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

Abstract

Two-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.

Photophysical Engineering of Fluorescent Proteins: Accomplishments and Challenges of Physical Chemistry Strategies

“…Another study based on considering the transition dipole moments was reported by Molina et al (2020) who developed a high-throughput, fluorescence microscope-based screening device (GIZMO) for screening bacterial cells by two-photon excited fluorescence. 47 The two-photon absorption crosssection can be used to examine the radiative rate and electric fields around the chromophore (the effects of internal electric fields on FP photophysics is discussed below). GIZMO is capable of screening 10 4 bacterial cells in ∼7 h, demonstrating great potential for this type of selection in the directed evolution of FPs.…”

“…Information from such studies can inform development of FPs for applications that are sensitive to the directions of the transition dipoles, such as FRET. Another study based on considering the transition dipole moments was reported by Molina et al (2020) who developed a high-throughput, fluorescence microscope-based screening device (GIZMO) for screening bacterial cells by two-photon excited fluorescence . The two-photon absorption cross-section can be used to examine the radiative rate and electric fields around the chromophore (the effects of internal electric fields on FP photophysics is discussed below).…”

“…Theoretical and experimental studies have examined the influence of electric fields and electrostatics on radiative and nonradiative rates. − ,,,, For example, TD-DFT calculations on the isolated GFP chromophore (Figure ) predict that electric fields can change the oscillator strength of an electronic transition and therefore the radiative rate constant. , Park and Rhee performed nonadiabatic molecular dynamics simulations on the GFP chromophore and found that electrostatic effects can outweigh steric factors impeding the twisting of the methine bridge between the phenoxy and imidazolinone rings, suppressing a major pathway for nonradiative relaxation . Drobizhev and co-workers investigated these issues by measuring two-photon absorption cross sections of RFPs .…”

Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

“…The customized optical instrument, called the GIZMO, was previously designed and implemented for screening of large libraries of FP mutants for their two-photon brightness. It was described in detail in [ 41 ]. Its complete CAD model as well as software and analysis programs can be found at github.com/rosanamolina/gizmo-paper (accessed on 23 December 2021).…”

A high-throughput multiparameter screen for accelerated development and optimization of soluble genetically encoded fluorescent biosensors

“…This poses a challenge for biosensor screening. Despite impressive advances in engineering new platforms for evolving fluorescent proteins and biosensors 7 , 11 – 14 , existing screens are still limited in the number of conditions that can be tested against the biosensor. Here we have surmounted these limitations, increasing screening content and throughput by orders of magnitude, by combining droplet microfluidics and automated two-photon fluorescence lifetime imaging (2p-FLIM).…”