Research on exosomes and other forms of extracellular vesicles (EVs) have rapidly expanded over the last two decades. These lipid-enclosed, nanoscale messengers are released from cells packed with diverse cargo and can travel long distances to modify the function of target cells. Found in abundant quantities in biological fluids like blood, there is great clinical interest in using EVs as diagnostic markers or altering their properties for therapeutic delivery. Tune in to find out more about what exosomes are, how researchers study them, and what challenges remain. This talk will highlight multi-laser nanoparticle tracking analysis (NTA) with the ViewSizer 3000 and what it offers in exosome research.
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Exosomes biomarkers mediating important biological process,especially in the systemic disease
diagnostics and therapeutics,yet the protective exosomal vesicle structure hinders rapid,simple detection of the harbored molecules.
Exosomes biomarkers mediating important biological process,especially in the systemic disease
diagnostics and therapeutics,yet the protective exosomal vesicle structure hinders rapid,simple detection of the harbored molecules.
Microsatellite instability testing is an important part in diagnostics in Metastatic cancer settings after the FDA has given approval for tissue agnostic indications in almost all solid cancers. MSI by PCR and MMR status by IHC is also helpful for evaluation of genetic risk in Colon and Endometrial cancers
Exosomes have specialized functions and play a key role in different physiological processes and pathological conditions. Consequently, exosomes have attracted increasing attention in their clinical applications for prognosis,
https://www.creative-biolabs.com/exosome/applications.htm
Automated histopathological image analysis: a review on ROI extractioniosrjce
IOSR Journal of Computer Engineering (IOSR-JCE) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of computer engineering and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications in computer technology. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
There are several important changes in the WHO 5th edition hemato-lymphoid with a paradigm shift towards genetic diagnosis along with morphological aspects. Precursor lesions of Clonal hematopoiesis, CHIP and CCUS are formally included, Changes include those in AML, MPN, JMML is now a part of MPN, MDS-MPN, ALAL etc.
Lung cancer is a major cause of cancer deaths with approximately 80% of cases accounting to nonsmall cell lung cancer (NSCLC) . In NSCLC target therapy, epidermal growth factor receptor (EGFR) is a promising candidate.
Microsatellite instability testing is an important part in diagnostics in Metastatic cancer settings after the FDA has given approval for tissue agnostic indications in almost all solid cancers. MSI by PCR and MMR status by IHC is also helpful for evaluation of genetic risk in Colon and Endometrial cancers
Exosomes have specialized functions and play a key role in different physiological processes and pathological conditions. Consequently, exosomes have attracted increasing attention in their clinical applications for prognosis,
https://www.creative-biolabs.com/exosome/applications.htm
Automated histopathological image analysis: a review on ROI extractioniosrjce
IOSR Journal of Computer Engineering (IOSR-JCE) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of computer engineering and its applications. The journal welcomes publications of high quality papers on theoretical developments and practical applications in computer technology. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
There are several important changes in the WHO 5th edition hemato-lymphoid with a paradigm shift towards genetic diagnosis along with morphological aspects. Precursor lesions of Clonal hematopoiesis, CHIP and CCUS are formally included, Changes include those in AML, MPN, JMML is now a part of MPN, MDS-MPN, ALAL etc.
Lung cancer is a major cause of cancer deaths with approximately 80% of cases accounting to nonsmall cell lung cancer (NSCLC) . In NSCLC target therapy, epidermal growth factor receptor (EGFR) is a promising candidate.
Picturing Science: An overview of Imaging TechnologiesDr. Sima Salahshor
In the past decades imaging technologies are increasingly used to model the dynamics and structure of biological systems. Biomedical imaging is now an integral part of biological and medical sciences and is used in both clinical practice and research. In this session some of the latest imaging technologies were reviewed.
Study of Mitotic Index and DNA profile when exposure to He-Ne laser and UVC r...IOSR Journals
In vitro, He-Ne laser show a modifying response of cells to ionizing radiations. So there is a need to show the effect of He-Ne laser (632.8nm), Ultraviolet radiation UVC (250nm) and He-Ne laser pre and post irradiation against the UVC radiation of Mitotic index of femur and in vivo to DNA of testis in Mice. In this study 100 albino male mice were divided into five groups, the first group Control which have (10) number of mice, the second group Laser which have (27) number of mice were divided into three groups different time periods (5, 10, 15 min), the third group Ultraviolet radiation (UVC) which have (9) number of mice and duration of exposure one hour, the fourth group laser (5, 10 and 15 min) + UVC (1h) which have (27) number of mice, with ½ hour time interval between the two irradiations and the finally group UVC (1h) + laser (5, 10, 15 min) which have (27) number of mice, with ½ hour time interval between the two irradiations was monitor the effect of radiation on mice according to the classification totals above after various time periods (7, 14, 21 days). Mitotic index as shown increase the percentage of Mononucleus and less increase of Dinucleus after exposure of the radiation according to the classification totals above. The He-Ne laser per-irradiation show a protection properties, which appeared the DNA damage against UVC light irradiation. But the He-Ne laser pre-irradiation against UVC irradiation farther more reduce the DNA testis damaging. UVC shows a damaging effect on the DNA. This damage was reduced by the He-Ne laser pre- irradiation. Thus Laser pre-irradiation may be attributed to the induction of endogenous of radio protectors or which may be involved in DNA damage repair.
CBCT is rapidly becoming the standard in 3D dental imaging. First generation CBCT was first used in 1982 (Mayo Clinic Biodynamics Research Laboratory) to perform angiography.
Hence, CBCT system was extended to other medical section, finding its best application in dentistry and maxilla-facial region study.
Although the CBCT principle has been in use for almost 2 decades, only recently—with the development of inexpensive x-ray tubes, high-quality detector systems and powerful personal computers—have affordable systems become commercially available.
CBCT is a compact, faster and safer version of conventional CT.
Using a coneshaped X-ray beam, the size of the scanner, radiation dosage and time needed for scanning are all dramatically reduced. CBCT scanners are systems that are able to provide 3D reconstructions that are based on the reformat of 2D images.
The scan is performed with a single 360 scan in which the x-ray source and a reciprocating area detector synchronously move around the patient’s head, which is stabilized by a head holder
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
CBCT is rapidly becoming the standard in 3D dental imaging. First generation CBCT was first used in 1982 (Mayo Clinic Biodynamics Research Laboratory) to perform angiography.
Hence, CBCT system was extended to other medical section, finding its best application in dentistry and maxilla-facial region study.
Although the CBCT principle has been in use for almost 2 decades, only recently—with the development of inexpensive x-ray tubes, high-quality detector systems and powerful personal computers—have affordable systems become commercially available.
CBCT is a compact, faster and safer version of conventional CT.
Using a coneshaped X-ray beam, the size of the scanner, radiation dosage and time needed for scanning are all dramatically reduced. CBCT scanners are systems that are able to provide 3D reconstructions that are based on the reformat of 2D images.
The scan is performed with a single 360 scan in which the x-ray source and a reciprocating area detector synchronously move around the patient’s head, which is stabilized by a head holder
Modern Particle Characterization Techniques Series: Laser DiffractionHORIBA Particle
This part two of the webinar series will introduce participants to basic experimental considerations when choosing laser diffraction for particle size analysis. The presentation will explain what makes laser diffraction a “modern technique.” Both wet and dry case studies will be shown along with brief demonstration videos.
In this webinar, you will learn:
- Method development
- Choosing an appropriate refractive index
- Understanding the analysis results
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Mastering the Processing Methods of Engineered ParticlesHORIBA Particle
This webinar will explain the development process for particles with specific attributes that can cause problems during production. Three case studies will be discussed: Engineered particles that protect omega-3 oil from oxidation using special microencapsulation methods; modified cellulose fibers with high water holding capacity; and engineered particles produced by melt atomization processes with unique attributes. The talk will focus on alternative processing methods, the importance of understanding the materials being used, and what can happen when you do not understand the functional properties that you are designing for.
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Modern Particle Characterization Techniques Series I: IntroductionHORIBA Particle
Particle characterization is a rich field that touches industries from mining to pharmaceutical production. There are a number of characterization techniques available to the modern analyst. Understanding them is key to selecting the right technique as well as gaining deeper insight into the meaning of measurement results.
This webinar is the beginning of a new series reviewing a number of modern measurement techniques. Dr. Michael Pohl, Vice President of HORIBA Scientific, will describe some common ideas in particle characterization along with common questions to ask when selecting a technique. Mike will also give a very brief overview of some modern techniques before subsequent webinars go into detail.
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Concentration and Size of Viruses and Virus-like ParticlesHORIBA Particle
Accurate concentration for virus and virus-like particles can be determined by multi-laser nanoparticle tracking analysis due to the fact they are nanoparticles. Other biologically relevant materials will have sizes that are close to those of viruses, whether they are protein aggregates that provoke an unwanted immune response or exosomes with a similar size and do not provoke an immune response.
In this webinar, Dr. Jeff Bodycomb will discuss the use of multi-laser nanoparticle tracking analysis (NTA) to determine the size distribution and concentration of these species, the latter which is correlated to viral infectivity.
Learn more about:
-How NTA determines concentration and size distribution
-Advantages and limits of the multi-laser technique
-Example measurement results
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The Value of Real-time Imaging: Integrating Particle Size Analysis onto Fluid...HORIBA Particle
The capability to measure critical quality attributes (CQA) such as particle size in real time reveals their functional relationships with the critical process parameters (CPP). The Eyecon2™ offers a true non-product contact, a real-time imaging system that can be used with dry and wet bulk solid processing equipment affording a digital maturity competitive edge. We will dive into how the imaging technology works, the basic principles of analysis for particle size detection, the methods of integration onto process equipment such as Fluid Beds, Twin Screw Granulators and Roller Compactor and discuss key applications where the value of real-time in-line particle size results archetype how the Eyecon2 enables transparency, agility and productivity that aligns with the Factory of the Future and Pharma 4.0.
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How and Why to Analyze Ceramic Powder ParticlesHORIBA Particle
Packing density, mechanical strength, and processing of ceramics are all affected by the size distribution of the powders. Therefore, particle size analysis is an important quality control step. Due to its wide size range and flexibility, laser diffraction is often the preferred method of analysis. Laser diffraction can be used for particles with sizes from 10’s of nanometers to millimeters. In this webinar, Dr. Jeff Bodycomb of HORIBA Scientific discusses particle analysis of ceramic particles, including electronic materials and common oxides. He will cover the basic principles of analysis, practical methods for obtaining good data, and example data.
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Interpreting Laser Diffraction Results for Non-Spherical ParticlesHORIBA Particle
Particle shape can have a profound impact on particle size distribution (PSD) measurements. In the case of Laser Diffraction, the shape and aspect ratio of particles alter the diffraction pattern used to determine PSD, which is calculated on the basis of equivalent spherical diameter. For instance, it has been established that the reported size of an ellipsoid is always smaller than the physical major dimension of the particle. Furthermore, when non-spherical particles align within a flowing sample, laser diffraction instruments typically report a bi-modal size distribution even in the case of monodisperse samples.
Equipped with only qualitative knowledge of particle shape, the particle analyst can resolve this inherent ambiguity and use laser diffraction to obtain quantitative information (such as aspect ratio) about non-spherical particles. This webinar explains the origin of this effect, describes how to interpret PSD data in such cases, and demonstrates several practical applications for measurements of crystals, bacteria, and clays.
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Particle Classroom Series VI: Method DevelopmentHORIBA Particle
Great results need a great method. In order to compare different lots of material or different manufacturing approaches, variation due to sample preparation should be minimized. Should the sample be run in suspension or as a dry powder? What salts or surfactants are needed for the suspension? How much energy should be applied and how? Systematically determining the answers to these questions is method development.
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Particle Classroom Series V: Sampling and DispersionHORIBA Particle
The goal of a particle analysis is to understand the properties of a material, whether it is the size distribution of particles that are manufactured today or the size distribution of particles in the truck that just arrived. Naturally, a particle analyzer only encounters a tiny fraction of that material, the sample.
In addition, particles can be bound together to form agglomerates that do not represent the underlying materials. The instrument will then measure the agglomerates, not its constituent particles. In this webinar, Jeff discusses how to improve data quality by obtaining a representative sample and effectively disperse the sample to remove or prevent agglomerates.
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Particle Size Analyses of Polydisperse Liposome Formulations with Multispectr...HORIBA Particle
During this webinar, Dr. Singh will discuss the significance of liposome size characteristics in medicine. He will discuss the challenges present in particle size measurement for heterogeneous size containing formulation (polydisperse). He will also discuss his recently published results on polydisperse bead and liposome formulations using DLS, conventional NTA, laser diffraction and a novel multispectral NTA measurement techniques.
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Particle Classroom Series IV: System VerificationHORIBA Particle
Confirming the performance of a particle analyzer is a critical step in ensuring and proving data quality. Join Dr. Jeff Bodycomb as he discusses performance expectations, confirming system performance, and recommended practices. This is part four of a six-part classroom series.
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Principio, Optimización y Aplicaciones del Análisis de seguimiento de Nanopar...HORIBA Particle
Este webinar tiene como objetivo presentar los fundamentos teóricos del análisis de seguimiento de nanopartículas (NTA), su optimización y su aplicaciones más recientes en la industria e investigación.
- Principio de la Técnica de Rastreo de Nanopartículas (NTA)
- Información que nos proporciona la técnica NTA
- Limitaciones de la técnica NTA
- Optimización
- Aplicaciones más recientes
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Surface area is an important physical property that influences the reactivity, dissolution, catalysis, and separation of materials. The surface area often must be carefully engineered and measured to optimize specific functions. In this Webinar, our applications lab will explain with real-world examples:
- Physical adsorption technique - BET theory
- Sample preparation – the start of a good measurement
- Calculating specific surface area from gas adsorption on solid surfaces
- Troubleshooting – what happens when things go wrong?
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Particle Classroom Series III: Refractive Index and Laser DiffractionHORIBA Particle
Modern laser diffraction particle analyzers use particle refractive index to accurately model the behavior of light inside of the particle. However, this presents the analyst with the challenge of choosing the correct value. In this Webinar, Dr. Jeff Bodycomb will discuss:
- Why do we need a refractive index value?
- What is refractive index?
- How do we choose refractive index values?
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How to Present and Compare Data Obtained by Particle Tracking Analysis and Ot...HORIBA Particle
This Webinar is for anyone who wants to understand how experimental data should be presented and compared properly while using histograms.
Dr. Kuba Tatarkiewicz examines methods for particle size distributions as obtained by particle tracking analysis, fluorescence, as well as micro-sedimentation. Choices of binning schemes that users can design themselves will be discussed with examples of how various parameters (like mode and D50) change with different binnings, especially for highly polydisperse colloids. Methods for comparison of particle size distributions will be presented and explained in practical terms.
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Why the University of Washington chose the HORIBA Laser Scattering Particle S...HORIBA Particle
Join our users at the University of Washington (UW) as they discuss how the HORIBA particle size analyzer is used in their undergraduate courses and how the instrument manages to support a wide range of applications. Some examples include polymers spheres, ceramic powders, soil, and rocks. In this Webinar, Materials Science and Engineering (MSE) graduate students, Michelle Katz and Tiffany Tang will demonstrate with case studies how other orthogonal methods such as optical microscopy, scanning electron microscopy and x-ray diffraction help them cross-validate their particle size and size distribution. You will also learn in their own words, why the UW MSE program chose the HORIBA particle size analyzer over other options for their undergraduate environment.
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Particle Classroom Series II: The Basics of Laser DiffractionHORIBA Particle
Particle size analysis by laser diffraction offers many advantages. The technique is fast, reliable, and can be used for analyzing a wide range of particle sizes. In laser diffraction scattering as a function of angle is measured and the data used to determine the particle size distribution. The technique can be used over a very wide range of particle sizes -- 10's of nm to 100's of microns. In addition it is very fast and reliable. In this webinar, Dr. Jeff Bodycomb will discuss:
Exactly what happens when light strikes a particle
Light intensity and how it effects the measurement
Fraunhofer vs. Mie
Real and imaginary refractive index values
This is a great introduction to someone who wants to understand the science behind the measurement.
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Particle Classroom Series I: Introduction to Particle AnalysisHORIBA Particle
If you're new to particle characterization, this is a webinar just for you! Dr. Jeff Bodycomb will discuss the basics of particles....why different size definitions will give you different results, various methods used to measure particles and why the method you use matters! This webinar will be the first in a series that will give you the knowledge you'll need to be the particle expert in your lab.
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Improved Visualization, Counting and Sizing of Polydisperse Nanoparticle Coll...HORIBA Particle
The ViewSizer® 3000 offers the ability to visualize nanoparticle colloids without requiring calibration standards or knowledge of any particle material properties, such as refractive index. It was developed by MANTA – the Most Advanced Nanoparticle Tracking Analysis – and offers the user an unprecedented ability to count and size highly polydispersed samples, such as milk, sea water, or blood plasma.
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Key Points to Achieving Successful Laser Diffraction Method DevelopmentHORIBA Particle
Unlock the secrets to the best measurement for particles. Topics covered include choosing appropriate accessories, selecting the best dispersing medium, assessing the effect of circulation pump speed, and evaluating the impact of using different imaginary refractive index values.
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Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.
Exosomes: Exploiting the Diagnostic and Therapeutic Potential of Nature’s Biological Nanoparticles
1. Exosomes: Exploiting the Diagnostic and Therapeutic
Potential of Nature’s Biological Nanoparticles
June 11, 2020
HORIBA Webinar Particle Characterization Series
Niaz Zafar Khan
MD/PhD Candidate
University of Maryland School of Medicine
Medical Scientist Training Program
Program in Neuroscience
2. The Lab for the Study of Central Nervous System Injury
Dr. Alan Faden Dr. Bogdan Stoica Dr. Junfang Wu Dr. Marta Lipinski Dr. David Loane
3. Loane and Faden (2010), Trends Pharmacol Sci
Primary Injury Secondary Injury
Pathophysiology of CNS Injury
Secondary injury contributes to progressive cell loss after neurotrauma.
4. Loane & Kumar (2015), Exp. Neurol.
Neuroinflammation after CNS Injury
Pro-inflammatory, microglial activation persists months after neurotrauma.
5. Kourembanas et al. (2015), Annu Rev Physiol
Extracellular Vesicles (EVs)
EVs are biological messengers that can transfer proteins, lipids, and nucleic acids.
7. Implications:
(1) Biomarker Potential
(2) Long-distance
communication
between organ systems
(3) EVs as drug delivery
carriers
Quinn et al. (2015), J Extracell Vesicles
EVs in Biological Fluids
8. Shah et al. (2018), N Engl J Med
EVs as Biomarkers for Disease
9. Holm et al. (2018), Trends Neurosci
Bidirectional Cellular Crosstalk through EVs
10. Kumar et al. (2017), J. Neuroinflammation
EVs and Neuroinflammation after TBI
Blood microparticles (MPs) of
microglial-origin analyzed by flow
cytometry after TBI.
Pro-inflammatory microglia release MPs
that can promote inflammatory activation.
Kumar et al. (2017), J. Neuroinflammation
11. • Advantages over synthetic
nanoparticle systems may
include:
– Can be Personalized
– Long circulating half-life
– Reduced immunogenicity
– Inherent targeting capabilities
– Ability to cross biological
barriers such as the blood-
brain barrier
Rufino-Ramos et al. (2017), J Controlled Release
EVs as Therapeutics
14. Sample Collection
Sample Storage
EV Isolation
-”Omic” analyses
Transcriptomic
Genomic
Proteomic
Lipidomic
EV Characterization
Quantitative and
Qualitative
Functional Assays
Pre-analytical variables
Workflow in EV Research
1
2
3
16. Rufino-Ramos et al. (2017), J Controlled Release
CD9, CD63, CD81
<50-200nm
50-1000nm
Classification of EVs
Original definitions based on biogenesis
>1000nm
/Microparticles
17. Classification of EVs
Original definitions based on biogenesis and physical separation
Centrifugation Steps
1. 1000g, 10 min
2. 2000g, 20 min
3. 10,000g, 30 min
4. 100,000g, 2 hr
Cell Debris
Apoptotic Bodies
Microvesicles
Traditional definitions
Exosomes
Current ISEV recommendations
• No current isolation protocol can purify based on
biogenetic origin
• Size is not an appropriate defining feature alone
• Describe EVs based on
• Physical characteristics
• Size: Large EVs, medium EVs, small EVs
• Biochemical characteristics
• Cell origin or stimulus condition
18. Large EV with “cup-
shaped” indentation
Likely small
lipoprotein
structure
EV Isolation
Ultracentrifugation has been the gold-standard procedure but lacks purity
500 nmCredit: Dr. Ru-ching Hsia, UMSOM EM Core Facility
20. Sample Collection
Sample Storage
EV Isolation
-”Omic” analyses
Transcriptomic
Genomic
Proteomic
Lipidomic
EV Characterization
Quantitative and
Qualitative
Functional Assays
Pre-analytical variables
Workflow in EV Research
1
2
3
21. EV Characterization Toolbox
Hartjes et al. (2019), Bioengineering (Basel)
Considerations in evaluating technology:
• EV Size
• EV Count
• EV Phenotype
• EV Morphology/Visualization
• Single EV or Bulk analysis?
• Isolation or Direct detection?
23. Flow Cytometry provides excellent phenotyping capability but size
resolution is a limitation, especially for small EVs
1300
Si
880
Si
590
Si
180
Si
240
Si
300
Si
F110
PS
F500
PS
Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FITC-A
Phenotyping EVs by Flow Cytometry
Erdbrügger et al. (2017), Cytometry A
Credit: Dr. Xiaoxuan Fan, UMSOM Flow Core Facility
25. Flow Cytometry provides excellent phenotyping capability but size
resolution is a limitation, especially for small EVs
Phenotyping EVs by Flow Cytometry
A B
A – White
B – Red
28. Nanoparticle Tracking Analysis for EVs
NTA has been used extensively in EV research since the mid-2000s
Erdbrügger et al. (2017), Cytometry A
29. Nanoparticle Tracking Analysis for EVs
NTA represented an important advance over DLS for polydisperse mixtures
Filipe et al. (2010), Pharm Res
30. Multi-Spectral Advanced NTA (MANTA) ViewSizer 3000
ViewSizer can use three lasers simultaneously to visualize nanoparticle samples
31. 180Si
F110PS
240Si
300Si
F500PS
590Si
880Si
1300Si
0 500 1000 1500
0
100
200
300
400
Particle Diameter (nm)
Countsperbin
ViewSizer 3000 Performance
1300
Si
880
Si
590
Si
180
Si
240
Si
300
Si
F110
PS
F500
PS
Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FITC-A
ViewSizer 3000 (Inverted)
Light Scatter
ViewSizer can accurately resolve a complex, polydisperse bead mixture.
32. 0 500 1000 1500
0
50
100
150
Particle Diameter (nm)
Countsperbin
F110PS
F500PS
F500
PS
F110
PS
ViewSizer 3000 (Inverted)
F110
PS
F110
PS
F110
PS
F110
PS
Fluorescence
ViewSizer 3000 Performance
1300
Si
880
Si
590
Si
180
Si
240
Si
300
Si
F110
PS
F500
PS
Abbreviations
F – Fluorescent
# – size in nm
PS – Polystyrene
Si – Silica
SSC-A
FITC-A
ViewSizer can identify fluorescent particles uniquely out of a polydisperse mix.
33. Influence of Laser Wavelength on Particle Detection
BLUE
ViewSizer 3000
GREEN
ViewSizer 3000
RED
ViewSizer 3000
0 50 100 150 200 250 300
0
1×107
2×107
3×107
4×107
Particle Diameter (nm)
DensityofPSD
(Particles/mL/nm)
Blue Only
Green Only
Red Only
All Lasers
0 50 100 150 200 250 300
0
10
20
30
40
50
60
70
80
90
100
Particle Diameter (nm)
CumulativeFrequency
Percent
EVs isolated from plasma require higher energy wavelengths for accurate analysis
34. ViewSizer 3000 Comparison with NanoSight LM10
0 50 100 150 200 250 300
0.000
0.005
0.010
0.015
Particle Diameter (nm)
DensityofPSD
(NormalizedUnits)
ViewSizer Red Only
ViewSizer All Lasers
NanoSight LM10
NanoSight LM10
RED
0 50 100 150 200 250 300
0
10
20
30
40
50
60
70
80
90
100
Particle Diameter (nm)
CumulativeFrequency
Percent
RED
ViewSizer 3000
Laser wavelength can significantly affect particle count and size distribution
35. Biggest Advantages
Accurate counting and sizing of individual nanoparticles
Fluorescence NTA may help distinguish real EVs from contaminants
Current Limitations
Conventional NTA requires a clean EV isolation procedure
Minimum size detected for biologics – 50nm?
Future Directions
What design features can be added to improve lower detection limit?
Can instruments be designed for multiplex phenotyping like flow cytometry?
Future Potential of NTA in EV Research
36. Bill Travers, Ph.D.
Sean Travers, Ph.D.
Jeff Bodycomb, Ph.D.
Julie Chen Nguyen, Ph.D.
Alan Faden, M.D.
Junfang Wu, M.D., Ph.D.
Steven Jay, Ph.D.
Mentor Team
…and the rest of the lab!
Acknowledgements
Funding Sources
RF1 NS110637-01 (JW/SJ)
R01 NS094527-03 (JW)
R01 2NR013601-07 (JW/AIF)
R01 NS110635-01 (JW/AIF)
R01 NS110567-01 (JW)
Kuba Tatarkiewicz, Ph.D.
37. • Latest MISEV guidelines (2018)
https://www.tandfonline.com/doi/full/10.1080/20013078.2018.1535750
• Original ISEV position statement (MISEV 2014)
https://www.tandfonline.com/doi/full/10.3402/jev.v3.26913
• Coursera Course “Basics on Extracellular Vesicles”
https://www.coursera.org/learn/extracellular-vesicles#about
• Extracellular Vesicle Club for latest advances in research
https://www.youtube.com/channel/UC0nhdTaTEUqpO8anXZqRdkQ
Resources to learn more about EVs/exosomes