Significance: Large space-bandwidth product is highly desirable in many biomedical imaging. Fourier ptychographic microscopy (FPM) is a computational imaging technique that can significantly increase the space-bandwidth product of a standard microscope. The illuminator of a Fourier ptychographic microscope is not flexible at present, and it is inconvenient to meet different imaging needs.
Aim: An illuminator based on a two-axis motorized rotation stage was presented to provide a more flexible illuminating way with the goal of meeting different imaging needs.
Approach: The illuminator adopts a concentric illuminating method to provide coherent illumination in any direction on the sample plane. The sampling pattern can be freely designed and changed according to the parameters of the imaging system. A dither removing algorithm was proposed to remove the potential dither influence introduced in the image acquisition process.
Results: The illuminator could be conveniently integrated into different imaging systems. The feasibility and flexibility were demonstrated by applying it to imaging systems with numerical aperture of 0.045 and 0.01. The resolution gain is about 4- and 13-fold, respectively. The effectiveness of the dither removing algorithm was validated in both simulation and experiment.
Conclusions: A more flexible illuminator for FPM was presented to meet different imaging needs. A dither removing algorithm was proposed to remove dither influence.
The refractive index of blood is a key biophysical parameter, which can reflect the physiological state. We measured the refractive index of whole blood and other components, such as serum, plasma, and hemoglobin, based on internal reflection by using a homemade apparatus in the spectral range of 400 to 750 nm. In addition to the hemoglobin solution, which has a Soret band about 420 nm and two Q-bands between 500 and 600 nm, the measurements of other samples are the normal dispersion curve. The results are approximated by the Cauchy equation and Sellmeier equation, and the correlation coefficients are more than 0.997.
We investigated the effect of thiazone, a widely used penetration enhancer, on in vitro porcine skin and muscle tissue by single-integrating sphere technique during optical clearing (OC) treatment. The results showed that thiazone induced an increase on the total transmittance of skin which led to a reduction in that of muscle in the spectral range from 400 to 800 nm. Small particles crystalized out from the thiazone-treated muscle were observed by microscopy imaging. With the help of x-ray diffraction measurement, we ascertained that the crystal was a single-crystal of thiazone, which mainly induced an increase of the scattering. Contrast transmittance measurements carried on the mixture of water, thizaone–propylene glycol solution showed that the free water in muscle could be the main reason for the thiazone crystallization. Therefore, during OC treatment of thiazone, the remarkable effect on skin and the noticeable inhibition effect on subcutaneous muscle tissue after penetrating into the skin should be considered. The experimental results provide such a reference for the choice of penetration enhancer.
We report the quantitative refractive index (RI) imaging of cocultured cells in their living environment by scanning focused refractive index microscopy (SFRIM). Mouse microglial cells and synovial cells are cocultured on the top surface of a trapezoid prism. The RI imaging of living cells is obtained in a reflection-type method. The RI information is deduced with the simple derivative total internal reflection method, where a complex retrieval algorithm or reconstruction process is unnecessary. The outline of each cell is determined according to the RI value compared with that of the immersion liquid. The cocultured cells can be discriminated in the RI image. The measurement is nondestructive and label-free. The experimental results prove that SFRIM is a promising tool in the field of biological optics.
The complex refractive index dispersion (CRID), which contains the information on the refractive index dispersion and extinction coefficient spectra, is an important optical parameter of biotissue. However, it is hard to perform the CRID measurement on biotissues due to their high scattering property. Continuous CRID measurement based on internal reflection (CCRIDM-IR) is introduced. By using a lab-made apparatus, internal reflectance spectra of biotissue samples at multiple incident angles were detected, from which the continuous CRIDs were calculated based on the Fresnel formula. Results showed that in 400- to 750-nm range, hemoglobin solution has complicated dispersion and extinction coefficient spectra, while other biotissues have normal dispersion properties, and their extinction coefficients do not vary much with different wavelengths. The normal dispersion can be accurately described by several coefficients of dispersion equations (Cauchy equation, Cornu equation, and Conrady equation). To our knowledge, this is the first time that the continuous CRID of scattering biotissue over a continuous spectral region is measured, and we hereby have proven that CCRIDM-IR is a good method for continuous CRID research of biotissue.
The physical changes of tissue are complicated to evaluate during optical clearing (OC) treatment. Monitoring the changes of optical parameters, including the complex refractive index (CRI), helps people better understand the OC process. From the imaginary part of CRI, we can deduce the extinction coefficient of tissue. Based on the total internal reflection method, the time-dependent CRI of porcine muscle during natural dehydration is well determined. Results show that the real RI increases continuously with the increase of dehydration time, whereas the extinction coefficient initially increases and then decreases. Finally, the extinction coefficient becomes much smaller than the initial value, which demonstrates that better tissue optical clarity is obtained. The change tendency of the extinction coefficient of tissue is used to qualitatively explain the dynamic change of transmittance of a natural dehydrated tissue. Consequently, CRI, especially its imaginary part, is a very useful optical parameter by which to evaluate the OC effect.
We adapt the improved scanning focused refractive-index microscopy (SFRIM) technique to the quantitative study of
biological tissues. Delicate refractive index (RI) imaging of a porcine muscle tissue is obtained in a reflection mode.
Some modifications are made to the SFRIM for better two dimension (2-D) observation of the tissues. The RI accuracy
is 0.002. The central spatial resolution of SFRIM achieves 1μm, smaller than the size of the focal spot. Our method is
free from signal distortion. The experimental result demonstrates that SFRIM is a potential technique in a wide field of
biomedical research.
We report the dynamic refractive index (RI) change of tissue under a stepped compression load using a custom-built pressure apparatus. Angle-dependent reflectance profiles of biotissue samples are recorded, and the RI values are resolved using the derivative total reflection method. These results are relevant for understanding the mechanism of mechanical optical clearing, for investigating tissue dynamics under mechanical stimuli, and for other biomedical applications.
We investigate the effect of tissue fluid on the measurement of complex refractive index (RI) of animal tissue. A new model is proposed and verified through experimental results of simulation samples made of glycerol and methyl-red-doped poly(methyl methacrylate). Coupled with polarized optical reflectance measurements performed on several kinds of animal muscle tissues, RIs were resolved using the new model. We find that the tissue fluid existing at the prism-sample interface is unavoidable. We also find that with a change of proportion of the tissue fluid, the RI of muscle tissue can still be measured using the new model.
Tissue-equivalent phantom is becoming widespread as a substitute in the biological field to verify optical theories, test
measuring systems and study the tissue performances for varying boundary conditions, sample size and shape at a
quantitative level. Compared with phantoms made with Intralipid solution, ink and other liquid substances, phantom in
solid state is stable over time, reproducible, easy to handle and has been testified to be a suitable optical simulator in the
visible and near-infrared region. We present accurate determination of the complex refractive index (RI) of a solid tissueequivalent
phantom using extended derivative total reflection method (EDTRM). Scattering phantoms in solid state were
measured for p-polarized and s-polarized incident light respectively. The reflectance curves of the sample as a function
of incident angle were recorded. The real part of RI is directly determined by derivative of the reflectance curve, and the
imaginary part is obtained from nonlinear fitting based on the Fresnel equation and Nelder-Mead simplex method. The
EDTRM method is applicable for RI measurement of high scattering media such as biotissue, solid tissue-equivalent
phantom and bulk material. The obtained RI information can be used in the study of tissue optics and biomedical field.
Herein, we report two different dual-periodic Photonic Crystals (PCs) in dichromated gelatin emulsion which are
fabricated by four-beam holography and double-exposure holography. The minibands with high Q-factors have been
evidently located in both two structures. By taking into account the non-uniform distribution of material, the numerical
results agree quite well with the experimental results. We also compared the band-edge lasing in single-periodic PC and
miniband lasing in Moiré dual-periodic PC. Due to extremely flat dispersion and large mode volume of the miniband,
high optical conversion efficiency in miniband lasing is achieved as compared with that of band-edge lasing. Such effect
may provide potential applications in low-threshold lasers and ultra-sensitive fluorescent probes in biological assays.
Refractive index of biotissue is a useful optical parameter in the biomedical field. An extended differential total reflection method is introduced to determine the complex refractive index. The real part is directly determined by differential of the reflectance curve, and the imaginary part is obtained from nonlinear fitting. The method is verified by a series of tissue-mimicking phantoms, porcine muscle and porcine adipose.
Raising the scanning speed of a detection system is indispensable for practical applications of biochips such as fast clinical diagnoses and high-throughput filtration. Compared with PMT-based confocal scanning system, CCD-based one can afford simultaneous illumination and detection of multiple pixels with high speed. The performance of imaging lens system plays an important role in CCD-based fluorescence collection and imaging systems. The sensitivity, speed and resolution of the detection system are controlled by numerical aperture, field and aberration of lens system respectively. One of the key technologies of fast biochip detection system refers to the design of the imaging lens system. In this paper, the main characteristics of CCD-based fluorescence collection and imaging system are analyzed in detail, and an imaging lens system is designed to meet the requirements of fast scanning. An optical design software ZEMAX is applied to design and optimize the imaging lens system. The system parameters such as modulation transfer function and field curvature and distortion are obtained. The imaging of standard biochip samples with cyanine (CY5) dye dots on glass substrates are realized by our manufactured lens system. Results show that our lens system is suitable for biochip scanning system with high speed and high sensitivity. Its system parameters are as: numerical aperture for 0.52, viewing field for 10 mm, working distance for 22.5 mm, spatial resolution for 10um and small aberrations. The limitations of the imaging lens system and the routes for further improvement are discussed.
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