Standard Optical Microscope Research Articles

A Passive Microfluidic Device for Chemotaxis Studies.

This work presents a disposable passive microfluidic system, allowing chemotaxis studies, through the generation of a concentration gradient. The device can handle liquid flows without an external supply of pressure or electric gradients, but simply using gravity force. It is able to ensure flow rates of 10 µL/h decreasing linearly with 2.5% in 24 h. The device is made of poly(methylmethacrylate) (PMMA), a biocompatible material, and it is fabricated by micro-milling and solvent assisted bonding. It is assembled into a mini incubator, designed properly for cell biology studies in passive microfluidic devices, which provides control of temperature and humidity levels, a contamination-free environment for cells with air and 5% of CO2. Furthermore, the mini incubator can be mounted on standard inverted optical microscopes. By using our microfluidic device integrated into the mini incubator, we are able to evaluate and follow in real-time the migration of any cell line to a chemotactic agent. The device is validated by showing cell migration at a rate of 0.36 µm/min, comparable with the rates present in scientific literature.

A Microforce-Sensing Mobile Microrobot for Automated Micromanipulation Tasks

This paper presents a microforce-sensing mobile microrobot ( $\mu $ FSMM) for use in automated micromanipulation tasks. The design consists of a planar vision-based microforce sensor end-effector, while the microrobot body is made of chemically etched nickel that is driven by an exterior magnetic field. With a known stiffness, the manipulation forces can be determined from observing the deformation of the end-effector through a camera attached to an optical microscope. After analyzing and calibrating the stiffness of a micromachined prototype, the mobility and in situ force-sensing capabilities are verified through real-time, closed loop, force controlled manipulation tests with automated path planning and navigation. The calibrated stiffness of the microforce sensor end-effectors fabricated is on the order of 10−3 N/m. The online (real time) force-sensing resolution is approximately $1.5~\mu \text{N}$ . The sensing range is 0–20 $\mu \text{N}$ along the two planar directions. In automated micromanipulation experiments with a microcomponent, the $\mu $ FSMM utilizes real-time force control to apply a prescribed force of $6~\mu \text{N}$ to a desired location on a fixed microobject. Similarly, in another automated micromanipulation experiment, the $\mu $ FSMM demonstrates the use of real-time force control to limit the manipulation forces experienced by the microobject to remain below a threshold of 12 $\mu \text{N}$ . Note to Practitioners —This paper was motivated by recent interest single-cell biological micromanipulation tasks that seek to understand the role of environmental forces on the mechanics of cell development (mechanobiology) and the biological mechanisms that control such behavior (mechanotransduction). In addition, tissue engineering applications require the safe micromanipulation of single cells to desired locations in the workspace for growing tissue scaffolds. The $\mu $ FSMM presented here can be easily inserted into existing biological testbeds to use for these aforementioned applications. The designed magnetic coil system is compatible with standard inverted optical microscopes, while a digital camera for real-time image processing is already standard in these testbeds. The developed software interface can be used to prescribe automated microforce controlled manipulations of single cells and tissues in the workspace to carry out these aforementioned tasks. This paper is also suitable for carrying out general automated micromanipulation and microassembly tasks with advanced manufacturing applications.

Measuring Fiber Orientation of Elliptical Fibers from Optical Microscopy

The orientation of fibers with elliptical cross-sections cannot be estimated using standard optical microscopy analysis methods in which the ratio of the minor-axis to the major-axis and orientation of the major-axis are directly used to determine the fiber spherical coordinates, θ and ϕ . A new method for estimating the orientation of fibers with elliptical cross-sections is presented and validated using both simulations and experiments. Fibers with elliptical cross-sections rather than circular possess a roll degree of freedom, which significantly affects the dimensions of projected cross-sections in viewing planes. The equations of the projected ellipse of an elliptic cylinder onto a viewing plane are determined in terms of typical spherical coordinate system angles, θ and ϕ , the roll angle, α , and the fiber semi-major and semi-minor diameters. Fiber angles are determined by numerical fitting of the developed equations to measured ellipses. An ambiguity in the determined angles is identified, and, in the special case of fiber bundles, a scheme is presented by which the ambiguity can be resolved. Validation experiments showed that the method is quite effective at estimating fiber orientation from micrographs when fiber cross-section dimensions are measured beforehand, and the additional ambiguity is resolved easily in the case of fiber bundles.

Morpho-anatomy of the inflorescence of Musa × paradisiaca

Bananas and plantains are herbaceous monocotyledonous plants belonging to the genus Musa, Musaceae, which has a widespread distribution around the world. Various parts of banana plant are commonly used in traditional medicines. Several species of Musa are reported to possess anti-inflammatory, anti-hyperglycemic and antidiabetic properties. This work is aimed at studying the morphological and anatomical characteristics of the inflorescences of Musa×paradisiaca L., that could contribute to the characterization of these species cultivated in Brazil. Plant materials were collected and prepared in accordance with standard optical microscopy techniques. Morphological characterizations were conducted using morphological descriptors for inflorescences, including some descriptors from International Plant Genetic Resources Institute for Musa spp. Microscope slides were prepared using glycol-methacrylate and were stained in toluidine blue. Main features observed for M.×paradisiaca inflorescence were amphistomatic bracts with tetracytic stomata, fiber caps next to the phloem, adaxial and abaxial uniseriate epidermis, and papillose on the abaxial face. Outer tepals have multilayer epidermis and vascular bundles aligned next to the abaxial face. Free tepal has unilayeredepidermis. Anthers are tetrasporangiate and the locules are separated by the septum. Ovary is inferior and trilocular with external unilayered and internal epidermis. The main morpho-anatomical characteristics of inflorescence of Musa×paradisiaca are highlighted in this study, contributing to provide more information about the characterization of this species cultivated in Brazil.

High-Resolution Quantitative Phase Imaging of Plasmonic Metasurfaces with Sensitivity down to a Single Nanoantenna.

Optical metasurfaces have emerged as a new generation of building blocks for multifunctional optics. Design and realization of metasurface elements place ever-increasing demands on accurate assessment of phase alterations introduced by complex nanoantenna arrays, a process referred to as quantitative phase imaging. Despite considerable effort, the widefield (nonscanning) phase imaging that would approach resolution limits of optical microscopy and indicate the response of a single nanoantenna still remains a challenge. Here, we report on a new strategy in incoherent holographic imaging of metasurfaces, in which unprecedented spatial resolution and light sensitivity are achieved by taking full advantage of the polarization selective control of light through the geometric (Pancharatnam-Berry) phase. The measurement is carried out in an inherently stable common-path setup composed of a standard optical microscope and an add-on imaging module. Phase information is acquired from the mutual coherence function attainable in records created in broadband spatially incoherent light by the self-interference of scattered and leakage light coming from the metasurface. In calibration measurements, the phase was mapped with the precision and spatial background noise better than 0.01 and 0.05 rad, respectively. The imaging excels at the high spatial resolution that was demonstrated experimentally by the precise amplitude and phase restoration of vortex metalenses and a metasurface grating with 833 lines/mm. Thanks to superior light sensitivity of the method, we demonstrated for the first time to our knowledge the widefield measurement of the phase altered by a single nanoantenna while maintaining the precision well below 0.15 rad.

Object plane detection and phase retrieval from single-shot holograms using multi-wavelength in-line holography.

Phase retrieval and the twin-image problem in digital in-line holographic microscopy can be resolved by iterative reconstruction routines. However, recovering the phase properties of an object in a hologram requires an object plane to be chosen correctly for reconstruction. In this work, we present a novel multi-wavelength iterative algorithm to determine the object plane using single-shot holograms recorded at multiple wavelengths in an in-line holographic microscope. Using micro-sized objects, we verify the object positioning capabilities of the method for various shapes and derive the phase information using synthetic and experimental data. Experimentally, we built a compact digital in-line holographic microscopy setup around a standard optical microscope with a regular RGB-CCD camera and acquired holograms of micro-spheres, E. coli, and red blood cells, which are illuminated using three lasers operating at 491nm, 532nm, and 633nm, respectively. We demonstrate that our method provides accurate object plane detection and phase retrieval under noisy conditions, e.g.,using low-contrast holograms with an inhomogeneous background. This method allows for automatic positioning and phase retrieval suitable for holographic particle velocimetry, and object tracking in biophysical or colloidal research.

Direct mineralogical imaging of economic ore and rock samples with multi-modal nonlinear optical microscopy

Multi-modal nonlinear optical (NLO) microscopy, including stimulated Raman scattering (SRS) and second harmonic generation (SHG), was used to directly image mineralogical features of economic ore and rock samples. In SRS/SHG imaging, ore samples generally require minimal preparation and may be rapidly imaged, even in their wet state. 3D structural details, at submicron resolution, are revealed tens of microns deep within samples. Standard mineral imaging based on scanning electron microscopy (SEM), with elemental analysis via energy dispersive X-Ray spectroscopy, was used to independently validate the mineral composition of the samples. Spatially-resolved SRS from dominant Raman-resonant bands precisely maps the locations of specific minerals contained within the samples. SHG imaging reveals locally non-centrosymmetric structures, such as quartz grains. Competing absorption and nonlinear scattering processes, however, can reduce contrast in SRS imaging. Importantly, the correlation between standard electron microscopy and multi-modal NLO optical microscopy shows that the latter offers rapid image contrast based on the mineral content of the sample.

Gray-level co-occurrence matrix analysis of chromatin architecture in periportal and perivenous hepatocytes.

Periportal hepatocytes (PPHs) and perivenous hepatocytes (PVHs) in standard optical microscopy appear to be morphologically identical. However, the functional properties of these two cell populations and their roles in liver lobules are not the same. Despite significant differences in gene expression between these two hepatocyte populations, it is still unclear whether the differences are present at the higher levels of chromatin organization. In this study, we present results, indicating that periportal and perivenous hepatocytes, when stained using toluidine blue histological dye, have different chromatin textural patterns quantified with gray-level co-occurrence matrix (GLCM) method. Hepatic tissue was obtained from ten male, healthy mice. Chromatin structures were analyzed using GLCM. For each structure, we measured the values of angular second moment, inverse difference moment, GLCM Contrast, GLCM Variance, and GLCM Sum Variance. The results indicate that there is a statistically significant difference in all GLCM mathematical parameters except the contrast. In addition, some chromatin GLCM features were in correlation with serum aminotransferase levels in perivenous, but not in periportal hepatocytes. To the best of our knowledge, this is the first study to test the nuclear morphological differences between hepatocytes using GLCM and to investigate the respective relation with serum liver enzymes.

Surface-Acoustic-Wave (SAW)-Driven Device for Dynamic Cell Cultures.

In the last few decades, new types of cell cultures have been introduced to provide better cell survival and development, with micro- and nanoenvironmental physicochemical conditions aimed at mimicking those present in vivo. However, despite the efforts made, the systems available to date are often difficult to replicate and use. Here, an easy-to-use surface-acoustic-wave (SAW)-based platform is presented for realizing dynamic cell cultures that is compatible with standard optical microscopes, incubators, and cell-culture dishes. The SAW chip is coupled to a standard Petri dish via a polydimethylsiloxane (PDMS) disc and consists of a lithium niobate (LN) substrate on which gold interdigital transducers (IDTs) are patterned to generate the SAWs and induce acoustic streaming in the dish. SAW excitation is verified and characterized by laser Doppler vibrometry, and the fluid dynamics is studied by microparticle image velocimetry (μPIV). Heating is measured by an infrared (IR) thermal camera. We finally tested this device with the U-937 monocyte cell line for viability and proliferation and cell-morphological analysis. The data demonstrate that it is possible to induce significant fluid recirculation within the Petri dish while maintaining negligible heating. Remarkably, cell proliferation in this condition was enhanced by 36 ± 12% with respect to those of standard static cultures. Finally, we show that cell death does not increase and that cell morphology is not altered in the presence of SAWs. This device is the first demonstration that SAW-induced streaming can mechanically improve cell proliferation and further supports the great versatility and biocompatibility of the SAW technology for cell manipulation.

Quantitative optical mapping of two-dimensional materials

The pace of two-dimensional materials (2DM) research has been greatly accelerated by the ability to identify exfoliated thicknesses down to a monolayer from their optical contrast. Since this process requires time-consuming and error-prone manual assignment to avoid false-positives from image features with similar contrast, efforts towards fast and reliable automated assignments schemes is essential. We show that by modelling the expected 2DM contrast in digitally captured images, we can automatically identify candidate regions of 2DM. More importantly, we show a computationally-light machine vision strategy for eliminating false-positives from this set of 2DM candidates through the combined use of binary thresholding, opening and closing filters, and shape-analysis from edge detection. Calculation of data pyramids for arbitrarily high-resolution optical coverage maps of two-dimensional materials produced in this way allows the real-time presentation and processing of this image data in a zoomable interface, enabling large datasets to be explored and analysed with ease. The result is that a standard optical microscope with CCD camera can be used as an analysis tool able to accurately determine the coverage, residue/contamination concentration, and layer number for a wide range of presented 2DMs.

Application of Palynomorph Darkness Index (PDI) to assess the thermal maturity of palynomorphs: A case study from North Africa

This study focuses on the thermal maturity assessment of Silurian-Devonian sediments from the Ghadamis Basin, North Africa, comparing optical and geochemical analyses of palynomorphs. In southern Tunisia, the investigated subsurface cored section comprises the Argiles Principales Formation of Silurian age. In Libya, the succession studied covers the Awaynat Wanin III and IV formations, assigned to the Late Devonian (Frasnian-Famennian).Geochemical approaches used to reconstruct thermal alteration of sediments necessitate advanced, relatively expensive analytical techniques. In this study, the effectiveness of the less costly, relatively simple approaches of visually assessing palynomorph colour to determine thermal alteration (i.e., SCI: Spore Colour Index, TAI: Thermal Alteration Index and PDI: Palynomorph Darkness Index) was evaluated.SCI and TAI are qualitative methods, strictly related to the operator's perception, which use ten and five point scales respectively, to characterize colour in terms of illustrated specimens and/or descriptions. In contrast, PDI is obtained from the measurement of the red, green and blue (RGB) intensities of light transmitted through palynomorphs, using standard optical microscopes and digital cameras.The palynomorph-based thermal alteration estimates were compared to Rock-Eval pyrolysis data from the same samples. This calibration showed a linear relationship between these quantitative parameters and PDI. These results show that PDI is more reliable than the SCI and TAI methods.

A simple device for microinjections, manipulations and measurements using an electromorphological chip under mi-crointerferometric control of the interface and membrane processes at the thickness range of 5-1000 nm at different angles

Micromanipulations, perfusions and measurements performed using glass microelectrodes filled with an electrolyte is a conventional technique for experimental morphological and membrane electrophysiological studies at a single cell and membrane surface level. The typical (effective) diameter of the end of the glass microelectrode is from 500 up to less than 100 nm, which prevents one from observing it using a standard optical microscope in accordance with the optical resolution criteria, since the diameter less than 500 nm is indistinguishable within the interference zone. Microprocessor programming of the puller (microforge) that provides pulling and tearing allows to obtain in certain regimes the adjusted diameter and shape of the micropipette tip, although this result is not fully controlled due to the above limitations. In this connection it is necessary to design the control devices for the micropipette tips both at the preparation and operation stages (intracellular or extracellular insertion). This method also should provide visualization of the processes occurring upon interaction of the microelectrode tip with the cell in real time, depending on the electrode type and state, which allows to level the artifacts arising with the systematic error frequency from the uncontrolled operation of the micropipette tip after different ways of the microelectrode filling with the electrolyte. We propose an installation scheme that solves the above problems by means of introducing an interferometric device for microscopic control of the microelectrode and micromanipulator or microperfusor, for the first time for a given type of optical instruments combined with the interferometric optical scheme.

20 Assessment of Motion and Kinematic Characteristics of Semen from Four Cattle Breeds Using Computer-Aided Sperm Analysis

Subjective semen evaluation using standard optical microscopy is the most common practice. Semen parameters routinely assessed are volume, concentration, progressive motility, and morphology. However, computer-aided sperm analysis (CASA) represents an objective evaluation, sperm assessment that are reproducible and reliable. Such semen parameters have not been evaluated in Afrikaner, Brahman, and Bonsmara bulls’ semen. The present study evaluated the sperm motion and kinematics characteristics of semen from stud Afrikaner, Brahman, Bonsmara, and Nguni bulls using CASA technology. The electro-ejaculator was used for semen collection from Afrikaner (n = 11), Brahman (n = 7), Bonsmara (n = 10) and Nguni (n = 16) bulls of known and proven fertility. Semen was collected following 4 days of resting period. The bulls ranged between 5 and 6 years of age. After collection, the semen samples were immediately transferred to a thermo-flask and maintained at 37°C for further evaluation in the mobile laboratory (Nedambale, 2014). The CASA-Sperm Class Analyzer® system (Microptic, Barcelona, Spain) was used to evaluate sperm motion, velocity, and kinematic parameters or characteristics of raw/fresh semen from 4 cattle breeds. Data were analysed using GenStat® statistical programme (VSN International, Hemel Hempstead, United Kingdom). Treatment means were compared using one-way ANOVA. The total sperm motility rate was similar for all breeds: Afrikaner (92.2 ± 4.2), Brahman (90.7 ± 9.0), Bonsmara (93.9 ± 4.0), and Nguni (96.0 ± 2.7). However, Brahman and Afrikaner bull semen had higher sperm cells moving in a progressive motility of 57.3 and 45.6%, respectively, compared with other breeds (P < 0.05). Nguni, Afrikaner, and Bonsmara had the highest sperm cells moving in a rapid movement of 73.7, 72.4, and 67.4% (P > 0.05), respectively. The bulls sperm trajectories had a variation, as they were recorded to be irregular and not linear (P < 0.05). The straight-line sperm velocity (µm s−1), wobbling %, and amplitude of lateral head displacement % was similar for the 4 breeds (P > 0.05). In conclusion, CASA technology was a useful technique for assessing differences in sperm motion and kinematic (motility and velocity characteristics) among different bull breeds.

Plasmonic Color Filters as Dual‐State Nanopixels for High‐Density Microimage Encoding

Plasmonic color filtering has provided a range of new techniques for “printing” images at resolutions beyond the diffraction‐limit, significantly improving upon what can be achieved using traditional, dye‐based filtering methods. Here, a new approach to high‐density data encoding is demonstrated using full color, dual‐state plasmonic nanopixels, doubling the amount of information that can be stored in a unit‐area. This technique is used to encode two data sets into a single set of pixels for the first time, generating vivid, near‐full sRGB (standard Red Green Blue color space)color images and codes with polarization‐switchable information states. Using a standard optical microscope, the smallest “unit” that can be read relates to 2 × 2 nanopixels (370 nm × 370 nm). As a result, dual‐state nanopixels may prove significant for long‐term, high‐resolution optical image encoding, and counterfeit‐prevention measures.

Computational imaging of sperm locomotion.

Not only essential for scientific research, but also in the analysis of male fertility and for animal husbandry, sperm tracking and characterization techniques have been greatly benefiting from computational imaging. Digital image sensors, in combination with optical microscopy tools and powerful computers, have enabled the use of advanced detection and tracking algorithms that automatically map sperm trajectories and calculate various motility parameters across large data sets. Computational techniques are driving the field even further, facilitating the development of unconventional sperm imaging and tracking methods that do not rely on standard optical microscopes and objective lenses, which limit the field of view and volume of the semen sample that can be imaged. As an example, a holographic on-chip sperm imaging platform, only composed of a light-emitting diode and an opto-electronic image sensor, has emerged as a high-throughput, low-cost and portable alternative to lens-based traditional sperm imaging and tracking methods. In this approach, the sample is placed very close to the image sensor chip, which captures lensfree holograms generated by the interference of the background illumination with the light scattered from sperm cells. These holographic patterns are then digitally processed to extract both the amplitude and phase information of the spermatozoa, effectively replacing the microscope objective lens with computation. This platform has further enabled high-throughput 3D imaging of spermatozoa with submicron 3D positioning accuracy in large sample volumes, revealing various rare locomotion patterns. We believe that computational chip-scale sperm imaging and 3D tracking techniques will find numerous opportunities in both sperm related research and commercial applications.

Microsphere-based super-resolution scanning optical microscope.

High-refractive index dielectric microspheres positioned within the field of view of a microscope objective in a dielectric medium can focus the light into a so-called photonic nanojet. A sample placed in such nanojet can be imaged by the objective with super-resolution, i.e. with a resolution beyond the classical diffraction limit. However, when imaging nanostructures on a substrate, the propagation distance of a light wave in the dielectric medium in between the substrate and the microsphere must be small enough to reveal the sample's nanometric features. Therefore, only the central part of an image obtained through a microsphere shows super-resolution details, which are typically ∼100 nm using white light (peak at λ = 600 nm). We have performed finite element simulations of the role of this critical distance in the super-resolution effect. Super-resolution imaging of a sample placed beneath the microsphere is only possible within a very restricted central area of ∼10 μm2, where the separation distance between the substrate and the microsphere surface is very small (∼1 μm). To generate super-resolution images over larger areas of the sample, we have fixed a microsphere on a frame attached to the microscope objective, which is automatically scanned over the sample in a step-by-step fashion. This generates a set of image tiles, which are subsequently stitched into a single super-resolution image (with resolution of λ/4-λ/5) of a sample area of up to ∼104 μm2. Scanning a standard optical microscope objective with microsphere therefore enables super-resolution microscopy over the complete field-of-view of the objective.

Microsphere-aided optical microscopy and its applications for super-resolution imaging

The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼200nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

Manufacturing and characterization of molybdenum pellets used as targets for 99mTc production in cyclotron

The method of 100Mo metallic target preparation for production of 99mTc by proton irradiation in 100Mo(p,2n)99mTc reaction was demonstrated. For this purpose, pressing of molybdenum powder into pellets and their subsequent sintering in reductive atmosphere were applied. The influence of parameters such as molybdenum mass and time of both pressing and sintering on the 100Mo target durability was investigated. Under the optimized conditions, 100Mo metallic pellet targets with density of 9.95±0.06g/cm3 were obtained. Morphology and structure of pressed pellets before and after sintering were studied by using standard optical microscope and Scanning Electron Microscope (SEM). Nanoindentation technique was used to investigate the mechanical properties such as nanohardness and Young modulus. Prepared 100Mo pellets were successfully irradiated with protons and 99mTc was efficiently isolated.

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up

BackgroundQuantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system’s functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories.MethodsA protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry.ResultsThe high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope– when coupled with appropriate software for image processing– might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described.ConclusionsThe experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

A simple 2D composite image analysis technique for the crystal growth study of L-ascorbic acid.

This work was destined for 2D crystal growth studies of L-ascorbic acid using the composite image analysis technique. Growth experiments on the L-ascorbic acid crystals were carried out by standard (optical) microscopy, laser diffraction analysis, and composite image analysis. For image analysis, the growth of L-ascorbic acid crystals was captured as digital 2D RGB images, which were then processed to composite images. After processing, the crystal boundaries emerged as white lines against the black (cancelled) background. The crystal boundaries were well differentiated by peaks in the intensity graphs generated for the composite images. The lengths of crystal boundaries measured from the intensity graphs of composite images were in good agreement (correlation coefficient "r" = 0.99) with the lengths measured by standard microscopy. On the contrary, the lengths measured by laser diffraction were poorly correlated with both techniques. Therefore, the composite image analysis can replace the standard microscopy technique for the crystal growth studies of L-ascorbic acid.