Method for optical excitation of fluorophore marked DNA and RNA

• US 6,528,802 B1
• Filed: 06/01/2001
• Issued: 03/04/2003
• Est. Priority Date: 07/29/1999
• Status: Expired due to Fees

• Current Assignee: CARL Zeiss JENA GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: CARL Zeiss JENA GmbH (Carl-Zeiss-Stiftung)
• Inventors: Fischer, Peter, Koenig, Karsten, Riemann, Iris, Halbhuber, Karl-Juergen
• Primary Examiner(s): Hannaher, Constantine
• Assistant Examiner(s): Gabor, Otilia
• Application Number:
  US09/806,375
• Time in Patent Office: 641 Days
• Field of Search: 250/459.1, 250/458.1, 250/201.2, 250/201.3, 250/307, 250/334.01, 250/340, 250/461.1, 250/461.2, 356/317, 435/6
• US Class Current: 250/459.1
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

A method for optical excitation of fluorophore-labeled DNA and fluorophore-labeled RNA, particularly of specific localizations of DNA and RNA labeled by fluorescence in situ hybridization (FISH). It is the object of the method to make possible in a simple manner a high-contrast simultaneous excitation of a plurality of FISH fluorophores which have different fluorescence characteristics and are to be detected and displayed three-dimensionally. The excitation and detection of fluorophores at a depth of the biological material greater than 100 micrometers must be ensured. The FISH fluorophores and DNA markers are excited to fluorescence by a multiphoton excitation simultaneously by pulsed and non-pulsed radiation at a single wavelength in the range between 700 nm to 1000 nm, preferably between 760 nm and 820 nm. A total of 20 commercially available FISH fluorophores and DNA markers were tested. Fluorophores which were excited simultaneously by multiphoton excitation were detected in all tested cases.

Claims

1. A method for optical excitation of fluorophore-labeled DNA and RNA in which a plurality of fluorophores with different fluorescence spectra which are to be detected are excited by radiation to fluorescence in the visible spectral range, said method comprising the step of exciting the fluorophores simultaneously by radiation of a single wavelength in a range of 700 nm to 1000 nm to induce multi-photon fluorescence excitation of the fluorophores.

2. The method according to claim 1, wherein the step of exciting the fluorophores is carried out with radiation of a wavelength between 730 nm and 820 nm.

3. The method according to claim 1, wherein the radiation for the multi-photon fluorescence excitation has an intensity of at least 100 MW/cm² and at most 100 TW/cm² by focusing in a spatially confined volume.

4. The method according to claim 1, wherein the step of exciting the fluorophores is carried out with radiation that is not pulsed.

5. The method according to claim 1, wherein the step of exciting the fluorophores is carried out with pulsed radiation.

6. A method for optical excitation of fluorophore-labeled DNA and RNA comprising the step of exciting a plurality of fluorophores with different fluorescence spectra simultaneously by radiation of a single wavelength in a range of 700 nm to 1000 nm to induce multi-photon fluorescence excitation of the fluorophores.

7. A method of observing samples using a multi-color fluorescence in situ hybridization technique, the method comprising:

labeling DNA and RNA of a sample with a plurality of fluorophores having different fluorescence spectra;

applying radiation of a single wavelength in a range of 700 nm to 1000 nm to the sample to induce multi-photon excitation of the fluorophores contained in the sample; and

detecting fluorescences of the plurality of excited fluorophores.

8. The method according to claim 7, wherein the step of applying radiation includes applying radiation of a single wavelength in a range between 730 nm and 820 nm.

9. The method according to claim 7, wherein the step of applying radiation includes applying a pulsed radiation having an intensity of at least 100 MW/cm² and at most 100 TW/cm² by focusing in a spatially confined volume.