Fluorescence Microscopy

Collagen orientation patterns in human secondary osteons, quantified in the radial direction by confocal microscopy

“…Confocal microscopy of the lamellar specimens employs bone's endogenous Xuorescence, also called primary or auto-Xuorescence (Price and Schwartz, 1956). Many biological materials exhibit auto-Xuorescence, the property of atoms and molecules to absorb light at particular wavelengths and after a brief interval subsequently to emit light of wavelength longer than the excitation (or absorption) wavelength (Allen, 1925).…”

“…Unlike the usual narrow emission spectrum of Xuorescent markers, auto-Xuorescence spectra are generally broad, extending over several hundred nanometers (Billinton and Knight, 2001). Because collagen type I, which represents 95% of bone's protein content (Prentice, 1967;Price and Schwartz, 1956), is reported in ligaments and tendons to show a Xuorescent spectrum that ranges from 300 to 700 nm (see for instance, Yova et al, 2001), confocal microscopy on lamellar specimens was preliminarily conducted with lasers of various wavelengths within the indicated range. Fluorescent excitation by argon-ion lasers of 405, 514, and 594 nm wavelengths and by a krypton laser of 568 nm wavelength, provides comparable images of unchanged quality of Xuorescent elements (Figs.…”

A review of photodetectors for sensing light-emitting reporters in biological systems

“…The sample (held in a cuvette or other holder) is excited with the light emitted from the excitation filter. The reporter absorbs photons and emits light in all directions, and only part of this light can be gathered [6]. It is passed through a long-pass or bandpass filter, called an emission filter, that filters out the excitation light from the light source.…”

“…is a strong function of wavelength and varies greatly depending on the material. The electron-hole pair generation rate at a depth from the illuminated side of the semiconductor layer (the depletion region in a junction-based device) is given by (6) in cm s , where is the internal quantum efficiency. By knowing the depth to which photons are likely to penetrate, the detector can be optimized for particular wavelengths by physically matching the detection region to regions of highest absorption.…”