In Situ Flow Cytometer Calibration and Single-Molecule Resolution via Quantum Measurement

Javier Sabines-Chesterking,Sergey V Polyakov,Ivan A Burenkov

doi:10.3390/s22031136

Abstract

Fluorescent biomarkers are used to detect target molecules within inhomogeneous populations of cells. When these biomarkers are found in trace amounts it becomes extremely challenging to detect their presence in a flow cytometer. Here, we present a framework to draw a detection baseline for single emitters and enable absolute calibration of a flow cytometer based on quantum measurements. We used single-photon detection and found the second-order autocorrelation function of fluorescent light. We computed the success of rare-event detection for different signal-to-noise ratios (SNR). We showed high-accuracy identification of the events with occurrence rates below even at modest SNR levels, enabling early disease diagnostics and post-disease monitoring.

Highlights

Flow cytometry is a commonly used optical technique to measure a wide range of cell properties in a high throughput manner by probing them with a laser as they flow through a flow chamber [1,2]
By introducing fluorescent markers to a sample, a flow cytometer can be used to detect the presence of target molecules and reveal the distribution of cells containing these molecules within a heterogeneous population [3,4]
We present a theoretical model for sensing fluorescent biomarkers at the single-molecule level based on the measurement of the second-order autocorrelation function of their emitted light

Summary

Introduction

Flow cytometry is a commonly used optical technique to measure a wide range of cell properties in a high throughput manner by probing them with a laser as they flow through a flow chamber [1,2]. Previous claims rely on a priori knowledge of sample concentration and statistical analysis of photonbursts, which do not necessarily arise from a single-emitter as large scatterers including bubbles within the flow cell could generate these bursts To this end, we present a theoretical model for sensing fluorescent biomarkers at the single-molecule level based on the measurement of the second-order autocorrelation function of their emitted light. We present a theoretical model for sensing fluorescent biomarkers at the single-molecule level based on the measurement of the second-order autocorrelation function of their emitted light This method provides in situ verification of single-molecule sensitivity from first principles. We demonstrate that our method can be used to discriminate between different numbers of biomarkers, with a high success rate

Method

Proposed Experimental Setup

Objective

Detection of Single Biomarkers

Conclusions