Low-cost and portable UV holographic microscope for high-contrast protein crystal imaging

Mustafa Ugur Daloglu,Aniruddha Ray,Derek Tseng,Calvin Brown,Blanca Chocarro-Ruiz,Laura M Lechuga,Michael J Collazo,Aydogan Ozcan,Duilio Cascio

doi:10.1063/1.5080158

Abstract

Imaging protein crystals and distinguishing them from salt crystals is an important task for protein crystallographers. The conventional tool used for this purpose is a dual-mode microscope composed of bright-field and ultraviolet (UV) induced fluorescence modes. The distinction between a protein and a salt crystal is made based upon the fluorescence response to the UV excitation, where most protein crystals absorb the UV excitation and emit fluorescence, unlike salt crystals. These dual-mode optical microscopes are sensitive; however, they are relatively bulky and expensive as they require UV-grade optics. As an alternative, here we demonstrate that on-chip UV holographic imaging offers a low-cost, portable, and robust technique to image and distinguish protein crystals from salt crystals, without the need for any expensive and bulky optical components. Only composed of a UV light-emitting-diode at 280 nm and a consumer-grade complementary metal–oxide–semiconductor image sensor de-capped and interfaced to a Raspberry Pi single-board computer, the necessary information from the crystal samples (placed very close to the sensor active area) is captured in the form of in-line holograms and extracted through digital back-propagation. In these holographic amplitude reconstructions, protein crystals appear significantly darker compared to the background due to the strong UV absorption, unlike salt crystals which do not show any contrast, enabling us to clearly distinguish between them. We believe that the on-chip UV holographic microscope could serve as a low-cost, sensitive, and robust alternative to conventional lens-based UV-microscopes used in protein crystallography.

Highlights

Protein crystallographers rely on a dual-mode optical microscope composed of bright-field and ultraviolet (UV) induced fluorescence modes to image protein crystals as well as to distinguish them from salt crystals that could form during the crystallization process
Composed of a UV light-emitting-diode at 280 nm and a consumer-grade complementary metal–oxide–semiconductor image sensor decapped and interfaced to a Raspberry Pi single-board computer, the necessary information from the crystal samples is captured in the form of in-line holograms and extracted through digital back-propagation. In these holographic amplitude reconstructions, protein crystals appear significantly darker compared to the background due to the scitation.org/journal/app strong UV absorption, unlike salt crystals which do not show any contrast, enabling us to clearly distinguish between them
Our portable on-chip imaging platform (Fig. 1) is built upon the versatile Raspberry Pi 3 board,[35] with its readily available 8 Megapixel complementary metal–oxide–semiconductor (CMOS) camera, all housed within a custom designed and 3D printed shell which holds a UV light-emitting diodes (LEDs) operating at 280 nm peak wavelength with a band-pass filter to block the side-band emissions.[31,32,36]

Summary

Introduction

Protein crystallographers rely on a dual-mode optical microscope composed of bright-field and ultraviolet (UV) induced fluorescence modes to image protein crystals as well as to distinguish them from salt crystals that could form during the crystallization process This distinction is mainly based on the response to the UV illumination, where most protein crystals absorb the UV light and emit fluorescence through tryptophan residues, unlike most salt crystals.[1,2] In addition to UV fluorescence,[3,4] the strong absorption of UV light within organic materials[5,6] has been utilized as an inherent contrast agent in imaging tissue samples,[7] cells,[8] intracellular nucleic acids and proteins,[9–12] viruses,[13] and protein aggregates,[14] making UV microscopy[15] an important tool for researchers. This, when integrated with the capabilities of portable and cost-effective on-chip holographic microscopy tools, enables a wide range of studies related to proteins, in the field of crystallography

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Conclusion