search.noResults

search.searching

note.createNoteMessage

search.noResults

search.searching

orderForm.title

orderForm.productCode
orderForm.description
orderForm.quantity
orderForm.itemPrice
orderForm.price
orderForm.totalPrice
orderForm.deliveryDetails.billingAddress
orderForm.deliveryDetails.deliveryAddress
orderForm.noItems
Knife-Edge Scanning Microscopy for Bright-Field Multi-Cubic Centimeter Analysis of Microvasculature


Michael J. Pesavento , Caroline Miller* , Katy Pelton , Molly Maloof , Corey E. Monteith ,


Venkata Vemuri , and Megan Klimen 3Scan, Inc. , 2122 Bryant St. , San Francisco , CA 94110 * mills@3scan.com


Abstract: Serial section microscopy has proven to be a powerful tool in examining the 3-dimensional (3D) nature of tissue; however, it is limited by the size of tissue samples that can be used and the manual process of acquiring data. The knife-edge scanning microscope (KESM) acquisition and analysis computational system resolves these issues by providing high-resolution light microscopy image data at multi-cubic centimeter scale. Using this system for quantifi cation of microvasculature provides the ability to examine, measure, and compare the 3D nature of vascular networks at whole organ scale and sub-micron resolution.


Introduction


Modern light microscopy has benefi ted from the use of serial section microscopy such as in the Allen Brain Atlas and the creation of new rodent models for Alzheimer’s disease research [ 1 , 2 ]. Typically, slides are prepared in the conven- tional manner, where images are collected using traditional light microscopy and digitally scanned. T e resulting images then must be computationally registered to one another to create a 3D image. However, numerous issues have inhibited widespread adoption of this method. T ese include the manual nature of slide preparation and staining, the introduction of artifacts from manual sectioning and mounting on slides, the time taken for whole-slide scanning an entire sample, the diffi cult nature of image registration and data handling, and the simple fact that it results in too much data for modern infrastructures to handle. T e knife-edge scanning microscope (KESM) combines sectioning and imaging into a single step, automates a large part of the traditional pathology workfl ow, and allows for a greater speed, precision, throughput, and scale at which tissues are digitized ( Figure 1 ). T e KESM achieves tissue data collection at a resolution of submicron pixels with a maximum sample volume of over 100 cm 3 , exceeding the depth off ered by confocal or 2-photon microscopy. To handle high-resolution data of over a terabyte per cm 3 , sophisticated data processing soſt ware is applied to model 3D tissue reconstructions, provide interactive image views, and apply quantitative analytics. T is allows quantitative analysis to be performed on 3D image stacks of whole mouse organs, which is extremely diffi cult, expensive, and time-consuming with traditional manual techniques. T e KESM was fi rst developed at the Brain Network Laboratory at Texas A&M as a neuroscience tool


14


to examine brain microvasculature and neuronal microcir- cuitry in 3D [ 3 ]. T e KESM technology provides extremely high-throughput processing, sectioning, and 3D image generation of samples. T is has attracted signifi cant interest from the research community, the drug development community, and academic pathologists who are looking for new methods to bring histopathology into the digital age. Multiple imaging modalities of KESM (mono- and tri-chromatic bright-fi eld plus multi-channel fl uorescence) are in development to bring this technology to a broader range of scientists, including clinical pathologists. T is article explains the design of the microscope, tissue preparation, how the KESM works, soſt ware reconstruction, some examples of data outputs, an application to mouse brain tissue, and the signifi cance of this novel imaging technique.


Materials and Methods Preparation of the tissue block . Mouse clearing, fi xation, and India ink perfusion are performed as follows. T e mouse is anesthetized using isofl urane and tested for toe pinch response prior to the beginning of the non-survivable surgical technique. A blunted 18-gauge needle is placed into the leſt ventricle of the heart, and the right atria is snipped to allow return fl ow. Mice are perfused at a pressure of 300 mmHg using a custom pressurized perfusion rig ( Figure 2 ). We have found that perfusing at high pressure ensures more complete staining of the microvascu- lature compared to lower-pressure perfusion techniques. T e mouse is fi rst perfused with phosphate buff ered saline (PBS) until the blood runs clear. Following clearing, the mice are perfused with 200 mL formalin; this long formalin perfusion provides excellent fi xation of the tissues and allows time for the vasculature to fi x in an “open” position. T is ensures complete


Figure 1 : Rethinking imaging workfl ow. (A) Current tissue imaging pathway, where tissue is embedded, sectioned, stained, and imaged, and each image must then be registered to one another. (B) KESM automation of this process to produce 3D stacks of images that only require concatenation.


doi: 10.1017/S1551929517000645 www.microscopy-today.com • 2017 July


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76