstract]ABSTRACT: This article describes the development and application of miniaturized two-photon-excited fluorescence microscopes ("two-photon fiberscopes"). Two-photon fiberscopes have been developed with the aim of enabling high-resolution imaging of neural activity in freely behaving animals. They use fiber optics to deliver laser light for two-photon excitation. Their small front piece typically contains a miniature scanning mechanism and imaging optics. Two-photon fiberscopes can be made sufficiently small and lightweight to be carried by rats and mice and to allow virtually unrestricted movement within a behavioral arena. Typically mounted to the animal's skull above a cranial window, two-photon fiberscopes permit imaging of cells down to at least 250 µm below the brain surface (e.g., in rat neocortex). In freely exploring animals, action-potential-evoked calcium transients can be imaged in individual somata of visual cortex neurons bulk-labeled with a calcium indicator. Two-photon fiberscopes thus enable high-resolution optical recording of neural activity with cellular resolution during natural behaviors.
Preview · Article · Oct 2013 · Cold Spring Harbor Protocols
"Combined, the promise is that, by searching the enormous chemical space of small molecules and of proteins, we would greatly extend the current repertoire of ROS indicators and ultimately achieve the same level of reliability as we have enjoyed while measuring intracellular Ca2+ with small-molecule probes such as fluo-3 , fura-2 , indo-1 , and fluorescent protein probes such as GCamp6  and GECOs, the palette with blue, improved green, and or red-shifted indicators . In synergy with the exponentially increasing numbers of indicator-expressing organisms and disease models, the booming technology for super-resolution and single-molecule imaging [113–115], and the trend for using miniature, plant-in devices to obtain images in conscious, free-moving animals [116–120], imaging ROS in vivo will serve as the most powerful force to transform the landscape and push forward the frontiers in ROS signaling. "
[Show abstract][Hide abstract]ABSTRACT: Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling-for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.
Full-text · Article · Jul 2013 · Journal of Molecular Medicine
"In this paper, we present data acquired with the dual-axes confocal microscopes as well as a miniature two-photon fluorescence microscope – , as shown in Fig. 2(c), and a commercially available confocal microendoscope , as shown in Fig. 1 "
[Show abstract][Hide abstract]ABSTRACT: Recent advances in optical imaging have led to the development of miniature microscopes that can be brought to the patient for visualizing tissue structures in vivo. These devices have the potential to revolutionize health care by replacing tissue biopsy with in vivo pathology. One of the primary limitations of these microscopes, however, is that the constrained field of view can make image interpretation and navigation difficult. In this paper, we show that image mosaicing can be a powerful tool for widening the field of view and creating image maps of microanatomical structures. First, we present an efficient algorithm for pairwise image mosaicing that can be implemented in real time. Then, we address two of the main challenges associated with image mosaicing in medical applications: cumulative image registration errors and scene deformation. To deal with cumulative errors, we present a global alignment algorithm that draws upon techniques commonly used in probabilistic robotics. To accommodate scene deformation, we present a local alignment algorithm that incorporates deformable surface models into the mosaicing framework. These algorithms are demonstrated on image sequences acquired in vivo with various imaging devices including a hand-held dual-axes confocal microscope, a miniature two-photon microscope, and a commercially available confocal microendoscope.
Full-text · Article · Oct 2010 · IEEE transactions on bio-medical engineering