Cornell University

Williamsville East High School

State University of New York

Auto-fluorescent spectra of leaf and pollen grain

— Cheng, WY; Cheng, VC; Cheng, P-c

In confocal and multi-photon microscopy, the intense auto-fluorescent associated with a specimen frequently hinders the identification of labeled fluorescent probe(s) and fluorescent proteins (e.g. GFP). The auto-fluorescent spectrum varies as the excitation wavelength changes; therefore, careful selection of excitation wavelength and the use of spectral imaging (available with number of modern confocal microscopes) can help the identification of fluorescent-probe-labeled structure(s).

As a reference to those using confocal and multi-photon fluorescent microscopy to select suitable excitation and detection wavelengths as well as detecting bandwidth, we have measured the auto-fluorescent spectra from the leaf of maize seedlings (Golden Beauty hybrid sweet corn, 1 week after emergence) (Figure 1) and pollen grain (Ohio 43) (Figure 2). The measurements were taken at excitation levels of 350nm, 380nm, 400nm, 448nm and 514nm. The shorter wavelength measurements may be used as a reference for multi-photon excitation (700–800nm of NIR from Ti-sapphire ultrafast laser). However, it is important to point out that the two-photon excited auto-fluorescent spectrum may be substantially different from the single-photon excited counterpart presented in this article (Cheng et al., (2001): Micron, 32, 661–669).

It is noted that, in leaf, both the spectrum and intensity of the auto-fluorescent may change according to illumination intensity as the plant adjusts its physiology; in addition, an increase in fluorescent intensity is frequently observed at the initial scanning in a confocal microscopy study as the specimen is exposed to high-intensity illumination. Subsequent decrease in fluorescent intensity is evident as a result of photo-induced damages.

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