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 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

Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue

1997 ◽  
Vol 3 (S2) ◽  
pp. 305-306
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Collection Efficiency ◽  
Three Dimensional ◽  
Spatial Filter ◽  
Input Power ◽  
Photon Absorption ◽  
Focal Volume ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM 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 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.

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

scholarly journals Dual Near Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging

2017 ◽  
Author(s):  
Jackson T. Del Bonis-O’Donnell ◽  
Ralph H. Page ◽  
Abraham G. Beyene ◽  
Eric G. Tindall ◽  
Ian McFarlane ◽  
...  
Keyword(s):  
Near Infrared ◽  
Photon Absorption ◽  
Deep Tissue ◽  
Biologically Relevant ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Tissue Phantom ◽  
Photon Excitation ◽  
Photon Absorption And Scattering ◽  
Two Photon Excitation

A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically-relevant context of the intact brain, for which photons must traverse the cranium, skin and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, we describe optimal excitation and emission wavelengths for through-cranium imaging, and demonstrate that near-infrared emissive nanosensors can be photoexcited using a two-photon 1560 nm excitation source. Dopamine-sensitive nanosensors can undergo two-photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. We further calculate the two-photon absorption cross-section and quantum yield of dopamine nanosensors, and confirm a two-photon power law relationship for the nanosensor excitation process. Finally, we show improved image quality of the nanosensors embedded 2 mm deep into a brain-mimetic tissue phantom, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under two-photon excitation. Our approach overcomes traditional limitations in deep-tissue fluorescence microscopy, and can enable neurotransmitter imaging in the biologically-relevant milieu of the intact and living brain.

scholarly journals A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

2015 ◽  
Vol 309 (11) ◽  
pp. C724-C735 ◽  
Author(s):  
Wen Tao ◽  
Michael Rubart ◽  
Jennifer Ryan ◽  
Xiao Xiao ◽  
Chunping Qiao ◽  
...  
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Practical Method ◽  
Two Photon Microscopy ◽  
Cellular Processes ◽  
Two Photon ◽  
Photon Excitation ◽  
Wide Range

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes.

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
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