TY - JOUR
T1 - High throughput screening with multiphoton excitation
AU - Lakowicz, Joseph R.
AU - Gryczynski, Ignacy
AU - Gryczynski, Zygmunt
PY - 1999
Y1 - 1999
N2 - Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantaneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two- photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.
AB - Fluorescence detection is extensively used in high throughput screening. In HTS there is a continuous migration toward higher density plates and smaller sample volumes. In the present report we describe the advantages of two-photon or multiphoton excitation for HTS. Multiphoton excitation (MPE) is the simultaneous absorption of two long-wavelength photons to excite the lowest singlet state of the fluorophore. MPE is typically accomplished with short but high-intensity laser pulses, which allows simultaneous absorption of two or more photons. The intensity of the multiphoton-induced fluorescence is proportional to the square, cube, or higher power of the instantaneous photon flux. Consequently, two-photon or multiphoton excitation only occurs at the focal point of the incident beam. This property of two-photon excitation allows the excited volume to be very small and to be localized in the center of each well in the HTS plate. We show that two-photon-induced fluorescence of fluorescein can be reliably measured in microwell plates. We also show the use of 6-carboxy fluorescein as a pH probe with two-photon excitation, and measure 4'-6-diamidino-2-phenylindole (DAPI) binding and two- photon-induced fluorescence. In further studies we measure the time-dependent intensity decays of DAPI bound to DNA and calcium-dependent fluorophores. Finally, we demonstrate the possibility of three-photon excitation of several fluorophores, including indole, in the HTS plate. These results suggest that MPE can be used in high-density multiwell plates.
UR - http://www.scopus.com/inward/record.url?scp=0033384509&partnerID=8YFLogxK
U2 - 10.1177/108705719900400610
DO - 10.1177/108705719900400610
M3 - Article
AN - SCOPUS:0033384509
SN - 1087-0571
VL - 4
SP - 355
EP - 361
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 6
ER -