Next generation quantitative phase imaging at versatile wavelengths

Abstract

Quantitative phase imaging (QPI) techniques are emerged as a major modality in biomedical imaging, because of their ability to provide label free quantitative phase information of biological samples. QPI provides extra image contrast by measuring the total phase difference information, especially in optically transparent samples, that is useful in further quantitative studies of sample. Interferometric techniques are widely used in phase imaging modalities such as digital holography, while their phase retrieval sensitivities can be affected by environmental conditions and vibrations. Mostly reported QPI techniques use the visible wavelength range of light source which suffers from less penetration depth and generates a speckle noise from the scattering sample that drastically degrades the image quality. Although there are different techniques based on image or signal processing such as; wave front shaping, structured light illumination and computational imaging which were reported in recent years to overcome the imaging through scattering media problem. Another way to reduce the speckle noise and increase the penetration depth in the sample by using longer wavelength range source. It has been shown that the 2-μm wavelength regime can provide higher penetration depth and less scattering from the sample which improves the image quality. Based on this concept here we have demonstrated a spectrally encoded QPI microscopy using 2-μm fiber laser. Several techniques are available to retrieve phase information using a non-interferometric optical system that only measures intensity images of the sample. These techniques are mainly iterative and based on multi-plane intensity measurements but simple in implementation. Due to simplicity in implementation, they are less sensitive to environmental conditions and vibrations. It has also been demonstrated in various publications that the laser source wavelength in QPI plays an important role in the imaging system’s performance. It has been shown that in the ultraviolet regime absorption increases with shorter wavelengths for proteins, DNA, and other molecules. In the infrared regime, absorption increases with longer wavelengths due to presence of the water content in the tissues. Moreover, near 650 nm to 1.3 μm wavelength window is called as therapeutic or diagnostic window where absorption is less. In this diagnostic regime near 1-μm wavelength window, the absorption is very small for various biological tissues, making it suitable for biomedical imaging for diagnostic or therapeutic applications. Here we have proposed and demonstrated to explore the advantages of this therapeutic wavelength window together with non-interferometric QPI for the measurement of refractive index, stress-strain on the cell membrane, mass, and volume of the sample. Moreover, two-photon absorption process also provides significant advantages, such as higher depth selectivity, high resolution, less photo-toxicity, and better localization in optical imaging. Here we are also leveraging the two-photon absorption for 1-μm wavelength regime QPI to improve its performance and in other words, demonstrated phase sensitive two-photon microscopy (PS2PM) or two photon QPI microscopy.