Massive image acquisition is required along the optical axis in the classical image-analysis-based autofocus method, which significantly decreases autofocus efficiency. A wavefront-sensing-based autofocus technique is proposed to increase the speed of autofocusing and obtain high localization accuracy. Intensities at different planes along the optical axis can be computed numerically after extracting the wavefront at defocus position with the help of the transport-of-intensity equation method. According to the focus criterion, the focal plane can then be determined, and after sample shifting to this plane, the in-focus image can be recorded. The proposed approach allows for fast, precise focus detection with fewer image acquisitions compared to classical image-analysis-based autofocus techniques, and it can be applied in commercial microscopes only with an extra illumination filter.
Erythrocyte morphology is an important factor in disease diagnosis, however, traditional setups as microscopes and cytometers cannot provide enough quantitative information of cellular morphology for in-depth statistics and analysis. In order to capture variations of erythrocytes affected by metal ions, quantitative interferometric microscopy (QIM) is applied to monitor their morphology changes. Combined with phase retrieval and cell recognition, erythrocyte phase images, as well as phase area and volume, can be accurately and automatically obtained. The research proves that QIM is an effective tool in cellular observation and measurement.
Quantitative interferometric microscopy is used in biological and medical fields and a wealth of applications are proposed in order to detect different kinds of biological samples. Here, we develop a phase detecting cytometer based on quantitative interferometric microscopy with expanded principal component analysis phase retrieval method to obtain phase distributions of red blood cells with a spatial resolution ~1.5 μm. Since expanded principal component analysis method is a time-domain phase retrieval algorithm, it could avoid disadvantages of traditional frequency-domain algorithms. Additionally, the phase retrieval method realizes high-speed phase imaging from multiple microscopic interferograms captured by CCD camera when the biological cells are scanned in the field of view. We believe this method can be a powerful tool to quantitatively measure the phase distributions of different biological samples in biological and medical fields.
Quantitative interferometric microscopy is an important method for observing biological samples such as cells and tissues. In order to obtain continuous phase distribution of the sample from the interferogram, phase extracting and phase unwrapping are both needed in quantitative interferometric microscopy. Phase extracting includes fast Fourier transform method and Hilbert transform method, etc., almost all of them are rapid methods. However, traditional unwrapping methods such as least squares algorithm, minimum network flow method, etc. are time-consuming to locate the phase discontinuities which lead to low processing efficiency. Other proposed high-speed phase unwrapping methods always need at least two interferograms to recover final phase distributions which cannot realize real time processing. Therefore, high-speed phase unwrapping algorithm for single interferogram is required to improve the calculation efficiency. Here, we propose a fast phase unwrapping algorithm to realize high-speed quantitative interferometric microscopy, by shifting mod 2π wrapped phase map for one pixel, then multiplying the original phase map and the shifted one, then the phase discontinuities location can be easily determined. Both numerical simulation and experiments confirm that the algorithm features fast, precise and reliable.
The paper proposed a simple large scale bio-sample phase detecting equipment called gravity driven phase detecting cytometer, which is based on quantitative interferometric microscopy to realize flowing red blood cells phase distribution detection. The method has advantages on high throughput phase detecting and statistical analysis with high detecting speed and in real-time. The statistical characteristics of red blood cells are useful for biological analysis and disease detection. We believe this method is shedding more light on quantitatively measurement of the phase distribution of bio-samples.
Single-shot quantitative interferometric microscopy (QIM) needs a high-accuracy and rapid phase retrieval algorithm. Retrieved phase distributions are often influenced by phase aberration background caused by both imaging system and phase retrieval algorithms. Here, we propose an improved phase aberration compensation (PAC) approach in order to eliminate the phase aberrations inherent in the data. With this method, sample-free parts are identified and used to calculate the background phase, reducing phase errors induced in samples and providing high-quality phase images. We now demonstrate that QIM based on this PAC approach realizes high-quality phase imaging from a single interferogram. This is of great potential for a real-time speedy diagnosis.