Research on Interferometric Scattering Microscopy and Spatial Coherence Control for Multiparticle Tracking

Background Introduction

Interferometric Scattering (iSCAT) microscopy has demonstrated unparalleled performance in the field of label-free optical imaging, especially in the detection and imaging of isolated nanoparticles and molecules. However, when imaging complex structures, such as biological cells, the superimposition of scattered fields from different sample positions can produce speckle-like backgrounds, significantly challenging the revelation of fine features. The research paper proposes that speckle backgrounds can be eliminated without sacrificing sensitivity by controlling the spatial coherence of illumination.

Source of Research

The main authors of this research paper include Mahdi Mazaheri, Kiarash Kasaian, David Albrecht, Jan Renger, Tobias Utikal, Cornelia Holler, and Vahid Sandoghdar. The research institutions involved are Max Planck Institute for the Science of Light and Friedrich-Alexander University Erlangen-Nürnberg. The paper was published in the journal “Optica” in July 2024.

Research Process and Methodology

Research Workflow

The research employed a method to eliminate the speckle background by controlling the spatial coherence of illumination, involving the placement of a rotating diffuser, a lens with adjustable focal length, and an aperture in the illumination path. Experiments demonstrated that this method maintains diffraction-limited imaging capabilities at high frame rates of 25 kHz and over a large field of view of 100 μm × 100 μm. By tracking over a thousand vesicles within a COS-7 cell in three dimensions (3D) and imaging the dynamics of the endoplasmic reticulum (ER) network, this method shows a significant advantage in combining label-free imaging, sensitive detection, and 3D high-speed tracking.

Specific Steps

1. Setting up the rotating diffuser and adjustable lens: The phase of the illumination beam was randomized effectively by placing a rotating diffuser and a lens of adjustable focal length in the illumination path and controlling the Numerical Aperture (INA) of the illuminating beam through the aperture. This suppressed the formation of IPSF rings and reduced the speckle background.
2. High frame rate imaging: Experiments imaged 40-nanometer gold nanoparticles (GNPs) at 25 kHz. The application of a rotating diffuser does not eliminate the IPSF ring at the center of the scattering signal, but significantly weakens the peripheral ring structures.
3. Sample selection: Selection of biological cell membranes, organelles such as the endoplasmic reticulum and mitochondria for testing, and recording and analyzing the dynamic behavior of the samples.
4. Data analysis: IPSF radial profiles were calculated and their changes over time were used to track the 3D trajectories of nanoparticles.

Main Results

Data Generation and Analysis

The experiment successfully realized control of the spatial coherence of the illumination beam, using a rotating diffuser, adjustable lens, and aperture, which effectively suppressed the speckle background during imaging. 3D tracking of multiple vesicles within COS-7 cells showed that useful information could be recorded over an extended time and a large field of view with high temporal resolution using optimized IPSF.

Conclusion and Significance

One conclusion drawn from the study is that controlling the spatial coherence of the light beam can significantly reduce the speckle background in interferometric scattering microscopy while increasing imaging speed. The method demonstrates immense potential for high-speed label-free imaging and nano-level 3D tracking.

The label-free microscope implemented with the TS-ISCAT method has high resolution and sensitivity and is capable of 3D dynamic tracking, which is of significant application value in the study of cell dynamics in biological science. The method also shows a broad range of potential applications in addressing light scattering issues.

Highlights

1. Manipulating spatial coherence: The method of suppressing speckle backgrounds by manipulating the spatial coherence of the light beam is innovative.
2. High frame rate and large field of view: Maintained diffraction-limited resolution at a frame rate of 25 kHz and a field of view of 100 μm × 100 μm.
3. Broad application potential: Demonstrated potential value in label-free 3D tracking and research of cell dynamics.

Additional Information

The research also explained how to fabricate standard samples with randomly distributed features to validate noise reduction effects and how to use deep learning networks, like SegNet, to enhance digital specificity in image-based organelle identification. These details further improve the comprehensiveness and accuracy of the research.

Through clever experimental design and rigorous data analysis, the research demonstrated how to significantly improve resolution and sensitivity of interferometric scattering microscopy in imaging biological samples, introducing new methods and directions for high-speed label-free imaging. The results lay a solid foundation for further applications in biological imaging and nanoscale structure research in the future.