Enhanced light collection in fluorescence microscopy using self-assembled micro-reflectors.

Zoltán Göröcs,Aydogan Ozcan,Euan Mcleod

doi:10.1038/srep10999

Abstract

In fluorescence microscopy, the signal-to-noise ratio (SNR) of the optical system is directly linked to the numerical aperture (NA) of the microscope objective, which creates detection challenges for low-NA, wide-field and high-throughput imaging systems. Here we demonstrate a method to increase the light collection efficiency from micron-scale fluorescent objects using self-assembled vapor-condensed polyethylene glycol droplets, which act as micro-reflectors for fluorescent light. Around each fluorescent particle, a liquid meniscus is formed that increases the excitation efficiency and redirects part of the laterally-emitted fluorescent light towards the detector due to internal reflections at the liquid-air interface of the meniscus. The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection. Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles. A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance. This method can be used as a sample preparation technique for consumer electronics-based microscopy and sensing tools, thus increasing the sensitivity of low-NA systems that image fluorescent micro-objects.

Highlights

Ratio for DNA microarrays[22]
Unlike its prior uses in digital holography for label-free bright-field imaging, where the enhancement of the scattered signal is achieved by depositing liquid nanolenses with a low surface contact angle, here we engineer these self-assembled vapor-condensed lenses to improve the sensitivity of fluorescence microscopy by using larger substrate contact angles to shape the meniscus as a reflector that increases the excitation efficiency and redirects laterally-emitted fluorescent light away from the sides and towards a detector located below the micro-reflector, thereby strongly enhancing the measured fluorescence signal for each micro-particle
To quantify the improvement due to the reflectors, we used in situ low-numerical aperture (NA) (~0.1) fluorescence microscopy to monitor the change in fluorescent signal levels from individual beads during the self-assembly of the micro-reflectors as the polyethylene glycol (PEG) vapor condenses

Summary

Introduction

Ratio for DNA microarrays[22]. Self-assembly of colloidal particles to act as an array of large NA lenses and increase the detected fluorescent intensity has been successfully used on surface-immobilized single fluorophores[23]. Unlike its prior uses in digital holography for label-free bright-field imaging, where the enhancement of the scattered signal is achieved by depositing liquid nanolenses with a low surface contact angle, here we engineer these self-assembled vapor-condensed lenses to improve the sensitivity of fluorescence microscopy by using larger substrate contact angles to shape the meniscus as a reflector that increases the excitation efficiency and redirects laterally-emitted fluorescent light away from the sides and towards a detector located below the micro-reflector, thereby strongly enhancing the measured fluorescence signal for each micro-particle (see Fig. 1). We compared our experimental results to numerical models implemented using ray tracing simulations, which helped us better understand how the size and shape of the liquid micro-reflectors enhance the collected fluorescent light through internal reflections at the air-PEG interface

Methods

Results

Conclusion