Cellular Dynamics Revealed by Digital Holographic Microscopy

With the development of new optical technologies (such as laser technology, confocal scanning microscope) and powerful numerical image processing (deconvolution technology, computational adaptive optics technology), optical microscopes have surpassed the traditional two-dimensional imaging technology and turned to the reproduction of high-resolution three-dimensional objects.

Digital Holographic Microscope (DHM)

Fig.1 The basic configuration of digital holographic microscopy (DHM). (Marquet, 2016)

Digital holographic microscope (DHM) is a new optical method that can provide real-time three-dimensional images of transparent living cells without the use of any contrast agent. DHM quantitatively studies cell dynamics by measuring the phase shift of living cells in the transmitted wavefront. Due to the high time stability of the phase signal (equivalent to N/1800) and low acquisition time (down to 20 μs), the dynamic monitoring of cells is visualized.

Working principle: The coherent light (A = 658 nm) generated by a VCSEL laser diode is split by a beam splitter (BS). The sample (S) was illuminated by a beam of light through the condenser (C). The microscope objective lens (MO) collects the transmitted light and forms an object wave (O), which interferes with the reference beam (R) to produce a hologram recorded by a digital CCD camera.

DHM Analyzes Neuron Network Activity

A distinctive feature of neural tissue is the intricate network of synaptic connections between neurons of different shapes. The development of technology that provides non-invasive resolution to local neural network activities is a prerequisite for a comprehensive study of the relationship between spatiotemporal activity patterns and neural network development and information processing.

• Use quantitative phase DHM (QP-DHM) to measure neuronal activity in transmembrane water movement imaging

At present, multi-modal microscopes, QP-DHM, and electrophysiological devices have been developed to study the early neuronal responses induced by glutamate in the primary culture of mouse cortical neurons. Using appropriate pharmacological tools to analyze the effects of agonists (such as glutamate) or antagonists (such as bitterness, furosemide, and bumetamide) on QP-DHM signal changes, provides a new approach for studying the functional properties of target membrane proteins.

Fig.2 Color-coded quantitative phase images of two neuronal cell bodies (“standard”) and around 10 min after the onset of the glutamate perfusion(“Excitotoxic”). (Marquet, 2016)

• Use DHM system to record spatiotemporal cell information

The shear DHM recorded the video rate data of living biological cells fluctuating in the axial film with nanometer sensitivity and then extracts features from the reconstructed phase map of each time segment cell for classification. The time-varying data of each extracted feature is input into a periodic bidirectional long-term memory (BI-LSTM) network, which classifies and recognizes cells according to the time-varying behavior of the cells for use in disease diagnosis.

Fig.3 Segmented digital holographic reconstruction of healthy red blood cells (a) and knife cell disease red blood cells (b). (O’Connor, 2020)

QP-DHM is an optical imaging technology that can obtain quantitative phase contrast images of transparent living cells from a single recorded hologram without using any dyes. The principle of interference applied to phase detection and reconstruction of digital holograms allows monitoring of cell dynamics with nano-axial sensitivity. In addition to measuring cell surface morphology and intracellular refractive index (RI), it can also measure various biophysical parameters, including dry mass, absolute volume, nano-scale and biomechanical properties of membrane fluctuations, water permeability.

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References

1. Marquet, P.; et al. Cellular dynamics revealed by digital holographic microscopy. Elsevier BV. 2016, 675-683.
2. O’Connor, T.; et al. Deep learning-based cell identification and disease diagnosis using spatio-temporal cellular dynamics in compact digital holographic microscopy. Biomedical Optics Express. 2020, 11(8): 4491-4508.