Beth Raff-fly.jpg
Microscopy Facilities
Included in this facility are both spinning disk and scanning confocal microscopes, a widefield microscope with deconvolution, widefield fluorescence microscopes without deconvolution, and transmission and scanning electron microscopes. This lab is supervised by Barry Stein.
Phone: (812) 855-7424
Fax: (812) 855-6082
Email: bstein@indiana.edu
Spinning disk confocal microscope (Yokogawa CSU10/Nikon TE 2000)
This spinning disk confocal fluorescence microscope is based on a Yokogawa CSU 10 Nipkow spinning disk that is used with a Nikon TE 2000 inverted microscope. The spinning disk confocal is capable of acquiring images at a rate of up to 300 frames per second, much faster than the usual laser scanning confocals. This makes possible the imaging of the movement of organelles and molecules inside living cells with high temporal as well as spatial resolution. A krypton-argon laser is used to excite fluorescence at wavelengths of 488 nm, 568 nm, and 647 nm. Images are acquired with a Cascade II 512B digital camera that is highly sensitive in both the visible and near infrared spectrums. The entire system is driven by Metamorph software. In conjunction with the spinning disk confocal is a Photonics Insutruments, Inc. MOSAIC digital diaphram system with a 488nm Argon laser. The Mosiac system allows for photoactivation or photobleaching of precise regions of living specimens.
Scanning Confocal Microscope (Biorad MRC600/Nikon Optiphot)
The BioRad laser scanning confocal fluorescence microscope is capable of generating much higher resolution than standard fluorescence microscopy. The MRC600 can create high resolution images at discrete successive depths in a thick specimen, avoiding the muddling effect of “out of focus information” from the rest of the specimen. The image analysis computer can then combine the successive two-dimensional images into a clear three-dimensional image that can be rotated and viewed from any angle. A krypton-argon laser is used to excite fluorescence at wavelengths of 488 nm, 568 nm, and 647 nm.
Transmission Electron Microscope (JEOL JEM1010)
The microscopy facility features a new JEOL JEM-1010 transmission electron microscope with a magnification range of x50 – x500K and an accelerating voltage range of 40KV – 100KV. This electron microscope provides an excellent means for t he ultrastructural analysis of biological samples, but it is also capable of selected area electron diffraction. Other features include a tilting goniometer sample stage for electron diffraction and 3-D imaging, a minimum exposure operation mode to preven t electron beam damage to fragile samples and a measurement function for the quantification of lengths and areas of structures within samples. The computer aided – menu driven operation makes it extremely user friendly. The facility also provides a staff EM technician who is responsible for maintaining the facility and for providing technical support and training for users.
Scanning Electron Microscope (JEOL JSM-5800LV)
The JSM-5800LV will operate at either high or at low vacuum allowing for the observation of samples with a high water content or low conductivity. The magnification range is from 18x to 300,000x. Resolution is 3.5nm at high vacuum and 5.5nm in low vacuum mode. Samples up to 8 inches in diameter can be viewed. Images can be recorded by 4×5 inch Polaroid or regular film, or digitized. Accelerating voltages of 0.3 to 30 KV are available.
Deconvolution Microscope
The Applied Precision DeltaVision Restoration Microscopy System for deconvolution microscopy is a wide-field microscope platform in which the light from a sample is collected from serial focal planes using a CCD camera. Deconvolution is a software technique that mathematically de-blurs images. Our current instrument is on an optimized Nikon TE-200 inverted microscope with a piezo electric Z-motor on the objective and a motorized stage. The sample is illuminated with a Hg arc lamp with filters for DAPI/UV, Green, Red, Far Red and Chlorophyll. This system is ideal for collecting high resolution images of fixed specimens.
Nikon E800 Epifluorescence Light Microscope with Metamorph Software
The Nikon E800 light microscope has Nomarski Differential Interference Contrast optics (DIC) to enhance contrast of samples. Fluoresence microscopy can be done with DAPI , FITC, TRITC, and Cy5 epifluorescence filter cubes or, for colocalization, a filter wheel that has the excitation filters for these labels and for chlorophyll. Images are acquired by a Hamamatsu Orca ER digital CCD camera controlled by MetaMorph sofware. MetaMorph also enables colocalization studies, morphometric analyses, and 2D deconvolution. Macros can be written to automate image acquisition as well as image analysis.
Sample Preparation: Leica Ultracut T Ultramicrotome, Sorvall Porter-Blum MT 2B, Denton DV502 Vacuum Evaporator, Balzers Critical Point Dryer, Polaron Sputter Coater, and all other standard prep. equipment.