Protein-crystal detection with a compact multimodal multiphoton microscope

Qing-Di Cheng,Hsiang-Yu Chung,Robin Schubert,Sven Falke,Christian Betzel,Celestin Nzanzu Mudogo,Shih-Hsuan Chia,Guoqing Chang,Franz X Kärtner

doi:10.1038/s42003-020-01275-8

Abstract

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.

Highlights

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray freeelectron lasers or high-intensity micro-focus synchrotron radiation sources
This indicates that the samples contain protein crystals as well as amorphous particles (THG positive and second-harmonic generation (SHG) negative). These results clearly demonstrated that micrometer-sized crystals of lysozyme, thaumatin, proteinase K, and thermolysin, which are more difficult to be detected applying only SHG, can be efficiently detected using the multiphoton microscope (MPM) instrument
To analyze the feasibility and detection sensitivity of the multimodal MPM system, we utilized different protein crystals grown in space groups with high symmetry

Summary

Introduction

There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray freeelectron lasers or high-intensity micro-focus synchrotron radiation sources. The size and dimensions of protein crystals required for diffractive imaging at high-brilliance micro-focus Xray sources is continuously decreasing, down to a few micrometers or even sub-micrometer regime[8,9] In this context, the demand to produce, detect, and, in particular, to monitor and image crystal suspensions upon preparation and just prior to data collection has substantially increased[10]. Novel and highly sensitive techniques and instruments are required to detect small crystals grown in vitro or in vivo, and capable to distinguish between salt and protein crystals To achieve this goal, we designed and developed an innovative multiphoton microscope (MPM) driven by a multicolor fiber-based ultrafast laser source.

Methods

Results

Conclusion