The Microscopy Laboratory exists to assist the researcher in generating high-resolution, high quality, microscopy-based data for publications and presentation at professional venues. A range of services is available for both experienced and inexperienced users.
Experienced users may use the lab's instruments after orientation by a staff member. Inexperienced users may choose to receive training in the use of the instruments, technical support in microscopy and image analysis, consultation in experimental design, or have us perform the microscopy for them as a service. The microscopy lab provides equipment, consultation, training, or labor for the following:
- Transmission Electron Microscopy (JEOL JEM-1230, digital & film)
- Confocal Microscopy (Zeiss LSM 510, upright & inverted platforms)
- Widefield Light Microscopy (multiple stations, upright & inverted platforms, digital [ORCA-ER, Spot2] & film)
- Live Cell Microscopy (FRAP, time-lapse, GFP imaging)
- Image Analysis & Output (Metamorph, deconvolution, 3-D, Photoshop, printers)
- Digital Macroscopic Imaging (transgenic animals, organs, 2-D gels, digitizing EM negatives)
All instruments and computers are located on the 3rd floor in the Vontz Center for Molecular Genetics.
The electron microscope is part of the department of pathology, located at 1204 MSB. The laboratory is equipped with a JEOL JEM-1230 transmission electron microscope. Transmission Electron Microscopy allows for high resolution ultrastructural analysis of ultrathin sections of biological, particle, and materials specimens.
The TEM is equipped with a AMT Advantage Plus 2K x 2K digital camera and software for digital image acquisition.Download User Guide (PDF)
The light microscopy facility is located in the Vontz Center for Molecular Studies Room 3403. The following are the services offered:
The most common reason to use a confocal is to obtain optical sections that have much less out-of-focus blur than images from widefield instruments. In addition, one can acquire a 3-D data set for volume determination or 3-D reconstruction.
Multi-tracking provides considerable improvement in the separation of similar dyes over that of a widefield microscope. Therefore, even if you do not need the optical sectioning ability of the confocal microscope, you may want to use it instead of a widefield microscope in order to ensure separation of dye pairs like FITC and rhodamine. Another reason to chose a confocal over a widefield microscope is to have precision in the overlay of images that are taken with different filters.
We currently maintain a LSM710 with an inverted microscope and aZeiss LSM 510 with an upright microscope. Only the LSM 710 can visualize near UV and therefore DAPI.
- LSM 510: A laser scanning confocal microscope for imaging up to three dyes plus a transmitted, DIC image. The LSM 510 includes 3-D software, multi-tracking, separate pinholes for each channel, and exquisite control over the region of interest (ROI). The available laser lines are 458, 488, 514, 543, 633 nm. This confocal does not have a UV laser, so contact us for alternatives to DAPI. Download User Guide (PDF)
- Zeiss LSM 710: A Zeiss Axio Observer Z1 inverted microscope is connected to a Zeiss LSM710 confocal. The available laser lines are 405, 458, 488, 514, 561 and 633nm. With the availability of a near UV laser this confocal can also visualize DAPI. In addition to the confocal images a DIC image can be acquired. Stage and objective heaters are available to aid live cell imaging. Download User Guide (PDF)
Widefield microscopy is conventional microscopy using either transmitted light or fluorescent light. We currently maintain a Nikon FX microscope with a Spot-camera, and an inverted Zeiss M100 with an Orca-ER camera for widefield microscopy. For users from the Department of Cancer and Cell Biology a Zeiss Axioplan Microscope with a Axiocam MRM (for fluorescence) and an Axiocam MRc5 (for brightfield microscopy) are available in addition.
- OrcaER Zeiss: A Zeiss Axiovert 100M infinity-corrected, inverted scope with DIC and epifluorescence is coupled to an Orca-ER cooled, B&W CCD camera. This system is more sensitive for fluorescence, has better optics, and is more automated than our other widefield scopes. It also has the unique (among our widefield scopes) ability to do automated Z-stacks and differential interference contrast (DIC) microscopy. Download User Guide (PDF)
- Axiovert100TV: This inverted scope is equipped for phase, brightfield, and fluorescence. The filter cubes are suitable for fluoresceine (GFP), rhodamine, and DAPI like dyes. The available objectives are 1.25x, 5x, 10x and a 32x long working distance objective. Additional objectives are available upon request. The filters and optics on this scope are not as advanced as the orcaerzeiss but it is useful for e.g. checking on transfection efficiency. A Zeiss Axiocam MRm is connected connected to the microscope and provides as resolution of up to 1388 x 1040 pixels. The camera is suitable for fluorescence imaging and provides only B&W images.
- Spotcam: A SpotII, cooled (color or B&W) CCD camera is coupled to a Nikon upright scope that is set up for epifluorescence, darkfield, and brightfield. Download User Guide for Brightfield (PDF) | Download User Guide for Fluorescence (PDF)
- Zeiss Axioplan with Axiocams: A Zeiss Axioplan Imaging 2e infinity-corrected upright scope with DIC and epifluorescence. Uses either a Color Zeiss Axiocam (for brightfield imaging) or a B&W Zeiss Axiocam for fluorescence. It also has an Apotome which provides structured illumination which allows subtraction of out-of-focus light. For use of members of the UC Department of Cancer Biology only. Download User Guide for Fluorescence (PDF) | Download Use Guide for Brightfield (PDF)
Digital cameras for conventional digital photography to macrophotography are available. We have a NikonD1X which can be used in combination with a microscope, a dissection microscope and a photostand. In additon a Canon PhotoPC 3000z and a Canon Powershot A640 are available.
We also offer help and advice with image processing questions.
Image J is a very useful free image processing and image analysis program, which has originally been developed by the NIH. It is based on Java and can get extended functions by additional Java-plugins. ImageJ can be be run on any platform. We recommend to download imageJ either from the Biophotonics Facility at McMaster University which maintains a manual as well as a version with quite a number of collated plugins. Another source of imageJ with a bundle of plugins is FIJI. An useful source for help with ImageJ is also Wiki-ImageJ.
Metamorph (Molecular Devices) is an industrial-strength image analysis program. Metamorph is located on the Analysiscpu system. Measurement is eased with journals (macros) that automate repetitive functions and by automatic transfer of measurements into an Excel spreadsheet. A few of Metamorph's abilities are Cell Counting, Morphometry, Colocalization and Quantitation of Intensity
Photoshop for MicroscopistsOne of the most common needs of the microscopist is to make a montage of multiple images. The goal of this tutorial is to teach the user how to make a montage of three images using Adobe Photoshop 7.0. The tutorial explains how to adjust intensity, resolution, and size. It explains how to crop, make scale bars, and add labels.Download the Tutorial (PDF)
This tutorial uses three images (green.tif, red.tif, and combo.tif) that can be found below. To download the files, right mouse click on each file and select "Save Target As." The images were acquired on a confocal microscope by Bob Hennigan. The samples were prepared by Robert Brackenbury and Amy Koshoffer.
Currently you can use 3D for LSM, ImageJ or Metamorph for Visualization of 3D structures.
- LSM Viewer (V.3.5.0)/LSM510 (V.3.2)/3D for LSM (V.1.4.2) - The LSM Image Browser can be downloaded for free and allows maximum intensity projection, slicing of stacks and also some measurements. A 3D visualization consisting of maximum intensity projections from different angles can be done using the 3D module in the LSM510 program (no free rotation). 3D rendering using the 3D for LSM program (both located on the confocal 510 system in Vontz 3403B) does something similar, but uses a gradient shading method. The options for adjustments are limited and side views can pixelate strongly.
Maximum intensity projection is used for fluorescent images, for brightfield images one would typically use the minimum intensity. The algorithm goes through a stack of images. For each pixel it compares its brightness in each of the images and stores the brightest value. These values get displayed in the final image. It has the function of an extended focus. In Photoshop maximum projection images can be generated by loading each source image into a different layer and using the "lighten" function instead of the "normal" function to show the different layers.
What are orthoslices? If images are recorded in different focal planes with a constant difference between the focal planes, the pixels of each image can be turned easily into a voxels The depth of each voxel corresponds to the distance in focus plane. The resulting 3D image can be viewed from different angles. Usually the ones orthogonal to the original view axis are chosen.
- Metamorph (v.6.3) - an image analysis program by Molecular Devices. It can load lsm files directly. It is mainly used for measuring a variety of image parameters. It's 3D reconstruction module, however, does not allow much flexibility, though the basic functions are available. No free rotation, 3D visualization by calculating projection views from different angles.
- ImageJ - a Java based free program. For 3D capabilities extra plugins are required. A plugin for reading lsm files is available too, so lsm files can get loaded directly. Different channels get loaded separately and have to be merged again. Views from different angles are calculated and assembled into an image stack (no free rotation). Image from some angles clearly pixelated.
- Amira - commercially available program (academic version ca. $3000, 2005). Can load lsm files directly. All visualizations can be freely rotated. Surface models can be constructed after image segmentation. Surface models can be assigned transparency values. Smoothing algorithms and interpolation available.Tutorial before first use necessary. One month trial version available.
- Analyze - commercially available program (academic version ca. $5000, 2005). Can load TIF files which contain stacks of images, but not lsm files. Possible to load and assemble stack from individual TIF files. Surfaces are not available directly from RGB images. It seems to be optimized for the evaluation and representation of grayscale images like tomographic images. Multiple measuring functions in addition to visualization. Tutorial use necessary, not very intuitive. One month trial licence available.
- BioimageXD - free open source software. Still beta version.