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


focused on the bevel of the diamond knife. A high-speed line-scan time-delay integration (TDI) camera (Dalsa Teledyne Piranha HS80-08k80) is coupled with the objective lens to capture line-scanned images of the tissue as it moves over the bevel of the knife. T e tissue is mounted inside the water bath and is moved across the bevel of the stationary knife by the motion of the high-precision mechanical air-bearing Aerotech stage ( XY precision = ±250 nm; Z precision = ±300 nm). T e stage moves along the X (vertical) axis to cut 2 mm-wide sections of tissue as thin as 0.5 µm. For tissue blocks wider than 2 mm, the KESM moves along the Y axis to take slices that are later stitched together using soſt ware to create a full face. Aſt er each face has been sectioned, the Z stage moves the tissue up at a fi xed slice thickness to start sectioning the next face. KESM control . An operator controls the KESM by logging into a web interface in a browser window on a laptop or tablet ( Figure 4 ). T e user interface allows the operator to set the physical cutting space in three dimensions ( XYZ ) (panel 1), depending on the size of the tissue block, and also allows the


Figure 2 : Perfusion rig. A homemade pressure system was used to perfuse the mice with a colloidal solution of carbon black, which renders their vasculature as black on a white tissue background. The whole system is pressurized to 300 mmHg. Mice are cleared with phosphate buffered saline (PBS) and fi xed with formalin; then the injection port is used to inject 10 ml of India ink into the vascular system.


fi lling of the vessels and capillary beds with the 10 mL colloidal solution of carbon (India ink) that follows. Organs of interest are dissected from the animal and leſt in formalin to further fi x overnight. T e following day tissue samples are processed and embedded in LR white resin; briefl y, the tissues are dehydrated to 100% ethanol and then subjected to increasing concentrations of LR white to 100% resin. Tissue and resin are then polymerized in a 65C oven for 24 hours. T e resulting block of resin and tissue is secured onto a custom sled for our microscope.


Sample imaging . T e KESM was designed to combine a microtome and a microscope into a single tool. T e KESM comprises three major components ( Figure 3A ): (1) the knife arm assembly, (2) the optics train and camera, and (3) the stage and water bath. T e knife arm assembly and optics train remain stationary during imaging. T e key feature of the KESM is the diamond knife arm assembly ( Figure 3B ), which functions as both a sectioning tool and the illumination source for the microscope. T e removable diamond knife is mounted on a fi xed knife arm with a channel running above and below. T e channel on the underside of the knife arm contains a fi ber optic cable that connects the LED light source to the back of the diamond knife, providing illumination that is transmitted through the tissue section to the line-scanning camera. T e channel above the knife arm creates a closed-loop water channel that pulls the tissue section away from the objective lens as the tissue is being sliced and directs the sectioned piece into a fi lter to be discarded.


Perpendicular to the knife arm assembly is the optics train with a 10× water-immersion objective (Zeiss 10 × /0.3 NA achromat)


2017 July • www.microscopy-today.com


Figure 3 : Overview of KESM mechanical components. (A) The knife-edge scanning microscope consists of (1) the knife arm assembly, (2) the optics train and sensor, and (3) the water bath and stages. (B) Schematic view of the (1) diamond knife, (2) the objective lens, (3) suction channel, (4) fi ber optic cable, (5) embedded tissue, and (6) precision XYZ stage.


15


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