Core Capabilities - Bindley Bioscience Center - Purdue University

Core Capabilities

Confocal Microscopy

Most suitable for creating 3D images of thicker (1-50 um) living or fixed samples labeled with multiple fluorophores. Non-fluorescent samples can also be visualized if they are reflective. Both of our Nikon confocals are equipped with the hybrid scanner (both galvanometer and resonant). Simultaneous photo-activation and ultrafast imaging using these two scanners allow acquisition of rapid changes after photo-activation and enables observation of intermolecular interaction. Dynamic intercellular events can be captured at video frame rates (30FPS) or faster. The resonant scanner can also be used to scan the samples quickly to find the area and focus of interest. The galvanometer scanner is typically used for the high resolution and high quality imaging which typically takes 1 sec to acquire. One of our Nikon confocals is equipped with the Spectral Imaging module for fast (approximately 1.0 sec) 32-channel acquisition.

Multiphoton Microscopy

Most suitable for creating 3D images of thick (50-500 um) living or fixed samples labeled with one or two fluorophores (blue/green). Our MP confocal system is equipped with the hybrid scanners making it suitable for both fixed (galvanometer) and living (resonant) samples. Multiphoton can also be useful for photo-uncaging, photo-polymerization and other processes that require high-power, short wavelength (350 nm) light.

Super Resolution

Super resolution microscopy is any light microscopy technique that can exceed diffraction-limited resolution (i.e., achieve a resolution <200 nm). Our super resolution system can perform two types of super resolution microscopy: Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM). Although both techniques use the same microscope, their operation is entirely different. STORM requires special fluorescent dyes and can achieve precision as high as 20 nm, typically on samples within ~10 um of the coverslip. SIM is compatible with all fluorescent dyes (within the blue, green and red channels) and can image up to 10 um deep, with resolution improvement at twice the diffraction limit. SIM also offers relatively quick, less than 1 sec, making it suitable for short live cell time lapse experiments. High-speed, multi-color TIRF is also possible.

Traditional Wide-field Modalities

Including bright-field, phase-contrast, differential interference contrast, polarized light (non-quantitative) and epifluorescence.

Sample Preparation

Methods for preparing biological samples for high-resolution microscopic observation are almost as diverse as the samples themselves. Please follow the manufactures protocol for staining technique. An very good reference to start sample preparations would be the reference; Epi-Fluorescence Microscopy. Webb DJ, Brown CM. Methods in molecular biology (Clifton, N.J.). 2013; 931: 29-59. The following are some general considerations that are broadly applicable to many kinds of samples. Please always use a high quality #1.5 coverslip over your specimen. Also, consider using an anti-fade product to reduce photo-bleaching. They typically can be purchased from a vendor, such as Thermofisher, that act as both a mounting medium and anti-fade reagent. We do not always recommend these anti-fade reagents when supplied with the UV dye. For Super Resolution imaging, please contact the core director for more sample-specific advice.

Image Processing

Images may be analyzed qualitatively (e.g., by looking at them) or quantitatively. In either case, some post-processing is almost always desirable prior to analysis. Of course, post-processing alters the raw data, so the user must understand (and describe in a methods section!) all of the processing steps that were performed.


Andy Schaber
Imaging Facility Director
Phone: (765) 496-3148

Purdue University, 610 Purdue Mall, West Lafayette, IN 47907, (765) 494-4600

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