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Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy
Published on: May 13, 2012
Design and implementation of algorithms for focus automation in digital imaging time-lapse microscopy
Allan J LeSage1, Stephen J Kron
1Center for Molecular Oncology, The University of Chicago, Chicago, Illinois 60637, USA.
Cytometry
|November 28, 2002
Summary
A new digital autofocusing system for Nomarski-DIC microscopy maintains focus on dynamic biological samples. This robust system tolerates drift and vibration, making it practical for time-lapse imaging.
Area of Science:
- Biophysics
- Microscopy
- Cell Biology
Background:
- Digital time-lapse microscopy with Nomarski-DIC demands autofocusing systems adaptable to dynamic cellular changes (shape, size, position) and environmental factors (drift, noise, hysteresis).
- Existing systems face challenges in maintaining focus on optically complex samples under varying conditions.
Purpose of the Study:
- To design and implement an autofocusing system capable of tracking subjects under dynamic conditions and maintaining focus within a critical threshold.
- To address the challenges of drift, noise, and hysteresis in digital microscopy for biological imaging.
Main Methods:
- Development and implementation of a novel autofocusing system utilizing proven and new algorithms for Nomarski microscopy.
- Performance evaluation through "virtual" experiments on recorded image stacks, simulating drift and sudden displacements to test algorithm response.
Main Results:
- A combination of a [1, -1] contrast function and an adaptive "warmer-colder" focusing algorithm was identified as optimal, balancing precision and noise tolerance.
- The system was successfully applied to record yeast cell growth kinetics over several hours in both single and multiple fields of view.
Conclusions:
- A robust digital autofocusing system has been successfully implemented for high-resolution imaging of optically complex samples.
- The system's demonstrated tolerance to drift and vibration makes it a practical solution for various time-lapse biological imaging applications.
