scholarly journals High throughput instrument to screen fluorescent proteins under two-photon excitation

2020 ◽  
Vol 11 (12) ◽  
pp. 7192
Author(s):  
Rosana S. Molina ◽  
Jonathan King ◽  
Jacob Franklin ◽  
Nathan Clack ◽  
Christopher McRaven ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Proteins ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals High throughput instrument to screen fluorescent proteins under two-photon excitation

2020 ◽  
Author(s):  
Rosana S. Molina ◽  
Jonathan King ◽  
Jacob Franklin ◽  
Nathan Clack ◽  
Christopher McRaven ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Agar Plate ◽  
Focal Volume ◽  
E Coli ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

AbstractTwo-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

scholarly journals Directionality of Two-Photon Excitation in Representative Fluorescent Proteins

2018 ◽  
Vol 114 (3) ◽  
pp. 171a-172a
Author(s):  
Olga Rybakova ◽  
Stepan Timr ◽  
Josef Lazar
Keyword(s):  
Fluorescent Proteins ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Measurement of two-photon excitation spectra of fluorescent proteins with nonlinear Fourier-transform spectroscopy

2010 ◽  
Vol 49 (17) ◽  
pp. 3323 ◽  
Author(s):  
Hiroshi Hashimoto ◽  
Keisuke Isobe ◽  
Akira Suda ◽  
Fumihiko Kannari ◽  
Hiroyuki Kawano ◽  
...  
Keyword(s):  
Fourier Transform ◽  
Fluorescent Proteins ◽  
Fourier Transform Spectroscopy ◽  
Excitation Spectra ◽  
Two Photon ◽  
Photon Excitation ◽  
Nonlinear Fourier Transform ◽  
Two Photon Excitation

scholarly journals Experimental Determination of Single- and Two-Photon Excitation Transition Moments in Representative Fluorescent Proteins

2016 ◽  
Vol 110 (3) ◽  
pp. 493a
Author(s):  
Josef Lazar ◽  
Prakash Shukla ◽  
Richard Chazal ◽  
Alexey Bondar ◽  
David von Stetten ◽  
...  
Keyword(s):  
Experimental Determination ◽  
Fluorescent Proteins ◽  
Two Photon ◽  
Transition Moments ◽  
Photon Excitation ◽  
Two Photon Excitation

scholarly journals High Throughput Screening with Multiphoton Excitation

1999 ◽  
Vol 4 (6) ◽  
pp. 355-361 ◽  
Author(s):  
Joseph R. Lakowicz ◽  
Ignacy Gryczynski ◽  
Zygmunt Gryczynski
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Focal Point ◽  
Incident Beam ◽  
Photon Flux ◽  
Multiphoton Excitation ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Two Photon Excitation

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 instantneous 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 of 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.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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