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

V. A. Matei, F. Feng, S. Pauley, Kirk Beisel, Michael G. Nichols, B. Fritzsch

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The β-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 β-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 β-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.

Original languageEnglish
Pages (from-to)33-43
Number of pages11
JournalBrain Research Bulletin
Volume70
Issue number1
DOIs
StatePublished - Jun 15 2006

Fingerprint

Galactosidases
Lighting
Lasers
Fluorescence
Light
Green Fluorescent Proteins
Epoxy Resins
Gene Expression
Bacterial Genes
Lac Operon
Aluminum Oxide
Information Storage and Retrieval
Titanium
Glycerol
Flow Cytometry
Proteins
Immunohistochemistry
5-bromo-4-chloro-3-indolyl beta-galactoside

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)

Cite this

Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance. / Matei, V. A.; Feng, F.; Pauley, S.; Beisel, Kirk; Nichols, Michael G.; Fritzsch, B.

In: Brain Research Bulletin, Vol. 70, No. 1, 15.06.2006, p. 33-43.

Research output: Contribution to journalArticle

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