Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance

Abstract

The beta-galactosidase protein generated by the bacterial LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The beta-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used beta-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633 nm light. Photoactivation is achieved through exposure to 730 nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730 nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.

Fig. 1

Imaging of the BCI precipitate of β-galactosidase is compared for transmitted light (A, B), epifluorescent light (C), confocal microscopy without (D, F) and with (E, G) 720 nm photoactivation. The BCI precipitate in cochlear hair cells absorbs rhodamine (568 nm) excitation fluorescence and can be electronically inverted to yield a signal, here false colored as green (C). Such imaging is comparable to either simple transmitted light (A) or transmitted light with a red rhodamine filter (B). Imaging the BCI precipitate with 488 nm excitation shows absorption of the background epifluorescent signal (D, F), while allowing nerve fibers stained using an Alexa 647-conjugated secondary antibody to be visualized (red fibers in D, F). Photoactivation with 720 nm excitation results in emission of a strong 488 nm-excited fluorescent signal in tissue areas of low BCI precipitate concentration (E, G). Note that high levels of BCI cause quenching, yielding a black hole in the fluorescence (G). Boxes in D are shown at higher magnification in E–G. Abbreviations: AC, anterior crista; HC, horizontal crista; OC, organ of Corti; U, utricle. Scale bar: 100 μm.

Fig. 2

These images show the appearance of BCI precipitates before (A–F) and after single (G–H) or multiple (G–N) scans using 720 nm excitation (19.5 mW average power at sample; 16 s scan per frame). The intrinsic background fluorescence emitted following 488 nm (A) and 543 nm (B) excitation shows various levels of absorption by BCI (A, B and zoomed in area in B shown in D, E), that is particularly obvious with the Hoechst stain (F). After a single excitation at 720 nm to image the Hoechst stain (F), the properties of BCI have changed. Instead of predominantly absorbing photons, 488 nm (G) and 543 nm (H) excitations produce a strong fluorescence emission. A second 720 nm exposure reveals cell nuclei which could not be seen in the first scan (F, I). Zooming out shows that the small area exposed to 720 nm light emits stronger fluorescent signals after 488, 543 and 720 nm excitation (J–L). A single 720 nm exposure of the entire field results in photoactivation (M, N). Using the lambda mode of the Zeiss 510 laser scanning microscope the maximal 488 nm-excited fluorescence emission is around 528 nm (O). Linear intensity profiles through the BCI precipitate (vertical lines in E, H) show the dramatic increase in fluorescent signal with 543 nm excitation (C). Bar: 100 μm.

Fig. 3

The plate illustrates the depth penetration (A, B) and wavelength dependency (C–E) of the photoactivation process. When using 40x (1.3 NA) lens and 730 nm excitation (2.6 mW average power at sample), the photoactivation process is limited to approximately 3 μm above and below the focal plane (A). Note that repeating the scan (B) produces a brighter signal after 488 nm excitation, but does not change the penetration beyond the ±3 μm already activated with the first scan. Imaging the Z-axis stack in lateral views reveals photoactivation only about 3 μm above and below the center focal plane (A^′, B^′). We estimated the best wavelength for photoactivation (C–E) using single near-infrared scans of small square regions at different positions along the cochlea (15.4–17.8 mW average laser power). Imaging the entire cochlea by zooming and illuminating with 488 (green, C), 543 (red, D) and 633 nm (blue, E) light reveals that the maximal photoactivation is achieved with 730 nm excitation. Scale bar: A^′, B^′, 10 μm; C–E, 100 μm.

Fig. 4

The images show that photoactivation is achieved in epoxy resin-embedded tissue sectioned at 10 μm (A, B) and in whole-mounted tissue exposed in phosphate-buffered saline (C, D). Note that only the intrinsic background fluorescence of the tissue is visible prior to photoactivation (A, C). After photoactivation, the previously invisible BCI precipitate (we selected very light labeling) turned into a brightly fluorescent product (488 nm excitation). This permits imaging of the BCI precipitate in areas of bdnf gene expression in hair cells (OHC) and supporting cells of the upper middle turn of an E18.5 embryonic cochlea. The increased imaging capability of the fluorescent signal allows imaging of the few Neurod1-LacZ-positive hair cells (arrow in C, D) in the organ of Corti (OC), in addition to the strongly labeled neurons of the spiral ganglion (SPGL) that were invisible prior to photoactivation. Other abbreviations: GER, greater epithelial ridge; OHC, outer hair cell. Scale bar: 20 μm.

Fig. 5

Fluorescence emission spectra of the photoactivated BCI precipitate at different excitation wavelengths. Photoactivation of BCI precipitate results in emissions with peaks near 528 and 663 nm. Excitation with 477, 488 or 517 nm (blue, green yellow vertical lines) causes prominent emission with a peak at 528 nm. Clearly, the emission is most profound after 488 nm excitation (green curve). In contrast, excitation with 543 or 633 nm (red and maroon vertical lines) yields emission near 633 nm with a second peak near 690 nm.

Fig. 6

The plate illustrates photobleaching and photoswitching by 488 and 633 nm excitation of BCI photoactivated product in epoxy resin. We first activated the Bdnf gene expression-mediated BCI precipitate (10 μm in epoxy resin) with a single 720 nm scan (40x/1.3 NA objective, 6 mW laser power output) leading to moderate fluorescence after 488 (A) or 633 (B) excitation. We then focused on the area indicated with a white square (C) and scanned 50 times with maximal 488 nm laser power. Subsequently, we took a single image using 488 and 633 nm excitation (C, D). While 488 nm exposure had resulted in noticeable bleaching, this had no effect on the 633 nm-excited fluorescence emission (D). We subsequently restored the 488 nm-excited fluorescence with a single 720 nm scan, followed by 25 scans of 633 nm, again with maximum laser power. The bleaching nearly eliminated the 633 nm-excited fluorescence (white square in F), but substantially increased the 488 nm-excited fluorescence (white square in E). We followed this with another scan at 720 nm, which brought the 633 nm-excited fluorescence back to normal while quenching the 488 nm-excited fluorescence emission at the same time (compare E with H). Another 720 nm scan further bleached the 488 nm emission close to the initial emission (compare A with I), whereas the emission elicited with 633 nm became much stronger (compare B with J). All imaging settings remained constant throughout the experiment. Scale bar: 20 μm.