This PDF file contains the front matter associated with SPIE Proceedings Volume 11254, including the title page, copyright information, table of contents, and author and conference committee lists.

Nanoparticles (NPs) from various metals (Zinc, Nickel, Cobalt, Copper) were designed and fabricated by direct synthesis using femtosecond laser ablation in liquids. Employing confocal microscopy with spectral detection and fluorescence lifetime imaging (FLIM), we have evaluated interaction of fabricated NPs with living Chlorella sp. algae by means of their naturally presented endogenous fluorescence. Live cell imaging was done in spectral region 500-550 nm and 650- 710 nm to evaluate the effect of NPs on both, the green and the red fluorescence that is derived from flavonoids/carotenoids and chlorophylls respectively. We observed fluorescence intensity decrease in the red spectral region by all but Ni NPs. The presence of NPs also lead to an increase in the blue fluorescence at 477-488 nm, possibly resulting from reflected light. Gathered observations constitute the first step towards creation of methodological approaches for fast natural biosensing of the effects of environmental pollution directly in live algae.

Whispering-gallery mode (WGM) microtoroid optical resonators have been used for highly sensitive biological and chemical sensing. With the help of auto-balanced detection and data processing techniques, frequency locked optical whispering evanescent resonator (FLOWER) has successfully reduced external noise and achieved detection of single macromolecules. FLOWER, however, can only analyze the size of particles and not their shape. To determine the shape of non-spherical particles interacting with different polarized WGM modes, we built a dual-FLOWER system to perform multi-mode locking. We demonstrate the ability of this system to detect particle shape by detecting gold nanorods and spheres.

Quantum imaging attempts to exploit the quantum features of light in order to enhance one or more aspects of classical imaging. Here we focus on two quantum imaging schemes – ghost imaging and interaction-free imaging – and moreover, on their combination. After a brief overview of the subject we report the main result: a laboratory demonstration of a new imaging scheme termed Interaction-Free Ghost Imaging. We then explain its merits for the task of imaging various structured objects. Next, we discuss an outgrowth of this scheme used for the purpose of nonlocal quantum erasure. We conclude by mentioning some related, low-dose schemes (both quantum and quantum-inspired) for X-ray and gammaray sources.

We present a nanoparticle (NP) enhanced imaging-based plasmonic biosensing technique using gold nanohole arrays (Au-NHAs) that enables highly sensitive protein detection with single analyte resolution in a one-step sandwich immunoassay. By digital quantification of individual Au-NP bound molecules on large area plasmonic surface, the portable biosensor enables rapid and robust detection of disease biomarkers from patient serum in hospital settings. Using mass-scalable deep-UV lithography (DUVL) sensor fabrication and simple LED based imaging set-up in a microarray format, our novel technology provides a powerful tool for clinical biomarker detection in a highly multiplexed manner.

In previous work we demonstrated a new method for shaping of pulsed IR (λ=1.55μm) laser probe beam in silicon. The shaping was done by a second pump pulsed laser beam at 0.532μm and 17ns pulse width which simultaneously and collinearly, illuminates the silicon surface with the IR beam. Following the Plasma Dispersion Effect (PDE), and in proportion to its spatial intensity distribution, the pump laser beam shapes the point spread function (PSF) by controlling the lateral transmission of the IR probe beam. In this paper we report on improvement by factor of 10 in the PSF of the probe beam. We use for the pump beam a pico-second laser at wavelength of 775nm. The use of shorter pulse width for the pump laser allows us to reduce the PSF of the probe beam to diameter of ~2μm, so far, which is smaller by factor of 10 from what we had before. Also, the penetration depth of the 775 nm pump beam in silicon is ~10μm compeer to ~1μm for the 0.532μm laser, which allows probe beam shaping inside the silicon. The use of the shaped probe beam in laser scanning microscopy allows imaging and wide range of contactless electrical measurements in silicon integrated circuits (IC) for failure analysis purposes. We propose this shaping method to overcome the diffraction resolution limit in silicon microscopy on and deep under the silicon surface depending on the wavelength of the pump laser and its temporal pulse width.

Conventional biological imaging typically relies on either spatial or temporal modulation which are limited by fluorophores undergoing photobleaching. As a result, imaging of biological processes for extended periods of time remains a challenge. To overcome this limitation, we introduce polarization modulation combined with nonbleaching plasmonic nanoparticles to boost signal-to-noise ratio and to eliminate strong background scattering. We show plasmonic nanoparticles can be extracted from the noisy background and cellular environment, where the visualization of nanoparticles has been previously hindered by the strongly scattering biological components. We demonstrate that such a reduction of background noise level greatly enhances the overall signal-to-noise ratio of nanoparticles within biological structures.

This study details the synthesis and time release properties of a paclitaxel-methylene blue drug incapsulated in a PLGA nanoparticle. The most important finding is that a redox switch of the methylene blue between the leuco and oxidized forms causes a change from photoacoustically silent to photoacoustically active states which was used to monitor drug release in vivo. We used this photoacoustic signal change to monitor drug release from paclitaxel-methylene blue encapsulated in PLGA in a murine model.

Techniques for imaging tissue acoustic vibrations are potentially important for clinical diagnosis; however, measuring the vibrations in two dimensions requires precise knowledge of both amplitude and phase across the entire tissue surface. Interferometric spectrally encoded endoscopy is ideal for addressing this challenge, allowing phase-sensitive three-dimensional vibration imaging with nanometric axial resolution. By measuring the mechanical and dynamical properties of the tissue with high spatial resolution, the system has been demonstrated promising for various clinical applications, including diagnosing hearing disorders in patients suffering from conductive hearing loss.

Recently, we realized a simple technique, which spontaneously bypasses the diffraction limit in a conventional confocal microscope by exploiting super-linear effects in nanoparticle bio-markers: super-linear excitation-emission (SEE) microscopy. Here, we present a theoretical framework and its practical implementation for optimizing and expanding this technique. We accurately predict the expected 3D super-resolution by accounting for all crucial parameters affecting the resolution: the empirically measured/modelled excitation-emission curve, the filling factor of the microscope objective back pupil, the polarization and the pinhole setting. The presented theoretical framework is a practical tool, which enables end-users to augment their own confocal microscopes with super-resolution capabilities.

We utilized these NIR QDs to demonstrate the utility of an image processing algorithm capable of determining the relative concentration ratios of two distinct emitters at various tissue depths. This algorithm is based on the dual probe imaging method, which we modified for use in wide-field imaging systems. The algorithm was validated using skin-mimicking tissue phantoms that were used to vary the effective imaging depth from 0-1.2 mm. The algorithm correctly determined concentration ratios of a two-color QD system for variations in concentration ≥ 10%, independently of depth. These contrast agents and the imaging approach are being adapted for use for in vivo targeted tumor imaging.ng adapted for use for in vivo targeted tumor imaging.

Fluorescence bioimaging in the second near-infrared spectral region (NIR-II, 900-1700 nm) can provide advantages of high spatial resolution and large penetration depth, due to low light scattering. In addition, since long-wavelength light with low photon energy is utilized as the excitation and less absorbed by the biological tissues, NIR-II fluorescence bioimaging also has low autofluorescence, as well as negligible photodamage towards biosamples. During the past few years, NIR-II fluorescence bioimaging has experienced rapid development. In this paper, we will introduce some recent research works about NIR-II fluorescence bioimaging in our research group, such as video-rate whole-body/brain/tumour angiography, organ imaging, as well as diagnosis and imaging guided treatment of tumour, on mice and monkeys.

One of the main goals in nanomedicine is the development of effective drug delivery systems. Polymeric nanoparticles (NPs) have been exploited as nanocarriers for their stability, handiness and biocompatibility. In this context, NPs have been coupled to molecular beacons (MBs), theranostic agents that conjugate the sensing of specific mRNA with a silencing activity. The aim of this work was to evaluate by confocal microscopy and fluorescence measurements: a) the involvement of endocytosis in the NP uptake; b) the NP fate at different times of cell incubation to verify their localization in lysosomes; c) the MB localization on the Endoplasmic Reticulum (ER) where the target mRNA is located. Results: a) PMMA-NPs promote endocytosis; b) strong co-localization of NP and lysosome tracker fluorescence after 2- 48 hours of incubation; c) co-localization of the MB fluorescence with the ER-marker signal after 90' of incubation. The data highlight the ability of the PMMA-NPs to promote the survivin-MB internalization by endocytosis and demonstrate that the PMMA-NPs are an appropriate delivery system capable of being eliminated by cells; these evidences also contribute to consider the MB as an effective tool for the intracellular sensing.

In this paper we propose a novel approach for remote speckle-based sensing of mechanical vibrations in the Hirudo medicinalis leech central nervous system (CNS) connective tissue. Using this method, spontaneous vibrations generated at the connective tissue following partial cut injury are continuously and remotely monitored. A laser beam illuminates the connective tissue and back scattered defocused patterns at the far field are captured by the camera. The spatialtemporal spontaneous vibrations of the connective are monitored by tracking the speckle spatial-temporal trajectory. After applying correlation-based analysis we were able to detect these vibrations of the connective tissue during recovery with respect to control measurements. This approach is the first step towards understanding the possible involvement of the tissue movements for the recovering process via mechanical vibrations sensing of the leech CNS connective tissue.

Controllable preparation of water-soluble multifunctional nanoprobes is of great significance for cancer early diagnosis. In this study, protein-modified hydrophilic copper sufide (CuS) nanotriangles with tunable absorption in the second near-infrared region are developed in the presence of halide ions. Further, DTPA-Gd3+ is conjugated on it by using the unique characteristics of the protein-protected nanotriangles. Specifically, the asobtained nanostructures are investigated as contrast agents for enhanced in vivo photoacoustic/magnetic resonance dual-modal tumor imaging. More importantly, in vitro and in vivo toxicity analysis are also performed, which show that the dual -modal nanoprobes are biocompatible for most of the cases. It is demonstrated that the novel asprepared protein-modified nanotriangles are able to work as a nanoplatform to construct dual-modal nanoprobes, which paves a new anenue for improving the photoacoustic/magnetic resonance imaging contrast in cancer detection. It should be pointed out that other functional blocks may also be linked on it, which makes it a general method to design multifunctional nanoprobes.

Macrophages are one of the most important candidates of innate immune response with pronounced phagocytic activity. As a part of the defense mechanism of our system, an increased level of particular chemokines initiate the activation and further differentiation of the macrophages into two major phenotypes, M1 (classically activated) and M2 (alternatively activated). M1 macrophages promote ‘pro-inflammatory’ activities, whereas, M2 macrophages show ‘anti-inflammatory’ activities. Now, normally a certain ratio of M1/M2 macrophages is maintained in healthy individuals. However, at certain disease conditions, a shift in the M1 to M2 or M2 to M1 macrophage population is observed. An assessment of the M1/M2 ratio would readily predict the health condition of the individual. Here, we propose a novel approach to identify M1 and M2 populations using simple flow cytometry and Gold nanorods (GNRs). According to reports, macrophages can readily internalize GNRs by phagocytosis. Now, this internalization of highly scattering GNRs will increase the cellular scattering of macrophages and thus can be identified by the Flow Cytometric technique. For the first time, we are reporting about the differential uptake of polyallylamine hydrochloride (PAH) coated GNRs by M1 and M2 macrophages (differentiated from THP1 cells). A 24 h incubation with the 100μg/ml PAH-GNRs results in a greater intake of PAH-GNRs by M2 cells compared to M1, which leads to an increased side scatter for M2 cells in Flow cytometry. Overall, this study opens a new avenue for simple identification of M1 and M2 cell types.

We present a nanofluidic optical device based on light-scattering microscopy, which enables label-free detection and quantitative analysis of individual biomolecules freely moving in solution. The key component is a nanochannel with subwavelength dimensions, which is imaged by dark-field microscopy. Due to interference between light scattered by the nanochannel and a biomolecule inside it, a single biomolecule can be directly detected. In addition, the molecular weight can be measured, both by tracking the Brownian motion and from the optical contrast. This is demonstrated for both single DNAs and proteins with molecular weights ranging down to tens of kDa.

In recent years, we demonstrated a new approach to super-resolution microscopy based on driving a nonlinear interaction with a Spatial Frequency Modulated Imaging (SPIFI). SPIFI is a line imaging technique that linearly sweeps all the frequencies supported by the band-pass of the objective lens. Here we introduce a new method of unrestricted super-resolution imaging based on driving saturated absorption in the specimen excitation. The saturated absorption drives harmonic distortions of the spatial frequencies used to illuminate the sample. These harmonics manifest themselves as temporal harmonic frequencies allowing for easy detection and separation of the super-resolution information in the far field.

We recently developed a digital cytometric assay using plasmonic nanoparticles to quantify the bound ovarian cancer marker (CA125) on single cells. We demonstrate the quantification capability of our novel digital cytometry method by discriminating the different MUC16 binding levels on the PBMCs between healthy donors and EOC patients. Furthermore, we tracked the amount of surface-bound MUC16 on the EOC patient’s PBMCs over 17 months with 1-month time intervals. Initial data from our cytometric technique shows it to be a quantitative tool that can provide new information connecting leukocyte-bound MUC16 to the time course of ovarian cancer.

Laser based photonic non-contact sensor can be used for continuous sensing of periodic vital bio signs by analyzing the temporal changes of the spatial back scattered secondary speckle patterns. This analysis allows extraction of nanovibrations of the inspected tissue (surface and in-depth) and associate it with various vital bio-signs. One of limitations for this type of sensing is the temporal bandwidth of the analyzing camera. It’s number of frames per second limits the maximal temporal bandwidth of the biological signals that can be sensed. I will show how to increase the temporal bandwidth in two ways. First by encoding the illuminating laser with orthogonal convolution codes-based patterns and using the fact that the to be sensed biological signals are periodic, in order to allow a significant enhancement of the bandwidth of the camera aiming to overcome the limiting factor induced by its low sampling rate. Second, I will show how sampling rate could be reduced when sensing blood flow-based bio-signs and by converting the temporal signals that are to be sensed into space. In the overall the proposed enhancements allow using much simpler sensors/cameras for extraction of biomedical information included in much higher temporal frequencies which can be much more informative for biomedical diagnosis and analysis.

Here we describe a lens free on-sensor imaging platform which is able to perform a high spatial resolution imaging over the large field of view. By measuring the geometrical shadow of the microscopic objects at different oblique illumination angles, we were able to measure the height of these objects. In addition, we were able to record bacterial bioluminescence signals directly on the CMOS chip. This is, to our knowledge, the first report of lens-less, on-sensor bioluminescence measurements.

While differential sensing has the potential to discriminate millions to trillions of analytes based on hundreds of sensing elements, most of the current differential sensor arrays have limited scalability, with only a handful of recognition elements demonstrated. Here we demonstrate a novel differential sensor array based on a class of organic/inorganic hybrid fluorophores, DNA-templated silver nanoclusters (DNA/Ag NCs), that not only has great scalability but also combine both recognition element and signal transduction element into one unit. Six DNA/Ag NC sensors were designed to classify the lysates of six breast epithelial and cancer cell lines with distinct metastatic potential. While MDA-MB-231 and MCF7 lysates showed significantly different fluorescence signatures upon interaction with the sensor array, other cell lines were also differentiable. Our method is simple, versatile, low-cost, reliable, and scalable.

Development of optically active nanomaterials for pH sensing at biological environments can offer many advantages for biomedical examinations and drug delivery applications. Raman spectroscopy, a rapid and low intrusive optical technique, has been widely used in biological studies. Since pH is an important variable in many pathological and physiological pathways, nanoscale Raman pH probes can expand opportunities for pH sensing and controlling at biological interfaces. In this study, Raman spectra of a pH responsive selfassembled mesophase in water was investigated upon varying the pH of the solution. Raman peaks of the mesophase can be addressed as pH dependent optical output, enabling the material to be used as a nanoplatform for Raman pH sensing.

RNAs play pivotal roles in many biological processes, such as translation, gene editing etc. Since RNAs are usually smaller in size than optical diffraction limit, it is impossible for optical microscopes to get deep insight of them. With the recent emergence of super-resolution optical imaging microscopies, it becomes possible to study the ultra-fine structure of such RNA in side cells. Different kind of fluorescent probes are used for RNA labeling and imaging inside cells. However, most of them cannot be used for live-cell super-resolution imaging, due to typical laser toxicity to live cells. Herein, we developed a new type of RNA-labeled probe, which can be used for STED imaging of RNA in living cells. We believe this probe is of great significance for studying biological behaviors of RNA in living cells.

We developed a unique temporal imaging scheme which is able to detect all the properties of an optical signal in a sub picosecond resolution. This includes imaging of the intensity, the phase, and the state of polarization as a function of time. We are aiming to utilize this advance temporal imaging scheme for studying ultrafast phenomena which were not investigated until now. In this proceeding we describe in details the experimental setup with all its components and parts beyond what we showed and explained in the paper.

Time-lenses in general proved to be useful for many applications and specifically when utilizing them for temporal imaging schemes where they can image ultrafast signals that cannot be detected by any electronic based device. Over the last few years, we demonstrated that when joining together several time-lenses into a single time-lens array, it is possible to gain more information on the input signal. Such as measuring temporal depth imaging, the state of polarization of the input signal as a function of time, and retrieving the phase dynamics. However, when designing an array of time-lenses, there is a trade-off between joining large number of small time-lenses, so each signal will interact with many time-lenses but each one has low resolution, and joining small number of large time-lenses, so each has better temporal resolution but on the expanse of interacting with smaller number of time-lenses in the array. We showed that one way to overcome this drawback is to overlap adjacent timelenses. Thus it is possible to both have large number of time-lenses without compromising on the size of each time-lens and obtaining high temporal resolution. In this proceeding, we overlap two time-lenses and measure the spectrum of the idler. We compare the numerical simulations of the frequency domain of the idler to the measured spectrum of the idler.

Near-infrared (NIR) diagnosis permits sensing changes in the optical properties on an air-tissue interface. While imaging is bound to the surface due to the high tissue scattering, many diagnosis methods, such as the photoplethysmograph (PPG) and pulse oximeter, focus on sensing instead of imaging. These methods are commonly used in cylindrical tissues, such as fingertips, or half infinite tissues, such as forehead. However, these methods are less common in endoscopic applications, where a cylinder of air is surrounded by tissue. Previously, we proposed measuring the full scattering profile (FSP), which is the angular intensity distribution, of cylindrical tissues. MC simulations revealed that the FSP has a fixed intensity point, named the iso-path-length (IPL) point, which does not depend on the reduced scattering coefficient. The location of this point depends solely on geometry. Using an analytics photon diffusion method, we have discovered that the IPL point exists in the endoscopic geometry as well. In this study, we present Monte Carlo (MC) simulations of the FSP in endoscopic geometry, where a cylinder of air is surrounded by tissue. This geometry, as the cylindrical geometry, has two IPL points; along the azimuthal direction and the longitudinal direction. We will demonstrate that while the position of the azimuthal IPL point depends on the diameter of the air hole, the position of the longitudinal one stays constant. The IPL point in endoscope applications may open a new path of diagnosis of change in cells of the digestive system.

Sensing physiological parameters by optical methods is very common because they are simple to use, they do not hurt the patient and they provide a real-time measurement. These parameters, such as oxygen saturation, are based on detection of light absorption through the tissue. The existing method uses multiple wavelengths to extract the absorption coefficient. However, the optical pathlength depends on the wavelength. Hence, the use of different light sources, which propagate through the tissue in different pathlengths, adds an inherent error to the calculation of the absorption coefficient. We suggest a new optical method for extracting the absorption coefficient of cylindrical tissues by a single wavelength. The method uses one wavelength only and therefore does not suffer from inherent errors. Our calculation is based on the iso-pathlength (IPL) point that we previously found. This phenomenon was discovered using a new characterization of cylindrical tissues, the full scattering profile, which is the light intensity distribution on the surface. In the IPL point, the light intensity remains constant for different scattering coefficients. We will use the light intensity measurement in this point in order to quantitatively extract the absorption coefficient.

The interaction of light with different materials has been studied for hundreds of years in various research fields e.g., material analysis, quantum physics, biomedical optics, etc.. This interaction is determined by the material's optical properties which are spectrally dependent. Spectroscopic methods examine the spectral dependence of the interaction, which reveals the material fingerprint. However, in opaque materials, their characterization is more complex and challenging due to the scattering. We have previously presented the iterative multi-plane optical property extraction (IMOPE) technique for characterizing materials using the reflection from turbid media. The reflection-based IMOPE, which estimates medium scattering properties, combines a theoretical model with an experimental setup and the multiple measurement GS algorithm. This technique was initially aimed for biomedical applications, where the red wavelengths are the desired radiation source due to higher penetration depths. However, as in spectroscopy, multiple wavelengths, rather than just one, can provide more accurate information about components within the sample. In this research, we introduce the spectral reflection-based IMOPE for extracting the reduced scattering coefficient of a medium in the blue regime.

A low-cost biosensor for glucose analysis was designed, fabricated and characterized. It was fabricated by using a DLPdirect light processing, 3D printed technique. The operation principle for the electro-optic device is based on the total internal reflection. The photosensitive resin is considered that is doped with nanoparticles such as quantum dots and carbon nanotubes as a waveguide. By using DLP 3D printing technique the device was fabricated. The operation principle was based on the optical phase shift. The entire structure of the device features a Mach-Zehnder Interferometer (MZI) that was numerically analyzed in a commercial software featuring finite element. The biosensor works in the transmission scheme. Mach-Zehnder interferometer is composed of a waveguide that encompasses the resin doped with nanodots that linked both input and output signals. In contrast, one arm is kept isolated and the other arm exposed to the medium in order to obtain the phase change at the MZI output. A laser-diode of 980 nm was considered as the source. The optical array features a single-mode optic fiber and a bidirectional coupler. While tested, several glucose concentrations (from 0% to 3%) were provided in order to determine the relationship between the glucose concentration and the output signal in order to increase the sensitivity. The future possibility of the aforementioned structure describes the use of several materials for biological-related applications are also discussed.

Multimodal imaging, integrating different imaging modalities, is emerging as a promising strategy for improving both preclinical and clinical imaging. Computed tomography (CT) and magnetic resonance imaging (MRI) are among the widely clinically-used imaging techniques. These imaging modalities are used for disease diagnosis and screening, each providing different and complementary information about the patient and the disease state. Nanoparticles are considered promising contrast agents for in vivo imaging applications, providing improved imaging and targeting capabilities compared to the commonly used contrast compounds. In this project, we present the development of a novel hybrid nanostructure, comprising of iron oxide core and gold shell nanoparticle for dual model CT and MRI imaging. The hybrid structures presented herein have potential to serve as a unique platform for precision imaging.

Cell replacement therapy is a feasible approach for vision restoration in several degenerative diseases of the outer retina in order to halt disease progression and recover lost vision. In this treatment photoreceptor precursors are transplanted in the subretinal space and later integrate within the retina. In order to evaluate the efficiency of cell transplantation for vision restoration, it is crucial to monitor cell survival and integration in the host retina over extended periods, therefore, longitudinal tracking of the transplanted cells in clinical and experimental setups is crucial. Here we present a technique for labeling photoreceptor precursors with gold nanoparticles prior to vitreous and subretinal space transplantation treatment. This labeling allow multimodal imaging using both computed tomography (CT), optical coherence tomography(OCT) and fluorescence fundus imaging. Our multimodal imaging technique enabled high-resolution longitudinal tracking of the transplanted cells for over a month, and quantification of cell survival in the retina and vitreous, without any toxic effects. These findings promote the use of nanoparticles for retinal cell tracking and its translational potential providing more efficient cell replacement therapies for clinical applications.

Bacterial infection and biofilm buildup is a major challenge for wound healing. In the present work we developed a method to track bacteria in vivo in order to analyze the progress of the healing. Regulated irrigation combined with negative pressure (RI-NPT) is a nonpharmaceutical therapeutic strategy for reducing bacterial load in acute and chronically infected wounds. Here, we analyzed RI-NPT hydrokinetics and efficacy of bacterial load reduction in wounds. Escherichia coli were loaded with gold nanoparticles, in order to make them "visible" and trackable using computed tomography (CT). Bacteria were tracked using CT in a low-flow rat wound model over time. CT tracking revealed that while regulated negative pressure-assisted wound therapy reduced bacterial load to a limited extent (5%), RI-NPT significantly increased bacterial outflow and clearance (by 45%). This nanotechnology-based approach demonstrates the ability to track bacteria in vivo at real time and also the ability of the RI-NPT technology to effectively reduce bacterial load.

The movement of vesicle in a living cell includes essential information for understanding the details of the intracellular transport. Although the vesicle tracking method has allowed us to understand precise movement of a single nanoparticle from the physical point of view, the whole cell-level transport has still not been clearly explained with the analysis of only a few representative vesicle movements. In this study, as an initial attempt to gain insight into cell-level vesicle transport, we adopted a computer vision technique to analyze the overall intracellular vesicle transport. In detail, we propose an algorithm to estimate and visualize the ow of the entire endocytic vesicles in terms of convergence and divergence with respect to the geometric cell center. In this algorithm, optical ow of the fluorescent nanoparticles in a living cell is computed using Lucas-Kanade method. Then, the direction of vesicle movement regarding the geometric center of the cell is calculated and mapped to visualize either converging or diverging movement, based on four-quadrant inverse tangent. With this suggested method, it is expected that we can gain insight into cell-level vesicle transport, which can help design and quantitatively evaluate various biomedical applications, including drug delivery.