Long-term quantitative phase-contrast imaging of living cells by digital holographic microscopy

The dynamic analysis of biological living samples is one of the particular interests in life sciences. An improved digital holographic microscope for long-term quantitative phase-contrast imaging of living cells is presented in this paper. The optical configuration is optimized in the form of a free-space-fiber hybrid system which promotes the flexibility of imaging in complex or semi-enclosed experimental environment. Aberrations compensation is implemented taking into account the additional phase aberration induced by liquid culture medium in long-term observation. The proposed approach is applied to investigate living samples of MC3T3-E1 and MLO-Y4 cells. The experimental results demonstrate its availability in the analysis of cellular changes.     

Dynamic and quantitative phase-contrast imaging of living cells under simulated zero gravity by digital holographic microscopy and superconducting magnet

The experiment of dynamic and quantitative phase-contrast imaging of living cells in simulated zero gravity environment were performed by using digital holographic microscopy (DHM) combined with a superconducting magnet (SM). The SM with large gradient high magnetic field was used to simulate zero gravity by levitating biological living samples. The proposed DHM system provided highly efficient and versatile means for dynamically and quantitatively phase-contrast imaging MC3T3-E1 cells. To our knowledge, the phase images of living cells undergoing modifications and division under simulated zero gravity were firstly obtained by using DHM-SM prototype.     

Common-path phase-shifting digital holographic microscopy: A way to quantitative phase imaging and superresolution

We present an experimental setup useful for complex amplitude evaluation and phase image quantification of three-dimensional (3-D) samples in digital holographic microscopy (DHM). It is based on a common-path interferometric configuration performed by dividing the input plane in two contiguous regions and by placing a translation grating near to the Fourier plane. Then, complex amplitude distribution of the sample under test is recovered with phase-shifting standard method obtained by moving the grating using a linear motion stage. Some experimental results of an USAF resolution test are presented for different numerical aperture (NA) microscope lenses. In a second part, the proposed setup is tested under superresolution purposes. Based on the object’s spectrum shift produced by off-axis illumination, we use time multiplexing to generate a synthetic aperture enlargement that improves the final image resolution. Experimental results for the case of a biosample (human red blood cells) and a commercial low NA microscope lens validates the suggested superresolution approach.     

Ultrasonic intensity microscopy for imaging of living cells

Ultrasound intensity microscopy was developed for in vivo imaging. This paper describes the preliminary results obtained using 300 MHz ultrasound intensity microscopy for in vitro characterization of cell cultures. The novelty of the approach lies in the fact that it allows remote, non-contact and disturbance-free imaging of cultured synovial cells and the changes in the cells’ properties due to external stimulants such as transforming growth factor beta-1 (TGF-β1). The intensity imaging method has potential for extracting mechanical cell properties and monitoring the effects of drugs.Ultrasound propagates through a thin specimen such as cultured cells and is reflected at the interface between the specimen and substrate. A two-dimensional distribution of the ultrasonic intensity, which is closely related to the mechanical properties, is visualized to analyze cell organs, such as the nucleus at the central part and the cytoskeleton at the peripheral zone. After stimulation with TGF-β1, the ultrasonic intensity at the actin zone was significantly increased compared with the control.     

Extended focused imaging of a microparticle field with digital holographic microscopy

We present a numerical technique for extended focused imaging and three-dimensional analysis of a microparticle field observed in a digital holographic microscope working in transmission. The three-dimensional localization of objects is performed using the local focus plane determination method based on the integrated amplitude modulus. We apply the refocusing criterion locally for each pixel, using small overlapping windows, to obtain the depth map and a synthetic image in which all objects are refocused independent from their refocusing distance. A successful application of this technique in the analysis of the microgravity particle flow experiment is presented.     

Deep-learning-based prediction of living cells mitosis via quantitative phase microscopy

We present a deep learning approach for living cells mitosis classification based on label-free quantitative phase imaging with transport of intensity equation methods. In the approach, we applied a pretrained deep convolutional neural network using transfer learning for binary classification of mitosis and non-mitosis. As a validation, we demonstrated the performances of the network trained by phase images and intensity images, respectively. The convolutional neural network trained by phase images achieved an average accuracy of 98.9% on the validation data, which outperforms the average accuracy 89.6% obtained by the network trained by intensity images. We believe that the quantitative phase microscopy in combination with deep learning enables researchers to predict the mitotic status of living cells noninvasively and efficiently.     

Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μм SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin (20 μм) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cytosol and nuclear regions, indicating the ability of this technique to map intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10°C for 90 min. The pH values were calibrated in vitro, using SNARF-1 acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central necrotic cells. The degree of involvement of acidification in muscle fatigue was investigated by simultaneously determining force generation and intracellular pH in individual fibres of an intact rat muscle. The investigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue protocol induced little change in cytosolic pH in the fibres. We show that the use of SNARF-1, in conjunction with confocal microscopy is a powerful technique for accurately mapping pH within single cells, multicellular tissues and intact organs, as well as for accurately recording dynamic changes in pH.     

Dynamic imaging with MRI contrast agents: quantitative considerations

Time-resolved MRI has had enormous impact in cognitive science and may become a significant tool in basic biological research with the application of new molecular imaging agents. In this paper, we examine the temporal characteristics of MRI contrast agents that could be used in dynamic studies. We consider "smart" T1 contrast agents, T2 agents based on reversible aggregation of superparamagnetic nanoparticles and sensors that produce changes in saturation transfer effects (chemical exchange saturation transfer, CEST). We discuss response properties of several agents with reference to available experimental data, and we develop a new theoretical model that predicts the response rates and relaxivity changes of aggregation-based sensors. We also perform calculations to define the extent to which constraints on temporal resolution are imposed by the imaging methods themselves. Our analysis confirms that some small T1 agents may be compatible with MRI temporal resolution on the order of 100 ms. Nanoparticle aggregation T2 sensors are applicable at much lower concentrations, but are likely to respond on a single second or slower timescale. CEST agents work at high concentrations and temporal resolutions of 1-10 s, limited by a requirement for long presaturation periods in the MRI pulse sequence.     

4-D imaging of fluid flow with digital in-line holographic microscopy

The large depth of field of digital in-line holographic microscopy (DIHM) with numerical reconstruction provides an ideal tool for the study of microfluidic phenomena. As indicators of the flow patterns we use latex microspheres and also red blood cells whose three-dimensional trajectories and velocities can easily be measured as a function of time with subsecond and micron resolution. We demonstrate the efficiency of DIHM by showing 3-D views of the flow patterns around big spheres in various geometric arrangements.     

Incoherent digital holographic spectral imaging with high accuracy of image pixel registration

Fresnel incoherent correlation holography(FINCH) is a unique three-dimensional(3 D) imaging technique which has the advantages of scanning-free,high resolution,and easy matching with existing mature optical systems.In this article,an incoherent digital holographic spectral imaging method with high accuracy of spectral reconstruction based on liquid crystal tunable filter(LCTF) and FINCH is proposed.Using the programmable characteristics of spatial light modulator(SLM),a series of phase masks,none of whose focal lengths changes with wavelength,is designed and made.For each wavelength of LCTF output,SLM calls three phase masks with different phase constants at the corresponding wavelength,and CCD records three holograms.The spectral images obtained by this method have a constant magnification,which can achieve pixel-level image registration,restrain image registration errors,and improve spectral reconstruction accuracy.The results show that this method can not only obtain the 3 D spatial information and spectral information of the object simultaneously,but also have high accuracy of spectral reconstruction and excellent color reproducibility.     

Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy

We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue.     

Refractive-index profiling of optical fibers with axial symmetry by use of quantitative phase microscopy

The application of quantitative phase microscopy to refractive-index profiling of optical fibers is demonstrated. Phase images of axially symmetric optical fibers immersed in index-matching fluid are obtained, and the inverse Abel transform is used to obtain the radial refractive-index profile. This technique is straightforward, nondestructive, repeatable, and accurate. Excellent agreement, to within approximately 0.0005, between this method and the index profile obtained with a commercial profiler is obtained.     

Differential axial contrast of optical sections: laser microtomography and quantitative 3D reconstruction

Specific features of the quantitative laser microtomography of biological samples are discussed. The method exhibits the main advantages of a confocal microscope (rapid measurement of a stack of parallel optical cross sections and accurate displacement of an object along the optical axis). A relatively high contrast is reached owing to the superposition of pairwise complementary images on neighboring cross sections. A simple and convenient algorithm for image processing does not require additional software and can be computerized using a conventional graphic editor. The applicability of the method is illustrated using volume measurements of a single cell of an early mouse embryo.     

Spectral unmixing combined with Raman imaging,a preferable analytic technique for molecule visualization

This article reviews spectral unmixing used as a new method for Raman imaging. Raman spectroscopy explores the vibrational information about specific chemical bonds in molecules, which can be used for label-free molecular visualization. However, chemical bonds are usually shared among different molecules, which results in closed or mixed Raman peaks of many molecules. Therefore, the acquired spectra cannot be directly used to reconstruct the Raman images, as pure component spectra are hidden under the acquired spectra. Spectral unmixing is an effective method to provide a meaningful spectrum of each component with no priori spectral information and to also reconstruct the compositional distribution images. This article summarizes some representative spectral unmixing approaches used for Raman imaging and many related researches. This review strives to introduce the combination of spectral unmixing and Raman imaging as an efficient analytic technique to characterize various constituents and make subtle understanding of those complex structures.     

Automated segmentation and quantitative study of retinal pigment epithelium cells for photoacoustic microscopy imaging

We develop an improved region growing method to realize automatic retinal pigment epithelium(RPE) cell segmentation for photoacoustic microscopy(PAM) imaging. The minimum bounding rectangle of the segmented region is used in this method to dynamically update the growing threshold for optimal segmentation. Phantom images and PAM imaging results of normal porcine RPE are applied to demonstrate the effectiveness of the segmentation. The method realizes accurate segmentation of RPE cells and also provides the basis for quantitative analysis of cell features such as cell area and component content, which can have potential applications in studying RPE cell functions for PAM imaging.     

Measurement of refractive index change induced by dark reaction of photopolymer with digital holographic quantitative phase microscopy

Light-induced refractive index change in photopolymer is quantified by a digital holographic microscope. The refractive index change is induced as the dark reaction which proceeds inside the photopolymer after a writing beam is stopped. Time-lapse phase distribution across the photopolymer is measured by the off-axis digital holographic microscope which enables us to retrieve the 2-D phase map from a single hologram. It is found that the initial phase profile does not coincide with the illumination intensity distribution. This observation suggests that the rate of the refractive index change in dark reaction is not proportional to the illumination intensity in case the exposure energy becomes high.     

一种使用高灵敏度荧光显微镜系统进行活细胞研究的方法

利用显微镜进行细胞观察和实验，一般的做法都是将细胞做成切片，细胞难免会受到损伤，从而影响实验及观察结果。为了使细胞研究更为准确，便于临床应用，开发、研制活细胞的观察工具是必由之路。以高灵敏度荧光显微镜为基础，合理设计了一种新型暗场照明器。这种新型暗场照明器的工作距离可达３００ｍｍ，完全可以满足为活细胞滴加试剂的需要，已采得经过荧光染色的活细胞图像。这一设计解决了一个非常实际的问题，为活细胞的医学研究奠定了坚实的基础。     

Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system

A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.