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A General Analytic Approach for Rapid Diagnostics by a Simple Algorithm for Fluorescence Single Molecule Counting.

Juiena Hasan¹, Sangho Bok¹

¹Department of Electrical and Computer Engineering, University of Denver, Denver, CO 80208, USA.

|May 26, 2026

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Accurate single molecule counting is achieved using a novel blinking analysis method. This approach simplifies detection and speeds up analysis for ultralow concentration biomolecule quantification.

Area of Science:

Biochemistry
Analytical Chemistry
Biophysics

Background:

Accurate quantification of biomolecules at low concentrations is difficult with traditional methods.
Single molecule fluorescence counting offers higher sensitivity but faces challenges in analysis and reproducibility.

Purpose of the Study:

To develop a simple, rapid, and general analytical framework for single molecule detection.
To overcome limitations of manual inspection and complex machine learning in fluorescence assays.

Main Methods:

A deterministic threshold algorithm exploiting fluorescence blinking temporal signatures.
Direct analysis of time-resolved fluorescence image stacks with median filter noise suppression.
Identification of single molecule events via frame-to-frame intensity transitions without training data.

Keywords:

blinking analysis digital molecular counting fluorescence microscopy plasmonic biosensing rapid diagnostics single molecule detection threshold-based algorithm

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Last Updated: May 28, 2026

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

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Published on: September 24, 2015

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Published on: March 19, 2014

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Published on: April 25, 2025

Main Results:

Reproducible concentration-dependent trends for Alexa Fluor 488, 647, and Rhodamine Red-X.
Standardized detection under challenging low signal and high background conditions.
Approximately 9-fold increase in analysis efficiency compared to manual counting, reducing time from ~3h to ~20min.

Conclusions:

The developed framework provides an interpretable and computationally lightweight strategy for fluorescence single molecule counting.
The method is experimentally adaptable and suitable for rapid diagnostics.
Establishes a foundation for automated localization, clustering, and real-time molecular analysis.