Two-Photon Multicolor FISH: A Versatile Technique to Detect Specific Sequences with in Single DNA Molecules in Cells and Tissues
✍ Scribed by Karsten König; Axel Göhlert; Thomas Liehr; Ivan F. Loncarevic; Iris Riemann
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 803 KB
- Volume
- 1
- Category
- Article
- ISSN
- 1438-5163
No coin nor oath required. For personal study only.
✦ Synopsis
Two-photon multicolor fluorescence in situ hybridisation (FISH) is presented as an advanced single molecule detection technique. Based on a two-photon excitation process it enables pinhole-free 3D laser scanning microscopy. Nonresonant two-photon absorption in a subfemtoliter excitation volume was realized with a femtosecond laser scanning microscope equipped with a high numerical aperture objective. Nonlinear near infrared laser excitation and multicolor FISH has been used to image a variety of specific DNA regions. This innovative single molecule technique was applied to visualize genomic regions in fiber-DNA, human metaphase chromosomes, interphase nuclei and histological sections. Intense 170 fs laser pulses at 800 nm and GW/cm 2 intensities have been employed to induce visible fluorescence of a variety of FISH fluorophores coupled to DNA probes.
In particular, two-photon excited FITC-labeled 40 kb probes and multicolor labeled centromeric probes that bind to repetitive sequences of 0.340-2.000 kb have been used to visualize subtelomeric and different centromeric regions in metaphase chromosome spreads. Spectrum Orange and Spectrum Green labeled bcr and abl gene probes that target a stretch of 300 kb and 650 kb, respectively, have been imaged in a single fiber-DNA molecule with an estimated number of fluorescent molecules of 75 -225 µm -1 .
Using the advantages of pinhole-free optical sectioning with submicron spatial resolution and multi-fluorophore single-wavelength-excitation, 3D images of multiple labeled centromeric regions of amniotic fluid cells in interphase have been obtained and used in diagnosis of trisomy 18. In addition, first 3D two-photon studies on centromer distribution in human kidney biopsies by labeling chromosomes X, Y and 4 with the FISH-fluorophores Spectrum Blue, Spectrum Green and Spectrum Orange have been performed. As demonstrated, two-photon Multicolor FISH has the potential to perform Multi-Gene-Imaging with high spatial resolution also in turbid tissue layers.