Handheld Microscope Research Articles

Multiphoton endoscopes for in vivo imaging of unstained tissues

The clinical assessment of tissue health currently involves a physician using a low-magnification white-light laparoscope/ endoscope that penetrates into the body to image tissues in situ. Biopsy samples are then extracted, processed into histopathology slides, and analyzed by a pathologist. Although effective, this practice results in delayed diagnosis, high procedural costs, patient discomfort, and inaccuracies because the biopsy samples are located in situ using low-magnification white-light imaging. A different technique—multiphoton microscopy—can acquire images of unprocessed, unstained tissues with resolution and detail comparable to standard histology.1, 2 Here, we describe our recent advances in imaging unstained tissues using multiphoton microscopy. The ability to acquire diagnostic-quality images solely from intrinsic tissue fluorophores, such as the enzyme cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide, and harmonic generations is a significant advantage for multiphoton imaging. Indeed, although it is customary in the laboratory to use exogenous contrast agents with this technique, there are currently very few of these dyes that are approved for use within a patient owing to concerns of dye toxicity. However, a wide variety of epithelial tissues can be imaged using compact multiphoton endoscopes (MPEs) that enter the body through a natural orifice or small surgical incision. These devices have the potential to be used clinically to assess surgical margins, either as replacements or guides for conventional surgical biopsies. Several groups have demonstrated small and flexible3–5 as well as larger rigid6, 7 MPEs. The larger rigid devices typically use rodlike gradient-index (GRIN) lenses that are attached to an external microscope. Since only the GRIN lens penetrates Figure 1. (a) Schematic of portable hand-held microscope with a gradient-index (GRIN) lens. (b) Image of intrinsic fluorescence (green) of molecules such as the enzyme cofactor nicotinamide adenine dinucleotide and second harmonic generation (SHG, gray) signal acquired in vivo from a rat kidney. PMT: Photomultiplier tube.

High-resolution resonant and nonresonant fiber-scanning confocal microscope

We present a novel, hand-held microscope probe for acquiring confocal images of biological tissue. This probe generates images by scanning a fiber-lens combination with a miniature electromagnetic actuator, which allows it to be operated in resonant and nonresonant scanning modes. In the resonant scanning mode, a circular field of view with a diameter of 190 μm and an angular frequency of 127 Hz can be achieved. In the nonresonant scanning mode, a maximum field of view with a width of 69 μm can be achieved. The measured transverse and axial resolutions are 0.60 and 7.4 μm, respectively. Images of biological tissue acquired in the resonant mode are presented, which demonstrate its potential for real-time tissue differentiation. With an outer diameter of 3 mm, the microscope probe could be utilized to visualize cellular microstructures in vivo across a broad range of minimally-invasive procedures.

In Vivo Micro-Image Mosaicing

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.

Handheld digital microscope with the latest LCD TV monitor

1. Dae-young Kim, MD, PhD 2. Hyung Suk Kim, MD (td8144{at}yahoo.co.kr) 1. Seoul, Korea In the graft dissection process of hair transplantation, optical microscopes are widely used as a method to lower follicle transection rates. However, long working hours at the microscope stand and limited

MEMS-based handheld confocal microscope for in-vivo skin imaging

This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800x600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 μm to 216 μm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 μm full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 μm. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror.

Estudio de la microcirculación cerebral en el traumatismo craneoencefálico mediante el sistema Side Stream Dark Field (SDF)

Posttraumatic tissular hypoxia can be due to multiple causes, including microcirculation disturbances, which can be studied with the SDF (Side Stream Dark Field) system. This system is based on a small hand-held microscope that eliminates directly reflected green polarised light from an organ surface using an orthogonal analyser. It offers clear images of red and white blood cells flow through microcirculation. Specific software is later used to determine the length and density of microvessels. We present a case of a TBI patient who required surgical evacuation of a brain contusion. Images of the microcirculatory bed were recorded with the SDF microscope and compared with a normal pattern obtained from another patient who was operated on for an unruptured cerebral aneurysm. Both imaging and quantitative analyses showed significant differences in the cerebral microcirculatory status in these patients. Total length and density of vessels were markedly reduced in the TBI patient. SDF imaging allows direct and non-invasive in vivo observation of cerebral microcirculation, and may allow us to deepen our knowledge of the pathophysiology of posttraumatic brain ischemia.

Three-dimensional in vivo imaging by a handheld dual-axes confocal microscope

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

Handheld subcellular-resolution single-fiber confocal microscope using high-reflectivity two-axis vertical combdrive silicon microscanner

We introduce a handheld single-fiber laser-scanning confocal microscope, incorporating a high-reflectivity two-axis silicon vertical combdrive microscanner, aimed at in vivo early detection of epithelial precancers. The approach adopted is motivated by need for a portable, economical, biopsy-free, early precancer screening technology in low-infrastructure environments. Our microelectromechanical system (MEMS) based handheld probe integrates the microscanners with miniature objective lens system and flexible electrical routing in a forward-imaging configuration, with 4.8 mm distal probe tip outer diameter for unrestricted imaging access in biological sites such as the oral cavity and cervix. Reflectance confocal videos of a USAF 1951 resolution target and biological samples were obtained over 200 microm x 110 microm field of view, with 0.80 and 9.55 microm lateral and axial resolution, at 3.5-5.0 frames per second. With improvements to objective numerical aperture, our probe can enable precise evaluation of nuclear size, density, nucleus-to-cytoplasm ratio and cell density, which are important visual identifiers of epithelial precancers.

Dermatoscopia: o método de análise de padrões

A incidência do melanoma cutâneo tem aumentado mundialmente e, por tratar-se de neoplasia bastante agressiva e de difícil tratamento em estádios mais avançados, o diagnóstico precoce é fundamental para a cura do paciente. A dermatoscopia surgiu como exame auxiliar in vivo, que tem papel fundamental na realização do diagnóstico precoce e amplifica a acurácia diagnóstica do melanoma. Para a realização do método, é necessário utilizar o dermatoscópio, aparato que permite aumentar a lesão, no mínimo, 10 vezes. A imagem obtida é interpretada utilizando-se o método diagnóstico da preferência do examinador. O método de Análise de Padrões é atualmente o mais utilizado e o que possui maior acurácia para o diagnóstico do melanoma cutâneo, tendo-se demonstrado confiável para o ensino de residentes em dermatologia. Baseia-se em padrões globais e específicos que permitem diferenciar as lesões melanocíticas das não melanocíticas (também importantes no diagnóstico diferencial com o melanoma cutâneo), assim como identificar lesões melanocíticas consideradas benignas, suspeitas ou malignas.

In vivo fibreoptic confocal imaging (FOCI) of the human ocular surface

Recent developments in the miniaturization of confocal imaging technology have resulted in the development of a hand-held confocal microscope probe. There are many structures of interest in the human eye that are within reach of a fluorescence-mode confocal microscope; this study assessed the feasibility of in vivo human ocular imaging. Safety analysis was undertaken to ensure that the laser light applied to the ocular surface structures constituted no threat to patient safety. A fibreoptic confocal imaging (FOCI) probe using an illumination wavelength of 488 nm was applied to the ocular surface of four volunteers after topical administration of sodium fluorescein. Stabilization of the probe on the ocular surface was difficult, but movement artefacts could be minimized to a satisfactory level in most subjects by a variety of procedures. High-quality images of conjunctival epithelial and goblet cells, lamina propria structures, accessory lacrimal glands, lacrimal ducts and superficial sclera were obtained. Lateral resolution was 1-1.5 microm and axial resolution was approximately 30 microm; individual erythrocytes could be seen in conjunctival vessels. The rete ridges and intervening epithelial components, including the probable location of corneal limbal stem cells, could be viewed, although it was not possible to distinguish cell subgroups. The study showed that fluorescence-mode imaging of the ocular surface is a viable and promising tool for assessment of diseases and processes involving superficial ocular structures. Refinement of equipment and techniques, particularly probe stabilization, is necessary to realize fully the potential of FOCI for ocular use.

FIELD EVALUATION OF THE MEADE READIVIEW HANDHELD MICROSCOPE FOR DIAGNOSIS OF INTESTINAL SCHISTOSOMIASIS IN UGANDAN SCHOOL CHILDREN

A novel, inexpensive handheld microscope, the Meade Readiview, was evaluated for field diagnosis of intestinal schistosomiasis by comparison of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) against conventional compound microscopy as part of a parasitologic survey in nine sentinel schools and a rapid mapping survey across 22 schools in Uganda. Fecal smears from 685 primary school children were examined and the overall prevalence of Schistosoma mansoni was 45%. However, prevalence by school ranged widely from 0% to 100%. For individual diagnosis the Readiview had a sensitivity of 85%, a specificity of 96%, a PPV of 95%, and an NPV of 88%. Due to the poorer movement control of the glass slide on the Readiview stage, fecal smears with less than four eggs could be overlooked. At the highest magnification (160x), egg-like objects could be confounding. Estimating prevalence by school was usually within +/- 7% of that of conventional microscopy. Since the Readiview is more robust and portable, both in size and weight, and one-tenth as expensive as the traditional compound microscope, a change in the logistics and costs associated with field infection surveillance is possible. This inexpensive microscope is a pragmatic alternative to the compound microscope. It could play an important role in the collection of prevalence data to better guide anthelmintic drug delivery and also empower the diagnostic capacity of peripheral health centers where compound microscopes are few or absent.

Eye health service in Greenland.

Eye health service in Greenland.

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Studies of surface microarchitecture using a hand-held video microscope in cases of cultured epithelial autografts.

The purpose of this study was to evaluate skin texture after cultured epithelial autografting (CEA) by focusing on the surface microarchitecture. From August 1993 through October 1995, CEA was performed to improve the texture of burn scars in 26 patients. The surface microarchitecture was studied using a hand-held video microscope on a long-term basis. In addition, biopsies were obtained from graft sites in 5 patients before CEA and at 1, 3, and 6 months after CEA. Before CEA, observation of the surface microarchitecture of the burn scars showed no skin fields or skin furrows. Histologically, the epidermis had no rete ridges. At 6 months after resurfacing with autologous cultured epithelium, it was possible to recognize skin fields and skin furrows in all patients. The shapes of the newly formed skin fields were similar but not identical to those at the donor site. Histological examination revealed that rete ridges were formed during the same time period. These findings suggest that the improvement in skin texture of burn scars after resurfacing with cultured epithelium depends on the reconstruction of the skin fields.