Optical Microscope System Research Articles

Development of an optical microscopy system for automated bubble cloud analysis.

Recently, the number of uses of bubbles has begun to increase dramatically, with medicine, biofuel production, and wastewater treatment just some of the industries taking advantage of bubble properties, such as high mass transfer. As a result, more and more focus is being placed on the understanding and control of bubble formation processes and there are currently numerous techniques utilized to facilitate this understanding. Acoustic bubble sizing (ABS) and laser scattering techniques are able to provide information regarding bubble size and size distribution with minimal data processing, a major advantage over current optical-based direct imaging approaches. This paper demonstrates how direct bubble-imaging methods can be improved upon to yield high levels of automation and thus data comparable to ABS and laser scattering. We also discuss the added benefits of the direct imaging approaches and how it is possible to obtain considerable additional information above and beyond that which ABS and laser scattering can supply. This work could easily be exploited by both industrial-scale operations and small-scale laboratory studies, as this straightforward and cost-effective approach is highly transferrable and intuitive to use.

Geometric effect on photonic nanojet generated by dielectric microcylinders with non-cylindrical cross-sections

We experimentally report the geometric effect on photonic nanojet generated by dielectric microcylinders with non-cylindrical cross-sections. The non-cylindrical cross-sections include truncated microcylinders and elliptical microcylinders. The truncated microcylinder is formed with the cutting thickness. The elliptical microcylinder is defined by ratio of minor to major axes. The specific spatial electromagnetic fields for microcylinders with different non-cylindrical cross-sections are studied by using finite-difference time-domain calculation. The direct imaging of photonic nanojets for microcylinders with different non-cylindrical cross-sections are performed with a scanning optical microscope system. The field distribution and location of photonic nanojet depending on the various spatial shapes are experimentally demonstrated. The photonic nanojet with long focal length and low divergence could be used to scan over a target to obtain a large area image when the non-cylindrical micro-media are coupled with a conventional optical microscope.

Light scattering model for individual sub-100-nm particle size determination in an evanescent field

In this paper, we propose an optical method for observation and determination of individual nanosized particles that adhere to an interface by applying an evanescent field. Subsequently, we developed a portable (∼350 mm in length) experimental apparatus equipped with an optical microscopy system for particle observation. The observed intensity is consistent with that calculated using a light scattering model of sub-100-nm particles in the evanescent field.

Microscope system with on axis programmable Fourier transform filtering

We propose an on-axis microscope optical system to implement programmable optical Fourier transform image processing operations, taking advantage of phase and polarization modulation of a liquid crystal on silicon (LCOS) display. We use a Hamamatsu spatial light modulator (SLM), free of flickering, which therefore can be tuned to fully eliminate the zero order component of the encoded diffractive filter. This allows the realization of filtering operation on axis (as opposed to other systems in the literature that require operating off axis), therefore making use of the full space bandwidth provided by the SLM. The system is first demonstrated by implementing different optical processing operations based on phase-only blazed gratings such as phase contrast, band-pass filtering, or additive and substractive imaging. Then, a simple Differential interference contrast (DIC) imaging is obtained changing to a polarization modulation scheme, achieved simply by selecting a different incident state of polarization on the incident beam.

Optical coherence photoacoustic microscopy (OC-PAM) with an intensity-modulated continuous-wave broadband light source

We developed an optical coherence photoacoustic microscopy system using an intensity-modulated continuous-wave superluminescent diode with a center wavelength of 840 nm. The system can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously. Compared to the system with a pulsed light source, this system is able to achieve OCT imaging with quality as high as conventional spectral-domain OCT. Since both of the OCT and PAM images are generated from the same group of photons, they are intrinsically registered in the lateral directions. The system was tested for multimodal imaging the vasculature of mouse ear in vivo by using gold nanorods as contrast agent for PAM, as well as excised porcine eyes ex vivo. The OCT and PAM images showed complimentary information of the sample.

Investigation of serrated chip morphology change regular in the burning of titanium alloys

Titanium alloys are regarded as difficult-machining materials due to their high strength at elevated temperature, relatively low thermal conductivity, small elastic modulus, and high chemical reactivity. It is prone to produce cutting burn defect in end milling. In order to investigate the variation regular of serrated chip morphologies and the relationship between chip macro morphologies and tool flank wear in the burning of milling titanium alloys TC4, a series of cutting experiments were conducted in this study. The chip morphology and cutting vibration characteristic in different tool wear stages were examined using an optical microscope, SEM, and vibration test system. In addition, the variation characteristics of serrated chips in the burning of milling titanium alloy TC4 were analyzed. The results revealed that the occurrence of tool flank wear can make an influence on serrated chips morphologies and surface color in the cutting burning process. Besides, the chip segment degree, shear angle, and width of adiabatic shear band increased rapidly as the cutting burning occurring. The present research could be potentially used to evaluate tool wear and surface quality in the burning of end milling titanium alloy process.

Autofocus by Bayes Spectral Entropy Applied to Optical Microscopy.

This study introduces a passive autofocus method based on image analysis calculating the Bayes spectral entropy (BSE). The method is applied to optical microscopy and together with the specific construction of the opto-mechanical unit, it allows the analysis of large samples with complicated surfaces without subsampling. This paper will provide a short overview of the relevant theory of calculating the normalized discrete cosine transform when analyzing obtained images, in order to find the BSE measure. Furthermore, it will be shown that the BSE measure is a strong indicator, helping to determine the focal position of the optical microscope. To demonstrate the strength and robustness of the microscope system, tests have been performed using a 1951 USAF test pattern resolution chart determining the in focus position of the microscope. Finally, this method and the optical microscope system is applied to analyze an optical grating (100 lines/mm) demonstrating the detection of the focal position. The paper concludes with an outlook of potential applications of the presented system within quality control and surface analysis.

Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures.

The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

Photonic nanojet induced modes generated by a chain of dielectric microdisks

High intensity and low divergence photonic nanojet-induced modes are observed in a chain of dielectric microdisks illuminated from the side with laser source of wavelengths 405nm, 532nm, and 671nm. The properties of transmission in a chain of dielectric microdisks are calculated by using finite-difference time-domain method. The spectra of propagation lightwave within the chains of SiO2 and Si3N4 microdisks are directly measured by a scanning optical microscope system with an optical-fiber probe. The photonic nanojet-induced modes are observed from the optical microscope images for the 3μm, 5μm, and 8μm microdisk chains on a silicon substrate. The incident lightwave can be periodically reproduced in chains of dielectric microdisks due to photonic nanojets. The theoretical and experimental results on the field-of-view, transmission, and scattering loss are reported. Understanding these nanojet-induced modes is critical for selecting suitable microdisks to construct optical waveguides and photonic circuits for efficient lightwave guiding.

Investigation on Axicon Transformation in Digital Holography for Extending the Depth of Focus in Bio-Microfluidics Applications

Limited depth of focus (DOF) is one main shortage in traditional optical microscopy systems that severely affect simultaneous visualization of objects at different depths in the same field of view. In this paper, we propose a novel method to enhance the DOF in digital holography that consists of applying a numerical axicon transformation to the hologram during the reconstruction process. The idea behind this is to exploit the well-known ability of an axicon lens to create long and narrow focal lines along the optical axis. By this approach, we demonstrate that it is possible to obtain an extended focused image in which objects located at different depths are simultaneously visualized in good focus. First, the proposed method is tested in a case study of three different wires, positioned on different planes and recorded in lensless configuration. A comparison with a common DOF extension approach based on cubic phase function is performed. Finally, experiments of motile cells, flowing in a microfluidic channel and at different depths, are investigated for demonstrating the effectiveness of the proposed approach in bio-microfluidics.

Fast subcellular optical coherence photoacoustic microscopy for pigment cell imaging.

We developed a fast ultrahigh resolution optical coherence photoacoustic microscopy (FU-OCPAM) system by combining two complementary imaging modes of optical coherence microscopy (OCM) and photoacoustic microscopy (PAM) for cellular/subcellular imaging. The system used optical scanning to realize fast imaging speed and provided ultrahigh resolution of 1.24 and 0.59 μm for OCM and PAM, respectively. We imaged the retinal pigment epithelium (RPE) to demonstrate the subcellular imaging capability of the FU-OCPAM system. The OCM and PAM images clearly showed the RPE cell morphology and reflected the complementary optical properties of scattering and absorption. A quantitative analysis of the RPE cells was made based on photoacoustic (PA) signals. The cell area mainly ranged from 80 to 300 μm2, and had a linear relationship with the sum intensity of PA signals which mainly reflected the melanin content of the cells. The morphology and the PA signal could be used to identify qualitatively and quantitatively the aging and healthy states of the RPE cells. The results show the potential applications in studying the real-time cellular response to external stimulations and the progress of aging and diseases at the cellular level with FU-OCPAM.

Wide-field, full-field optical coherence microscopy for high-axial-resolution phase and amplitude imaging

An original single-objective, full-field optical coherence microscopy system is reported that is capable of imaging both the phase and the amplitude of semi-transparent samples over a field of view of 17.5 mm×17.5 mm with an axial sectioning resolution of 1.5 μm. A special stack acquisition arrangement ensures optimal reachable imaging depth. Several phase-shifting interferometry algorithms for phase measurement with broadband light are compared theoretically and experimentally. Using the phase information, noninvasive depth-resolved topographic images of multilayer samples are produced to characterize each layer by measuring their defects and curvature with a nanometric scale precision. Using the amplitude information, tomographic images with a constant detection sensitivity of ∼80 dB through the entire field of view are obtained and applied to biological specimens.

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

Performance Comparison of Two Ellipse Fitting-Based Cell Separation Algorithms

Cells in a culture process transform with time and produce many overlapping cells in their vicinity. We are interested in a separation algorithm for images of overlapping cells taken using a fluorescence optical microscope system during a cell culture process. In this study, all cells are assumed to have an ellipse-like shape. For an ellipse fitting-based method, an improved least squares method is used by decomposing the design matrix into quadratic and linear parts for the separation of overlapping cells. Through various experiments, the improved least squares method (numerically stable direct least squares fitting [NSDLSF]) is compared with the conventional least squares method (direct least squares fitting [DLSF]). The results reveal that NSDLSF has a successful separation ratio with an average accuracy of 95% for two overlapping cells, an average accuracy of 91% for three overlapping cells, and about 82% accuracy for four overlapping cells.

Acoustic signal characteristics of laser induced cavitation in DDFP droplet: Spectrum and time-frequency analysis

Cavitation has great application potential in microvessel damage and targeted drug delivery. Concerning cavitation, droplet vaporization has been widely investigated in vitro and in vivo with plasmonic nanoparticles. Droplets with a liquid dodecafluoropentane (DDFP) core enclosed in an albumin shell have a stable and simple structure with good characteristics of laser absorbing; thus, DDFP droplets could be an effective aim for laser-induced cavitation. The DDPF droplet was prepared and perfused in a mimic microvessel in the optical microscopic system with a passive acoustic detection module. Three patterns of laser-induced cavitation in the droplets were observed. The emitted acoustic signals showed specific spectrum components at specific time points. It was suggested that a nanosecond laser pulse could induce cavitation in DDPF droplets, and specific acoustic signals would be emitted. Analyzing its characteristics could aid in monitoring the laser-induced cavitation process in droplets, which is meaningful to theranostic application.

Investigation on the influence of tool wear upon chip morphology in end milling titanium alloy Ti6Al4V

Titanium alloys are widely utilized in aerospace, automotive, biomedical and chemical engineering, etc., thanks to their excellent combination of high-specific strength, fracture, corrosion resistance characteristics, etc. However, titanium alloys are difficult-to-machine materials. Tool wear is one of the bottlenecks restricting their machining efficiency. A systematic study on the relationships among tool wear, chip morphology, and cutting vibration is inadequate. In this study, chip morphology and cutting vibration characteristics under different tool wear stages are examined using optical microscope, SEM, and vibration test system. The mechanism of tool wear in end milling titanium alloy is also investigated. Results indicate that with the progression of tool wear, the chip segment degree becomes more and more serious. The mechanism for this phenomenon is probed. Tool wear progression enlarges the cutting vibration which causes the friction force on tool/chip interfaces to increase, and this aggravates chip edge wear accordingly. On the contrary, the increase of chip segment degree induces the progression of cutting vibration and tool wear. Therefore, the aim of the present research is to investigate the sophisticated relationship. This will benefit for improving cutting efficiency and guaranteeing machining quality in end milling titanium alloy.

Plasmonic lens with multi-circular-slit apertures for improvement of light utilization efficiency

A plasmonic lens (PL) is one of the promising photonic devices utilizing the surface plasmon wave. Recently, we evaluated the focusing characteristics of PLs consisting of a single-spiral-slit aperture of several µm diameter, and attained a tightly focused beam with a subwavelength spot size in the far-field region. In this study, we designed and fabricated a PL composed of concentric multi-circular-slit apertures for 405 nm wavelength to increase the light utilization efficiency compared with the conventional PLs with a single-spiral-slit aperture. We have simulated the electric field distribution of the newly developed PL, and confirmed that the light intensity increased more than twofold at the focal spot compared with the conventional PL. Also, the focal spot with subwavelength size was maintained by phase adjustment of the surface plasmon wavelength inside the multiple circular slits of different slit widths. We have measured the focusing characteristics of the PL using the near-field scanning optical microscopy (NSOM) system and confirmed a half-wavelength spot size (200 nm) at 1.3 µm above the PL surface.

Development of a Portable Optical Interferometry Microscope System

Optical interferometry methods are widely used for measuring microdisplacement with nanometer accuracy. However, most commercially available optical interferometry systems are large and expensive for manufacturing applications. In this study, we report the development of a low-cost portable optical interferometry microscope for factory use. The light source was a tungsten–halogen white lamp with an optical filter. The microscope has an objective lens with a numerical aperture of 0.3, a magnifying power of 10, and field depth of 3.056 μm. Interference images were collected with an NTSC CCD-video camera. The resolution of the interference image is 320 × 240 pixels and stored in BMP format. To obtain phase shifted interferometry images, a piezoelectric actuator was used to monitor the table movement along the optical axis. The total cost of all system parts is approximately 7000 to 8000 US dollars. To evaluate the basic performance of the developed interferometry microscope, we measured a steel ball, the penetration mark of a Rockwell scale hardness indenter, and a gauge block surface with a bump. The developed interferometry microscope can measure continuous and gently sloping surfaces. The processing time is approximately 10–20 s.

Inhibition of martensitic formation and mechanical properties of the TZ20 alloy during hot rolling

The microstructure and mechanical properties of TZ20 (Ti–20Zr–6.5Al–4V) alloy after single-pass hot rolling with reductions from 30% to 50% are investigated through XRD, Olympus optical microscope, TEM and Instron testing system. The annealed specimen is composed of major α phase and small amounts of β phases. However, hot-rolled specimens followed by water quenching consist of major β phase and a few α″ martensites. Results also show that hot deformation can inhibit the formation of α″ martensite. The fraction of α″ martensite in hot-rolled specimen decreases with the increasing of rolling reduction from 30% to 50%. Tensile tests show that the yield strength of hot-rolled specimens increases with the increasing of rolling reduction. Conversely, the total elongation decreases with reduction. However, the variation of tensile strength with rolling reduction is inconspicuous. The strengthening mechanism of TZ20 alloy after hot deformation is also discussed. This study is important in understanding the martensitic transformation, microstructural evolution and strengthening mechanism of metals and alloys and helps in adjusting and controlling the microstructure and properties of the TZ20 alloy.

Analysis system of submicron particle tracks in the fine-grained nuclear emulsion by a combination of hard x-ray and optical microscopy.

Analyses of nuclear emulsion detectors that can detect and identify charged particles or radiation as tracks have typically utilized optical microscope systems because the targets have lengths from several μm to more than 1000 μm. For recent new nuclear emulsion detectors that can detect tracks of submicron length or less, the current readout systems are insufficient due to their poor resolution. In this study, we developed a new system and method using an optical microscope system for rough candidate selection and the hard X-ray microscope system at SPring-8 for high-precision analysis with a resolution of better than 70 nm resolution. Furthermore, we demonstrated the analysis of submicron-length tracks with a matching efficiency of more than 99% and position accuracy of better than 5 μm. This system is now running semi-automatically.