Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

<p>Abstract</p> <p>Background</p> <p>Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.</p> <p>Results</p> <p>Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications.</p> <p>Conclusions</p> <p>Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM.</p

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

Author Posting. © Optical Society of America , 2020. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Molina, R. S., King, J., Franklin, J., Clack, N., McRaven, C., Goncharov, V., Flickinger, D., Svoboda, K., Drobizhev, M., & Hughes, T. E. High throughput instrument to screen fluorescent proteins under two-photon excitation. Biomedical Optics Express, 11(12), (2020): 7192-7203, https://doi.org/10.1364/BOE.409353.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.National Institute of Neurological Disorders and Stroke (F31 NS108593, U01 NS094246, U24 NS109107); Howard Hughes Medical Institute

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

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750&ndash;800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~P&alpha; with &alpha; &gt; 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization

Deciphering the molecular mechanism responsible for GCaMP6m's Ca²⁺-dependent change in fluorescence

<div><p>The goal of this work is to determine how GCaMP6m’s fluorescence is altered in response to Ca²⁺-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca²⁺ is with a four-state model, in which a Ca²⁺-dependent change of the chromophore protonation state, due to a shift in pK_a, is the predominant factor. The pK_a shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca²⁺ and interacts with the M13 peptide. The absolute pK_a values for the Ca²⁺-free and Ca²⁺-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.</p></div

Photophysical parameters for GCaMP6m.

<p>Photophysical parameters for GCaMP6m.</p

Contributions to ΔΔG and ΔpK_a from important charged amino acids in GCaMP2 and GCaMP6m.

<p>Contributions to ΔΔG and ΔpK_a from important charged amino acids in GCaMP2 and GCaMP6m.</p

The changes in ΔpK_a between recent GCaMP generations.

<p>The changes in ΔpK_a between recent GCaMP generations.</p