Surgical oncology is guided by examining pathology that is prepared during or after surgery. The preparation time for Mohs surgery in skin is 20-45 minutes, for head-and-neck and breast cancer surgery is hours to days. Often this results in incomplete tumor removal such that positive margins remain. However, high resolution images of excised tissue taken within few minutes can provide a way to assess the margins for residual tumor. Current high resolution imaging methods such as confocal microscopy are limited to small fields of view and require assembling a mosaic of images in two dimensions (2D) to cover a large area, which requires long acquisition times and produces artifacts. To overcome this limitation we developed a confocal microscope that scans strips of images with high aspect ratios and stitches the acquired strip-images in one dimension (1D). Our “Strip Scanner” can image a 10 x 10 mm<sup>2</sup> area of excised tissue with sub-cellular detail in about one minute. The strip scanner was tested on 17 Mohs excisions and the mosaics were read by a Mohs surgeon blinded to the pathology. After this initial trial, we built a mobile strip scanner that can be moved into different surgical settings. A tissue fixture capable of scanning up to 6 x 6 cm<sup>2</sup> of tissue was also built. Freshly excised breast and head-and-neck tissues were imaged in the pathology lab. The strip-images were registered and displayed simultaneously with image acquisition resulting in large, high-resolution confocal mosaics of fresh surgical tissue in a clinical setting.
Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm 2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called “strip mosaicing,” which was demonstrated on a 10-×-10 mm 2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm 2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm 2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.
Confocal mosaicing microscopy is a developing technology platform for imaging tumor
margins directly in fresh tissue, without the processing that is required for conventional
pathology. Previously, basal cell carcinoma margins were detected by mosaicing of
confocal images of 12 x 12 mm<sup>2</sup> of excised tissue from Mohs surgery. This mosaicing
took 9 minutes. Recently we reported the initial feasibility of a faster approach called
"strip mosaicing" on 10 x 10 mm<sup>2</sup> of tissue that was demonstrated in 3 minutes. In this
paper we report further advances in instrumentation and software. Rapid mosaicing of
confocal images on large areas of fresh tissue potentially offers a means to perform
pathology at the bedside. Thus, strip mosaicing confocal microscopy may serve as an
adjunct to pathology for imaging tumor margins to guide surgery.
Minimally invasive surgical (MIS) techniques, such as laparoscopic surgery and endoscopy, provide reliable disease
control with reduced impact on the function of the diseased organ. Surgical lasers can ablate, cut and excise tissue while
sealing small blood vessels minimizing bleeding and risk of lymphatic metastases from tumors. Lasers with wavelengths
in the IR are readily absorbed by water causing minimal thermal damage to adjacent tissue, ideal for surgery near critical
MIS techniques have largely been unable to adopt the use of lasers partly due to the difficulty in bringing the laser into
the endoscopic cavity. Hollow waveguide fibers have been adapted to bring surgical lasers to endoscopy. However, they
deliver a beam that diverges rapidly and requires careful manipulation of the fiber tip relative to the target. Thus, the
principal obstacle for surgical lasers in MIS procedures has been a lack of effective control instruments to manipulate the
laser in the body cavity and accurately deliver it to the targeted tissue.
To overcome this limitation, we have designed and built an endoscopic laser system that incorporates a miniature dual
wedge beam steering device, a video camera, and the control system for remote and /or robotic operation. The dual
wedge Risley device offers the smallest profile possible for endoscopic use. Clinical specifications and design
considerations will be presented together with descriptions of the device and the development of its control system.
Imaging large areas of tissue rapidly and with high resolution may enable rapid pathology at the bedside. The limited field of view of high-resolution microscopes requires the merging of multiple images that are taken sequentially to cover a large area. This merging or mosaicing of images requires long acquisition and processing times, and produces artifacts. To reduce both time and artifacts, we developed a mosaicing method on a confocal microscope that images morphology in large areas of excised tissue with sub-cellular detail. By acquiring image strips with aspect ratios of 10:1 and higher (instead of the standard ∼1:1) and "stitching" them in software, our method images 10×10 mm2 area of tissue in about 3 min. This method, which we call "strip mosaicing," is currently three times as fast as our previous method.
One-dimensional linear detector arrays have been used in the development of microscopes. Our confocal line
scanning microscope electronics incorporate two printed circuit boards: control board and detector board. This
architecture separates control electronics from detection electronics allowing us to minimize the footprint at
microscope detector head. The Field Programmable Gate array (FPGA) on the control board generates timing and
synchronization signals to three systems: detector board, frame grabber and galvanometric mirror scanner.
The detector is kept away from its control electronics, and the clock and control signals are sent over a differential
twisted-pair cable. These differential signals are translated to single ended signals and forwarded to the detector at the
microscope detector head. The synchronization signals for the frame grabber are sent over a shielded cable. The
control board also generates a saw tooth analog ramp to drive the galvanometric mirror scanner. The analog video
output of the detector is fed into an operational amplifier where the white and the black levels are adjusted. Finally the
analog video is send to the frame grabber via a shielded cable.
FPGA-based electronics offer an inexpensive convenient means to control and synchronize simple line-scanning
Intravital microscopy of cancer is a well established tool that provides direct visualization of the tumor cycle. It traditionally involves one of several strategies: invasive subcutaneous (SC) implantation of tumors followed by surgical opening of skin flaps for imaging, techniques utilizing skin fold chambers and implanted optical windows or intradermal injections under 200μm from the skin surface.
All of these techniques allow the use of fluorescent proteins as markers for biologically significant constituents. However, observation methods utilizing skin-flaps, skin-fold chambers and optical windows are invasive and tend to alter the immune environment of the tissue and/or limit the duration of studies that can be performed. If implanted correctly, intradermally injected tumors can be minimally invasive, will not require biopsies or surgical intervention to observe and are accessible for direct transdermal imaging with a number of in vivo modalities.
We present our work in the development of a small animal intravital microscopy workstation that allows the acquisition of different contrast imaging modalities: reflectance confocal, wide field epifluorescence, multiphoton and second harmonic generation (SHG). The images are acquired pair-wise simultaneously and sequentially in time. The aim of our instrumentation is to gather all information generated by the single probing beam via the reflected or back-scattered signal, SHG signal and various fluorescence signals.
Additionally, we also present our development of a microscopic tissue navigation technique to mark, label and track sites of interest. This technique enables us to revisit sites periodically and record, with different imaging contrasts, their biological changes over time.
A new generation of confocal laser microscope, designed to image the human skin in vivo, improves the resolution, contrast and spectroscopic facilities as compared to the previous Tandem Scanning Microscope (TSM) prototype. The new device has been built with an Oz module (Noran) equipped with the skin contact device, assuming a perfect stability of skin images in the horizontal plane. The Z displacement of the objective lens, mounted directly on the Oz module, is assumed by a piezo motor with a course of 350 micrometer. Moreover, the Oz module has been suspended on articulated arms to reach any part of the human body. The power of the Argon/Krypton laser source has been limited to 2.mW to secure safety and provides three visible wavelength: 488, 568, and 647 nm. The facility of instantly checking wavelength during in depth exploration of the skin optimizes the resolution and contrast of images as compared with the white light used in the TSM. Consequently, better image quality of the epidermis is obtained in the blue region with unexpected details of corneocytes and keratinocytes. The papillary dermis comprising the vascular network is advantageously observed with the red light. The fluorescence channel detector gives additional information on the penetration of fluorescent probes through the skin barrier. Optical sections are digitized (512 X 480 X 8 bit) at video rate, providing easy and fast measurements of the thickness of epidermal layers. The Silicon Graphics workstation generates a transparent volume of living human skin in less than 5 minutes. This powerful and convivial new design for imaging the in vivo human skin opens up new promises in skin research and in vivo skin pharmacology.
Near field scanning optical microscopy of thin birefringent samples is described. The system utilized is the linear polarizing near field microscope, resulting in a pure birefringence image of the sample. The sign of the birefringence is also preserved. Two specific classes of sample are studied. These include thin sections of Kevlar fibers, and polymer dispersed liquid crystals. Results are correlated with simultaneously obtained topographic images. Based on experimental observations, the relative strength of the optical indices of the structures is determined
Images of a microlithographic sample obtained using a new near field scanning optical microscope (NSOM) that uses force regulation of the sample-tip separation are presented. The NSOM is a research instrument fitted with a metal covered glass tip probe that defines a small aperture at the sharp end. The aperture is estimated to be on the order of 100 nanometers in diameter resulting in a resolution exceeding that of diffraction limited systems. This form of microscopy can be done both in the transmission and the reflection modes. The force regulation mechanism produces a simultaneously obtained scanned force microscope image of the topography thus permitting correlative imaging of the sample. The samples are imaged in transmission and reflection near field optical format, with white light and with coherent light. The results are compared with other forms of IC imaging and characterization, namely scanned force microscopy and scanning electron microscopy.
A novel atomic force microscope (AFM) is used to image a microlithographic sample. The AFM operates in the non destructive non-contact mode, uses glass tips as opposed to tungsten or silicon, and has an optical interferometric detection system. Its estimated lateral resolution is under 10 nanometers and much better in the z direction. A sample consisting of chrome features on quartz was produced for measurements using AFM and electric probe techniques. The features are single and grouped lines on the order of 1 micrometers incorporated into an electric probe pad layout. Dimensions of these features are determined from the AFM images by relating their sizes in pixels to the excursions of the scanners during the formation of the images. These results are compared with measurements obtained through electric probing techniques.
Near field scanning optical microscopy (NSOM) provides a number of unique capabilities for high resolution imaging. In this regard, a fundamental aspect of the technique is its ability to retain much of the characteristics available in diffraction limited optical probing. Results are presented on the use of near field scanning optical microscopy (NSOM) in imaging a variety of samples, using different contrast mechanisms. The approaches adopted are based on the recently introduced simultaneous, non-contact, near field optical microscope with atomic force regulation. Amongst the techniques discussed are linearized polarizing microscopy, as well as amplitude, and phase, interference contrast imaging modalities.
KEYWORDS: Near field scanning optical microscopy, Near field optics, Atomic force microscopy, Scanning probe microscopy, Blood, Optical microscopes, Microscopes, Thin films, Integrated circuits, Microscopy
The design and theory of operation of a new form of near field scanning optical microscope are presented. In this system, the tip/sample distance regulation is achieved in a feedback system utilizing the topography information derived from the attractive force sensed between the tip and the sample. The technique affords the possibility of correlative microscopy. Results are presented on imaging blood smears and thin film integrated circuits.