Our Vision:

We are dedicated to developing next-generation Quantitative Phase Microscopy (QPM) technologies that offer exceptional sensitivity and imaging speed. Our research focuses on engineering robust QPM systems capable of operating in both reflection and transmission modes. This dual-mode capability enables the flexible imaging of a wide range of biological samples, from transparent single cells to complex, thick, and scattering tissue and material samples. This system will be optimized for both stationary or slowly moving biological specimens, such as U2OS and HeLa cells, and fast-moving samples, including fish keratinocytes and sperm cells.

By pushing the boundaries of phase sensitivity and acquisition speed, our goal is to unlock real-time, label-free visualization of dynamic biological processes with nanoscale precision. These advancements will not only enhance fundamental biological discovery but also support translational applications in disease diagnostics, drug screening, and tissue engineering. Our efforts are laying the foundation for a new class of imaging tools that combine precision, speed, and versatility to meet the needs of both research and biomedical applications.

Dynamic Speckle Illumination Quantitative Phase Microscopy

These studies collectively establish dynamic speckle illumination (DSI), also known as a pseudo-thermal light source, as a versatile and high-performance illumination strategy for advanced interference-based imaging systems, particularly in quantitative phase microscopy (QPM) and optical coherence/diffraction tomography (OCT/ODT).

DSI provides a light source with low spatial coherence and high temporal coherence, enabling high spatial phase sensitivity while preserving a wide field of view and high imaging throughput. This configuration supports single-shot QPM with improved contrast and phase resolution, overcoming traditional limitations of laser and broadband light sources. It has been successfully applied to high-resolution imaging of nanostructures, biological tissues, and live cells, including applications in histopathology and cell morphology studies.

Furthermore, these studies provide a theoretical and experimental foundation for understanding interference fringe formation under DSI and demonstrate how it enables stable phase imaging even with non-identical objective lenses. This removes constraints imposed by traditional balanced interferometric setups and opens up scalable, flexible configurations for both research and clinical applications.

A systematic investigation into the coherence properties of DSI reveals that its longitudinal coherence length (LC) becomes independent of the laser’s temporal coherence length when the source size exceeds a threshold. This makes it possible to tailor the LC length based on source geometry, allowing high axial resolution in OCT imaging without requiring broadband sources or dispersion compensation. For instance, an axial resolution of 650 nm was achieved using monochromatic light with optimized spatial coherence conditions.

References:

1. Ahmad, Azeem, Vishesh Dubey, Nikhil Jayakumar, et al. "High-throughput spatial sensitive quantitative phase microscopy using low spatial and high temporal coherent illumination." Scientific reports 11, no. 1 (2021): 15850. Link 

2. Ahmad, Azeem, Nikhil Jayakumar, and Balpreet Singh Ahluwalia. "Demystifying speckle field interference microscopy." Scientific Reports 12, no. 1 (2022): 10869. Link 

3. Ahmad, Azeem, Tanmoy Mahanty, Vishesh Dubey, et al. "Effect on the longitudinal coherence properties of a pseudothermal light source as a function of source size and temporal coherence." Optics letters 44, no. 7 (2019): 1817-1820. Link 

4. Usmani, Kashif, Azeem Ahmad, Rakesh Joshi, et al. "Relationship between the source size at the diffuser plane and the longitudinal spatial coherence function of the optical coherence microscopy system." Journal of the Optical Society of America A 36, no. 12 (2019): D41-D46. Link 

Common-path Quantitative Phase Microscopy

These works present the development of advanced quantitative phase microscopy (QPM) systems designed for high temporal stability, wavelength-independence, and scalable field of view—key attributes for reliable and flexible live-cell imaging.

One approach introduces a common-path, single-element QPM system optimized for stability and ease of integration. The system delivers ~15 mrad temporal phase stability without requiring vibration isolation and supports imaging across multiple wavelengths and magnifications. It enables accurate quantification of minute membrane fluctuations in human red blood cells (RBCs), demonstrating its potential for biomedical diagnostics and disease monitoring.

Building on the principle of common-path interferometry, another optical configuration incorporates a calcite crystal-based 4f setup to enable dynamic phase imaging of live cells. This system achieves high temporal stability (~20 mrad) and supports time-lapse imaging over long durations. Experiments on human RBCs and fibroblast cells reveal its ability to capture subtle sub-cellular dynamics, making it a powerful tool for live-cell quantitative imaging.

Additionally, a single-shot quantitative differential phase contrast microscopy (Q-DPCM) system is developed using a calcite beam displacer. This compact and partially coherent setup enables real-time, differential, and quantitative phase imaging using only one interferogram, offering high spatial phase sensitivity. The system supports dynamic monitoring of intracellular structures, such as nucleoli and lipid granules, in living cells like HeLa and U2OS over extended periods.

References:

1. Saxena, Anuj, Azeem Ahmad, Vishesh Kumar Dubey, Hong Mao, et al. "Single-shot quantitative differential phase contrast microscope using a single calcite beam displacer." Applied Optics 63, no. 32 (2024): 8350-8358. Link 

2. Saxena, Anuj, Azeem Ahmad, Vishesh Dubey, et al. "Dynamic quantitative phase imaging using calcite crystal-based temporally stable interferometer." Journal of Modern Optics 70, no. 19-21 (2023): 973-982. Link 

3. Ahmad, Azeem, Vishesh Dubey, Ankit Butola, et al. "Highly temporal stable, wavelength-independent, and scalable field-of-view common-path quantitative phase microscope." Journal of Biomedical Optics 25, no. 11 (2020): 116501-116501. Link 

White light Interference Microscopy

These works present advancements in low-coherence interference microscopy (LCIM) for achieving highly sensitive quantitative phase imaging in practical laboratory conditions. A phase shifting interferometry approach is introduced that enables sub-nanometer height sensitivity without relying on calibrated piezoelectric transducers or vibration-isolated environments. By analyzing continuous temporally phase-shifted interferograms with a Fourier-based algorithm followed by a phase-shifting algorithm, accurate phase maps are reconstructed even under environmental fluctuations. This enables precise, label-free imaging of nanostructures and biological samples, including red blood cells and optical waveguides.

In parallel, a flexible Linnik interferometer-based LCIM configuration is developed using non-identical objective lenses in the object and reference arms. This unbalanced setup removes the constraint of using identical optics, allowing high numerical aperture lenses in the imaging path and enabling easy magnification changes without reconfiguring the system. To ensure phase accuracy, advanced iterative algorithms and principal component analysis are applied to recover clean, speckle-free phase images. Demonstrated on various test targets and biological specimens such as HeLa cells, this approach supports scalable, high-resolution imaging with enhanced optical flexibility.

References:

1. Ahmad, Azeem, Vishesh Dubey, Ankit Butola, et al. "Sub-nanometer height sensitivity by phase shifting interference microscopy under environmental fluctuations." Optics Express 28, no. 7 (2020): 9340-9358. Link 

2. Ahmad, Azeem, Anowarul Habib, Vishesh Dubey, et al. "Unbalanced low coherence interference microscopy." Optics and Lasers in Engineering 151 (2022): 106932. Link 

Mirau interferometry

These works present a novel light source design that combines spatial, angular, and temporal diversity to synthesize pseudo-thermal light illumination with unique coherence properties. By carefully engineering these degrees of freedom, the resulting light exhibits a significantly reduced longitudinal spatial coherence length while preserving a narrow temporal frequency spectrum. This enables high axial resolution optical sectioning using standard low numerical aperture objectives, making the approach practical and accessible. The same light source is applied to quantitative phase imaging of biological samples, where its low spatial coherence effectively suppresses speckle and unwanted interference fringes that typically arise with conventional laser sources. Additionally, the use of a highly monochromatic yet spatially incoherent source removes the need for dispersion compensation, which is often required when working with broadband illumination. Overall, this strategy achieves the advantages of both broadband and laser-based systems, offering stable, high-resolution, and artifact-free imaging, particularly well-suited for multilayered biological and industrial samples.

References:

1. Ahmad, Azeem, Vishesh Dubey, Gyanendra Singh, et al. "Quantitative phase imaging of biological cells using spatially low and temporally high coherent light source." Optics letters 41, no. 7 (2016): 1554-1557. Link 

2. Ahmad, Azeem, Vishal Srivastava, Vishesh Dubey, et al. "Ultra-short longitudinal spatial coherence length of laser light with the combined effect of spatial, angular, and temporal diversity." Applied Physics Letters 106, no. 9 (2015). Link 

Development Projects

Reflection-mode Quantitative Phase Microscopy (QPM) system

We are developing a reflection-mode, non-common-path Quantitative Phase Microscopy (QPM) system based on a Linnik interferometer configuration and illuminated with dynamic speckle illumination, also known as a pseudothermal light source (PTLS). This illumination strategy provides high temporal coherence with low spatial coherence, significantly reducing coherent noise while preserving strong interference contrast over a large field of view, thereby delivering phase image quality comparable to that of incoherent sources such as halogen lamps or LEDs.

The system is realized in two configurations: a non-polarizing Linnik interferometer for multi-shot phase retrieval (~0.5 phase maps per second) and a polarization-based Linnik interferometer for single-shot phase recovery at high frame rates (up to 75 phase maps per second), while maintaining diffraction-limited phase accuracy through phase-shifting interferometric algorithms. The reflection-mode geometry is particularly advantageous for imaging samples on opaque substrates, such as optical waveguides, or biological specimens like cells and tissue sections mounted on reflective surfaces. Moreover, the reflection setup provides twice the phase sensitivity compared to transmission-mode systems due to the double-pass of light through the sample.

However, a key limitation of non-common-path interferometric systems lies in their vulnerability to environmental fluctuations such as mechanical vibrations, thermal drifts, and air turbulence. Since the object and reference beams travel along separate physical paths, even slight instabilities can introduce phase noise and degrade temporal stability, posing challenges for high-precision or long-duration imaging of dynamic samples.

To mitigate these effects, we are actively investigating common-path QPM architectures, which inherently suppress environmental disturbances and offer enhanced temporal stability. These ongoing developments are described in the following section.

Transmission-mode Quantitative Phase Microscopy (QPM) system

In progress ...

Common-path transmission and reflection QPM                                            

In progress ...

Applications

In progress...