Tryptophan fluorescence excitation is most efficient at 280 nm wavelength. The other aromatic aminoacids, tyrosine, phenylalanine and histidine, could only be excited at shorter wavelengths. Therefore they are of minor importance for in situ intrinsic fluorescence crystal detection (Figure 2).

The opacity of glas coverslips and sealing films reduce the light intensity significantly (Figure 3). Howerver, the characteristics of a filtered mercury arc lamp spectrum compensates the weak opacity and is still sufficient to excite tryptophan fluorescence.

Tryptophan fluorescence is shown in three images of the same glucose isomerase crystal as sitting drop in a 96 Well, CrystalQuick COC plate (greiner bio-one, 609820). All three images were taken using the same exposure time and light sensitivity. On top, the crystal was covered with a quartz cover slip (suprasil). The relative excitation spectrum intensity was calculated for three different proteins (product of the transmission spectrum with the specific molar absorption of the protein) shown on the left side. In the middle the crystal was covered with a standard polymeric film and below with a common glas cover slip. The opacity for wavelengths below 300 nm is significantly reduced when the crystal is covered by glas. However, the appearance of the intrinsic fluorescence seems almost identical, when illuminated with a filtered mercury arc lamp spectrum.

Trace Fluorescence Imaging

Covalently fluorescence labeled proteins with the fluorophore carboxyrhodamine: Absorption and emission maxima: λex 522 nm, λem 550 nm. The colored light source of XtalLight100C provides a green light spectrum (green curve) and the emission maximum and the absorption peak of carboxyrhodamine match exactly. 

However part of the green light spectrum overlays with the fluorescence spectrum of carboxyrhodamine, therefore a bandpass filter 525 nm with 20 nm spectral width is used for excitation and a 550 nm longpass filter is used to separate the fluorescence light.

Summarized spectra of carboxyrhodamine emission and excitation and the applyied optical filters.

Carboxyrhodamine stained crystals with a 0.5 to 2 % labeling efficiency exite
with 515 – 535 nm spectral width, exposure time: 1.2 s.