Imaging, Staining, and Markers

Abstract

This chapter overviews the main principles and devices used to image cells and tissues – the concepts students must know to accomplish their team projects. The main principles behind light absorption and fluorescence, emission/excitation spectra, use of bandpass filters, and selection of target-specific dyes are explained and briefly discussed. This is followed by an explanation of different types of microscopes that are most commonly used in tissue engineering, including compound, confocal, transmission, and scanning electron-based microscopes. Lastly, we discuss a few technical aspects of the imaging process, including proper positioning of live and fixed samples, dye aliquoting, and Mowiol fixation.

Self-Check Questions

• Q.7.1. You stained cell samples with mitochondria-specific dye and want to observe individual organelles, which are approximately 1–2 micron in size. The most appropriate objectives will be

  • A. 2×

  • B. 10×

  • C. 20×

  • D. 100×

• Q.7.2. You are trying to get a single image of a 2 × 2 mm piece of engineered tissue. The most appropriate objectives will be

  • A. 2×

  • B. 10×

  • C. 20×

  • D. 100×

• Q.7.3. Use online spectra finder to determine if a single excitation source can be used to image sample co-stained with _____ antibody and a nuclear stain ___.

  • A. FITC & TO-PRO-3

  • B. FITC & 7-AAD

  • C. TRITC & TO-PRO-3

  • D. TRITC & 7-AAD

• Q.7.4. Based on 7-AAD spectra, the following set of filters (excitation/dichroic/emission) should be suitable to image samples stained with it:

  • A. 480 nm short-pass, 500 nm, 600–650 nm band-pass

  • B. 540–570 nm, 600 nm, 620 nm long-pass

  • C. 480 nm long-pass, 650 nm, 600–650 nm band-pass

  • D. 500 nm long-pass, 550 nm, 600 nm short-pass

• Q.7.5. Choose the correct statement.

  • A. A confocal microscope uses a beam of electrons to create crisp images of tissue at different depths.

  • B. Mowiol aliquots cannot be stored and must be prepared fresh.

  • C. The use of acetoxymethyl ester AM form of the dye enables to store stained cells for at least a month at room temperature.

  • D. The surface of a sample to be observed using a 63× oil objective with ∞/0.17 marking must be less than 170 microns away from the objective lens.