Adaptation of a visible wavelength fluorescence microplate reader for discovery of near-infrared fluorescent probes.

Steven M Swasey,Stacy M Copp,Hunter C Nicholson,Elisabeth G Gwinn,Petko Bogdanov,Alexander Gorovits

doi:10.1063/1.5023258

Abstract

We present an inexpensive, generalizable approach for modifying visible wavelength fluorescence microplate readers to detect emission in the near-infrared (NIR) I (650-950 nm) and NIR II (1000-1350 nm) tissue imaging windows. These wavelength ranges are promising for high sensitivity fluorescence-based cell assays and biological imaging, but the inaccessibility of NIR microplate readers is limiting development of the requisite, biocompatible fluorescent probes. Our modifications enable rapid screening of NIR candidate probes, using short pulses of UV light to provide excitation of diverse systems including dye molecules, semiconductor quantum dots, and metal clusters. To confirm the utility of our approach for rapid discovery of new NIR probes, we examine the silver cluster synthesis products formed on 375 candidate DNA strands that were originally designed to produce green-emitting, DNA-stabilized silver clusters. The fast, sensitive system developed here discovered DNA strands that unexpectedly stabilize NIR-emitting silver clusters.

Highlights

The sensitive, high-speed and high-throughput studies enabled by fluorescence microplate readers have greatly benefitted understanding of biomolecular1–3 and cellular processes,2,4 biomedical studies of tissues,5,6 and development of biotechnologies.7,8 At visible wavelengths, dye molecules functionalized with suitable linkers are commonly used as fluorescent probes because they are available in a wide palette of wavelengths with high quantum yields, good photostabilities, and low toxicity
To confirm the utility of our approach for rapid discovery of new NIR probes, we examine the silver cluster synthesis products formed on 375 candidate DNA strands that were originally designed to produce greenemitting, DNA-stabilized silver clusters
Dye molecules functionalized with suitable linkers are commonly used as fluorescent probes because they are available in a wide palette of wavelengths with high quantum yields, good photostabilities, and low toxicity

Summary

Introduction

The sensitive, high-speed and high-throughput studies enabled by fluorescence microplate readers have greatly benefitted understanding of biomolecular and cellular processes, biomedical studies of tissues, and development of biotechnologies. At visible wavelengths, dye molecules functionalized with suitable linkers are commonly used as fluorescent probes because they are available in a wide palette of wavelengths with high quantum yields, good photostabilities, and low toxicity. Visible wavelength fluorescence microplate readers are highly evolved and widely available. The key elements in such systems are rapid mechanical drives that couple the individual, low volume sample “wells” in a microplate to an excitation light source and to a fluorescence detection system, enabling fast studies across arrays of up to 384 or even 1536 samples. Stringent separation of the excitation light from the sample well fluorescence is necessary because the fluorescence signal is typically many orders of magnitude dimmer. This separation is accomplished using dichroic mirrors together with optical filters and/or dispersive elements (gratings). The spectrally selected fluorescence is detected with a sensitive detector, most commonly a photomultiplier tube (PMT)

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Results

Conclusion