Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

Rufeng Li,Binjie Qin,Baowei Fei,Hong Xu,Yibei Wang

doi:10.3390/s17112685

Abstract

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Highlights

Advances in microscopy and fluorescence tools have pushed the quantitative biological research for biomolecules at nanometer and sub-nanometer scales [1,2,3]
The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073
alkaline phosphatase (AP)-labeled nanoparticle concentration measurement is of great importance for quantitative biomolecular analysis and measurement

Summary

Introduction

Advances in microscopy and fluorescence tools have pushed the quantitative biological research for biomolecules at nanometer and sub-nanometer scales [1,2,3]. It is difficult to count AP-labeled nanoparticles directly from fluorescent images since AP-labeled nanoparticles are too small to detect and are closely clustered. To solve this problem, a widely-used technology called the droplet microfluidics technique has been used to encapsulate the individual AP-labeled nanoparticle in monodispersed micro-droplets [5]. The micro-droplets encapsulating AP-labeled nanoparticles will emit remarkable fluorescent signals via the enzymatic reaction between the APs and the corresponding fluorogenic substrates within droplets. We call these micro-droplets fluorescent micro-droplets and the others empty micro-droplets. Micro-droplet detection is necessary to analyze the AP-labeled nanoparticle concentration

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Conclusion