This document describes an experiment using laser scanning confocal microscopy to study the Golgi apparatus and endoplasmic reticulum in plant cells. Arabidopsis thaliana cells were labeled with fluorescent proteins to tag the organelles of interest. A Leica SP5 confocal microscope along with image analysis software was used to capture z-stack images of the specimens and measure properties of the Golgi apparatus like size. Point spread function measurements of fluorescent beads were performed to allow for image deconvolution. The experiment aimed to better understand confocal microscopy and the movement and structure of the Golgi apparatus and endoplasmic reticulum in plant cells.
Compound Microscopes An Essential and Affordable ApparatusAlisha Roy
Compound microscopes are used to view transparent specimens not visible to the naked eye. There are several types of compound microscopes including standard light microscopes, inverted microscopes, stereo microscopes, monocular microscopes, digital microscopes, UV microscopes, and fluorescence microscopes. Standard light microscopes enlarge images 10x with the eyepiece and further with 4x, 10x, 40x, or 100x objectives, while inverted microscopes prevent specimens from touching high-powered lenses and allow examination of thicker samples.
The stereo microscope is an optical microscope variant designed for low magnification observation of surface samples. It has three key parts: a viewing head/body that houses optical components, a focus block that attaches the head to the stand, and a luminous stand that supports the microscope. Stereo microscopes provide an erect, three-dimensional perspective and are used for applications like insect dissection, microsurgery, watchmaking, and commercial inspection tasks.
This document provides information about the different levels of biological organization:
- The basic level is the cell, which is the smallest unit considered living. Cells make up tissues, organs, and organ systems.
- Organisms are composed of either a single cell (unicellular) or multiple organ systems (multicellular).
- Populations consist of groups of the same species living in an area. Multiple interacting populations form a community.
- Communities within a geographic region along with abiotic factors constitute an ecosystem or biome. All biomes together encompass the biosphere, the highest level of biological organization.
This document describes the components and workings of a compound microscope. A compound microscope uses multiple lenses, including an objective lens and ocular lens, to magnify specimens. The objective lens collects light from the specimen and forms a primary magnified image, which is further magnified by the ocular lens for viewing. Key parts include the mechanical base and arm, illuminating mirrors and condenser, and magnifying objective and ocular lenses. Compound microscopes can magnify specimens up to 1000x and are useful laboratory and educational tools for examining biological and mineral samples.
Compound microscope is the highly used instrument for the purpose to watch / observed any micro organism. It is the basic instrument for the analysis of the micro object.It used in the various fields such as biology, physics, chemistry,forensic science,geology etc.
The document discusses the compound microscope. It describes how a compound microscope uses two lenses, an objective lens near the specimen and an eyepiece lens, to magnify the specimen. It details the various parts of a compound microscope, including the stage, objective lenses, eyepiece, condenser, and diaphragm. It explains that changing the objective lens allows higher magnification and that oil immersion objectives provide the highest magnification of 100x.
Parts of the microscope and their functionsSimple ABbieC
Convex lenses are curved glass that are used in microscopes and glasses to bend and focus light. A microscope uses two convex lenses, an objective lens that gathers and magnifies the light from the specimen, focusing the image inside the body tube. The ocular lens at the top of the microscope then further magnifies this image for viewing. Turning the nose piece changes the objective lens, altering the magnification of the specimen.
Compound Microscopes An Essential and Affordable ApparatusAlisha Roy
Compound microscopes are used to view transparent specimens not visible to the naked eye. There are several types of compound microscopes including standard light microscopes, inverted microscopes, stereo microscopes, monocular microscopes, digital microscopes, UV microscopes, and fluorescence microscopes. Standard light microscopes enlarge images 10x with the eyepiece and further with 4x, 10x, 40x, or 100x objectives, while inverted microscopes prevent specimens from touching high-powered lenses and allow examination of thicker samples.
The stereo microscope is an optical microscope variant designed for low magnification observation of surface samples. It has three key parts: a viewing head/body that houses optical components, a focus block that attaches the head to the stand, and a luminous stand that supports the microscope. Stereo microscopes provide an erect, three-dimensional perspective and are used for applications like insect dissection, microsurgery, watchmaking, and commercial inspection tasks.
This document provides information about the different levels of biological organization:
- The basic level is the cell, which is the smallest unit considered living. Cells make up tissues, organs, and organ systems.
- Organisms are composed of either a single cell (unicellular) or multiple organ systems (multicellular).
- Populations consist of groups of the same species living in an area. Multiple interacting populations form a community.
- Communities within a geographic region along with abiotic factors constitute an ecosystem or biome. All biomes together encompass the biosphere, the highest level of biological organization.
This document describes the components and workings of a compound microscope. A compound microscope uses multiple lenses, including an objective lens and ocular lens, to magnify specimens. The objective lens collects light from the specimen and forms a primary magnified image, which is further magnified by the ocular lens for viewing. Key parts include the mechanical base and arm, illuminating mirrors and condenser, and magnifying objective and ocular lenses. Compound microscopes can magnify specimens up to 1000x and are useful laboratory and educational tools for examining biological and mineral samples.
Compound microscope is the highly used instrument for the purpose to watch / observed any micro organism. It is the basic instrument for the analysis of the micro object.It used in the various fields such as biology, physics, chemistry,forensic science,geology etc.
The document discusses the compound microscope. It describes how a compound microscope uses two lenses, an objective lens near the specimen and an eyepiece lens, to magnify the specimen. It details the various parts of a compound microscope, including the stage, objective lenses, eyepiece, condenser, and diaphragm. It explains that changing the objective lens allows higher magnification and that oil immersion objectives provide the highest magnification of 100x.
Parts of the microscope and their functionsSimple ABbieC
Convex lenses are curved glass that are used in microscopes and glasses to bend and focus light. A microscope uses two convex lenses, an objective lens that gathers and magnifies the light from the specimen, focusing the image inside the body tube. The ocular lens at the top of the microscope then further magnifies this image for viewing. Turning the nose piece changes the objective lens, altering the magnification of the specimen.
Light microscopy uses visible light and magnifying lenses to examine small objects not visible to the naked eye. There are several types of light microscopes, including bright-field, dark-field, phase-contrast, fluorescence, and differential interference contrast microscopes. These microscopes employ different optical techniques to produce high-contrast images of cells and intracellular structures that provide insight into biology.
The document outlines the main parts of a microscope including the eyepiece, body tube, nosepiece, objectives, arm, stage, stage clips, diaphragm, coarse and fine adjustment knobs, light source, and base. It provides instructions for using a microscope, such as carrying it with two hands, using lens paper for cleaning, and adjusting the coarse focus knob to bring the slide into view. Magnification is calculated by multiplying the eyepiece magnification by the objective magnification.
Microscopes are tools that magnify objects too small to see with the naked eye. They have various parts including objectives, an eyepiece, stage, and light source. There are three main types - simple, compound, and electron microscopes. Compound microscopes use two sets of lenses to magnify objects up to 200 times, while electron microscopes can magnify objects up to 300,000 times using electrons rather than lenses. Microscopes are used by scientists to study living and non-living tiny specimens.
The document outlines the main parts of a microscope including the eyepiece, body tube, nosepiece, objectives, arm, stage, stage clips, diaphragm, coarse and fine adjustment knobs, light source, and base. It provides instructions for using a microscope, such as carrying it with two hands, using lens paper for cleaning, and adjusting the coarse focus knob to bring the slide into view. Magnification is calculated by multiplying the eyepiece magnification by the objective magnification.
This document discusses experiments performed with microscopes and the examination of cells and carbohydrates. In the first exercise, specimens including a letter "e", skin hair, and mosquito larva are observed under compound and trinocular microscopes at different magnifications. The second exercise explores microscope magnification and how specimens appear larger through calculation of linear magnification. The third exercise examines animal cells from cheek, blood, and egg samples and plant cells from onion and monocot/dicot specimens under the microscope. Key animal and plant cell parts are also described. Finally, the fourth exercise tests whether common foods contain simple or complex carbohydrates through color changes when exposed to iodine solution.
These lectures has prepared for postgraduate student (Ophthalmology) according to the curriculum of Bangladesh College of Physician and Surgeons (BCPS) and Bangabondhu Sheikh Mujib Medical University (BSMMU) Bangladesh
The document discusses the history and components of the microscope. It describes how Antony van Leeuwenhoek is credited with developing the first microscope in the 16th century and discovering bacteria and cells. The main types of microscopes are described as optical, electron, and scanning probe microscopes. Optical microscopes are further divided into simple and compound microscopes. Compound microscopes use two lenses, the objective and eyepiece lenses, to magnify specimens, while simple microscopes use only one lens. The parts of a compound microscope and how it works to produce a magnified image are also outlined.
This document provides an overview of microscopy, including:
1. It defines microscopy as using an instrument called a microscope to view objects too small to see with the naked eye.
2. It describes some key parts and types of microscopes like compound, phase contrast, dark ground, and electron microscopes.
3. It explains concepts like magnification, resolution, and aberration that are important for microscopy.
The document provides an overview of microscopes, including their history and uses. It describes the key parts and functions of compound light microscopes, such as the mechanical and optical systems including objectives, eyepieces, stages, and condensers. Different types of lenses and their magnifications are discussed. The document also covers using microscopes properly, including focusing techniques and using immersion oil with high magnification objectives.
The early microscope was called simple microscope consisted of biconvex lenses and were essentially magnifying glassesto see microbes a compound microscope which has two lenses between the eye and the object this system magnify the object illumination system sun mirror lamp ensure adequate light is available for viewing
Bright field microscopefirst to be used in laballow light to pass directly through to the eye without being deflected by an intervening opaque in the condenser
The basic frame of microscope consists of abase ,stage to hold slides, an arm for carryingthe stage may have two clips or a movable mechanical stage to hold the slidelight source in the base
Condenser consisted of several lenses which concentrate light on slide by focusing it into conehas iris diaphragm control size and angle of cone lightoptimal light will reach to slide
Revolving nose piece holding three or four objective lenses
Eye piece lenses 10 x to 12.5 xif a microscope has only one ocular lens called mononuclear microscopebinocular microscope has two ocular lens
One can focus the image bymoving the tube closer to slide orthe stage closer to the objective lensusing coarse adjustment low power lenses 4x to 10xfine adjustment for focusing oil immersion or high powercoarse adjustment knop move the stage or move lenses longer distance
The area seen through microscope is called the field vision
The magnification power of microscope depends on the power of objective lenses used with the ocular
Compound microscope have three or four objective lenses mounted on nose piecescanning 4x low power 10xhigh –dry 40-44x oil immersion 97x-100x highest magnificationmust be used with immersion oilthe optical system could be built to magnify more than 1000x of microscope but the resolution would be poor
Magnification provided each lens is stamped on the barrel
The total magnification of objective lensesis calculated by multiplying the magnification of ocular usually 10x by the magnification of objective lensesmost important one in microbiology oil immersion lenses must used with oil power 97x- 100x
resolution limit or resolving powerit is a function of its numerical aperturethe wave length of light the design of condenserthe maximum resolution of best microscope with oil immersion lenses is 0.2 millimicron this means two objects that are 0.2 millimicron apart can be seen clearly as separate entities objects closer than that will be seen like single object bright field microscope
This presentation summarizes key concepts about microscopes and microscopy. It defines important terminology like magnification, resolution, resolving power, and numerical aperture. It explains that magnification increases apparent size but resolution is the ability to distinguish between two points. Numerical aperture depends on the angle of light collection and refractive index. The limit of resolution is calculated as the wavelength of light divided by two times the numerical aperture, allowing objects 0.2 micrometers apart to be distinguished under optimal conditions.
The document discusses the history and development of microscopy from the 14th century to the late 19th century. It describes how early microscopes used simple lenses and had magnifications between 10x-80x, while Leeuwenhoek achieved up to 300x magnification. The compound microscope was developed in the mid-17th century, improving detail visibility. Later advances included achromatic lenses, oil immersion, and condensers to further enhance resolution. Key optical principles such as refractive index and the factors affecting resolution and its relationship with magnification are also summarized.
This document outlines three services for real estate transactions: Protocol forms completed online with instant title checks and available contract packs; Express Search delivers a full contract pack to a buyer's lawyer within 24 hours of an offer; Gazeal It provides a digital handshake agreement between parties subject to a 14 day cooling off period with no overhead costs and a fully digital workflow that saves months in the transaction process.
George Enos Simbili is a Kenyan chef seeking employment. He has over 15 years of experience working in kitchens for schools, hotels, and conference centers in Kenya. Simbili has a diploma in food and beverage production from City & Guilds. He is currently the head cook at The Riara Group of Schools, where he supervises staff and coordinates menus. He has received awards for his performance and commitment from previous employers.
This short document contains 3 stock photos with captions and text encouraging the reader to create their own Haiku Deck presentation on SlideShare. In a few words, it promotes making slideshows on Haiku Deck and sharing them on SlideShare.
The Art of Learning International Ltd is a UK-based learning and technology company that helps people and organizations transform and thrive by making learning a natural part of their daily lives. They create "addictive" learning cultures by linking digital technologies, high-quality content, and world-class coaches to focus on change and transitions in both personal and professional contexts. They offer a range of courses and delivery solutions including videos, gamification, and social learning to support organizations and individuals through career and life transitions.
Scott Harding has over 25 years of experience in automotive sales and management. He founded Magnum Jewelers which expanded to 4 locations with over $1.2 million in annual revenue before selling and moving to Florida. In Florida, he began a successful career in automotive sales, quickly becoming a top salesman and accepting a management position. Throughout his career he has exceeded sales goals and led teams as a successful manager, trainer, and mentor.
Light microscopy uses visible light and magnifying lenses to examine small objects not visible to the naked eye. There are several types of light microscopes, including bright-field, dark-field, phase-contrast, fluorescence, and differential interference contrast microscopes. These microscopes employ different optical techniques to produce high-contrast images of cells and intracellular structures that provide insight into biology.
The document outlines the main parts of a microscope including the eyepiece, body tube, nosepiece, objectives, arm, stage, stage clips, diaphragm, coarse and fine adjustment knobs, light source, and base. It provides instructions for using a microscope, such as carrying it with two hands, using lens paper for cleaning, and adjusting the coarse focus knob to bring the slide into view. Magnification is calculated by multiplying the eyepiece magnification by the objective magnification.
Microscopes are tools that magnify objects too small to see with the naked eye. They have various parts including objectives, an eyepiece, stage, and light source. There are three main types - simple, compound, and electron microscopes. Compound microscopes use two sets of lenses to magnify objects up to 200 times, while electron microscopes can magnify objects up to 300,000 times using electrons rather than lenses. Microscopes are used by scientists to study living and non-living tiny specimens.
The document outlines the main parts of a microscope including the eyepiece, body tube, nosepiece, objectives, arm, stage, stage clips, diaphragm, coarse and fine adjustment knobs, light source, and base. It provides instructions for using a microscope, such as carrying it with two hands, using lens paper for cleaning, and adjusting the coarse focus knob to bring the slide into view. Magnification is calculated by multiplying the eyepiece magnification by the objective magnification.
This document discusses experiments performed with microscopes and the examination of cells and carbohydrates. In the first exercise, specimens including a letter "e", skin hair, and mosquito larva are observed under compound and trinocular microscopes at different magnifications. The second exercise explores microscope magnification and how specimens appear larger through calculation of linear magnification. The third exercise examines animal cells from cheek, blood, and egg samples and plant cells from onion and monocot/dicot specimens under the microscope. Key animal and plant cell parts are also described. Finally, the fourth exercise tests whether common foods contain simple or complex carbohydrates through color changes when exposed to iodine solution.
These lectures has prepared for postgraduate student (Ophthalmology) according to the curriculum of Bangladesh College of Physician and Surgeons (BCPS) and Bangabondhu Sheikh Mujib Medical University (BSMMU) Bangladesh
The document discusses the history and components of the microscope. It describes how Antony van Leeuwenhoek is credited with developing the first microscope in the 16th century and discovering bacteria and cells. The main types of microscopes are described as optical, electron, and scanning probe microscopes. Optical microscopes are further divided into simple and compound microscopes. Compound microscopes use two lenses, the objective and eyepiece lenses, to magnify specimens, while simple microscopes use only one lens. The parts of a compound microscope and how it works to produce a magnified image are also outlined.
This document provides an overview of microscopy, including:
1. It defines microscopy as using an instrument called a microscope to view objects too small to see with the naked eye.
2. It describes some key parts and types of microscopes like compound, phase contrast, dark ground, and electron microscopes.
3. It explains concepts like magnification, resolution, and aberration that are important for microscopy.
The document provides an overview of microscopes, including their history and uses. It describes the key parts and functions of compound light microscopes, such as the mechanical and optical systems including objectives, eyepieces, stages, and condensers. Different types of lenses and their magnifications are discussed. The document also covers using microscopes properly, including focusing techniques and using immersion oil with high magnification objectives.
The early microscope was called simple microscope consisted of biconvex lenses and were essentially magnifying glassesto see microbes a compound microscope which has two lenses between the eye and the object this system magnify the object illumination system sun mirror lamp ensure adequate light is available for viewing
Bright field microscopefirst to be used in laballow light to pass directly through to the eye without being deflected by an intervening opaque in the condenser
The basic frame of microscope consists of abase ,stage to hold slides, an arm for carryingthe stage may have two clips or a movable mechanical stage to hold the slidelight source in the base
Condenser consisted of several lenses which concentrate light on slide by focusing it into conehas iris diaphragm control size and angle of cone lightoptimal light will reach to slide
Revolving nose piece holding three or four objective lenses
Eye piece lenses 10 x to 12.5 xif a microscope has only one ocular lens called mononuclear microscopebinocular microscope has two ocular lens
One can focus the image bymoving the tube closer to slide orthe stage closer to the objective lensusing coarse adjustment low power lenses 4x to 10xfine adjustment for focusing oil immersion or high powercoarse adjustment knop move the stage or move lenses longer distance
The area seen through microscope is called the field vision
The magnification power of microscope depends on the power of objective lenses used with the ocular
Compound microscope have three or four objective lenses mounted on nose piecescanning 4x low power 10xhigh –dry 40-44x oil immersion 97x-100x highest magnificationmust be used with immersion oilthe optical system could be built to magnify more than 1000x of microscope but the resolution would be poor
Magnification provided each lens is stamped on the barrel
The total magnification of objective lensesis calculated by multiplying the magnification of ocular usually 10x by the magnification of objective lensesmost important one in microbiology oil immersion lenses must used with oil power 97x- 100x
resolution limit or resolving powerit is a function of its numerical aperturethe wave length of light the design of condenserthe maximum resolution of best microscope with oil immersion lenses is 0.2 millimicron this means two objects that are 0.2 millimicron apart can be seen clearly as separate entities objects closer than that will be seen like single object bright field microscope
This presentation summarizes key concepts about microscopes and microscopy. It defines important terminology like magnification, resolution, resolving power, and numerical aperture. It explains that magnification increases apparent size but resolution is the ability to distinguish between two points. Numerical aperture depends on the angle of light collection and refractive index. The limit of resolution is calculated as the wavelength of light divided by two times the numerical aperture, allowing objects 0.2 micrometers apart to be distinguished under optimal conditions.
The document discusses the history and development of microscopy from the 14th century to the late 19th century. It describes how early microscopes used simple lenses and had magnifications between 10x-80x, while Leeuwenhoek achieved up to 300x magnification. The compound microscope was developed in the mid-17th century, improving detail visibility. Later advances included achromatic lenses, oil immersion, and condensers to further enhance resolution. Key optical principles such as refractive index and the factors affecting resolution and its relationship with magnification are also summarized.
This document outlines three services for real estate transactions: Protocol forms completed online with instant title checks and available contract packs; Express Search delivers a full contract pack to a buyer's lawyer within 24 hours of an offer; Gazeal It provides a digital handshake agreement between parties subject to a 14 day cooling off period with no overhead costs and a fully digital workflow that saves months in the transaction process.
George Enos Simbili is a Kenyan chef seeking employment. He has over 15 years of experience working in kitchens for schools, hotels, and conference centers in Kenya. Simbili has a diploma in food and beverage production from City & Guilds. He is currently the head cook at The Riara Group of Schools, where he supervises staff and coordinates menus. He has received awards for his performance and commitment from previous employers.
This short document contains 3 stock photos with captions and text encouraging the reader to create their own Haiku Deck presentation on SlideShare. In a few words, it promotes making slideshows on Haiku Deck and sharing them on SlideShare.
The Art of Learning International Ltd is a UK-based learning and technology company that helps people and organizations transform and thrive by making learning a natural part of their daily lives. They create "addictive" learning cultures by linking digital technologies, high-quality content, and world-class coaches to focus on change and transitions in both personal and professional contexts. They offer a range of courses and delivery solutions including videos, gamification, and social learning to support organizations and individuals through career and life transitions.
Scott Harding has over 25 years of experience in automotive sales and management. He founded Magnum Jewelers which expanded to 4 locations with over $1.2 million in annual revenue before selling and moving to Florida. In Florida, he began a successful career in automotive sales, quickly becoming a top salesman and accepting a management position. Throughout his career he has exceeded sales goals and led teams as a successful manager, trainer, and mentor.
The document discusses the benefits of entrepreneurship for college students. It outlines the author's experience with three startups in social good, college societies, and digital arts consulting. The author details five skills learned through these experiences - having a vision and plan, public speaking, problem solving, time management, and networking to build a business. The document promotes entrepreneurship for college students and provides the author's contact information for further discussion.
The document discusses The Art of Learning International Ltd, which helps people and organizations learn and change during transitions. It offers various online and blended courses on topics like career changes, personal development, management, and more. The courses use personalized and bite-sized learning approaches with videos, materials, and social learning. The company created its Taolin learning platform to make change easier through lasting, cost-effective and personalized learning that is always ahead of the curve.
The document outlines the activities of a summer internship at a retail company from June to August. It includes orientation, training on ShopperTrak software, shadowing employees, analyzing store performance reports, updating metrics reports, and presenting final projects. The intern gained experience in areas like data analysis, report generation, store visits, and learning retail operations through this structured internship program.
This short document promotes the creation of presentations using Haiku Deck on SlideShare. It displays three stock photos from users kaybee07, missbutterflies, and bibendum84 to inspire new presentations. The document concludes by encouraging the reader to get started making their own Haiku Deck presentation.
Characterization methods - Nanoscience and nanotechnologiesNANOYOU
This document discusses characterization methods for nanomaterials, specifically microscopy and spectroscopy techniques. It describes scanning tunneling microscopy (STM) and atomic force microscopy (AFM) which allow imaging at the atomic scale. STM works by measuring tunneling current between a tip and conductive sample, which is translated into topographic images. It has been instrumental in advancing nanoscience by enabling visualization of materials at the nanoscale. The document also briefly mentions other microscopy and spectroscopy methods for nanomaterial analysis such as electron microscopy, X-ray techniques, and Raman spectroscopy.
Realisation of a Digitally Scanned Laser Light Sheet Fluorescent Microscope w...James Seyforth
This document describes the design and implementation of a digitally scanned laser light sheet fluorescence microscope (DSLM) at King's College London. The author elaborates on the fundamental physics and theoretical framework of DSLM and presents the optical setup, which includes a laser for illumination, digitally scanned galvanometers to control the light sheet, and a piezoelectric flexure objective scanner. Initial testing of the microscope used fluorescent beads and found a lateral resolution of 837 nm and axial resolution of 2470 nm, close to the estimated system resolution but with some systematic errors. Further imaging was limited by software and equipment issues.
Spectrophotometry is used in Biology to plot optical density curves (to determine the concentration of biochemicals) or to conduct a cell count for a suspension.
Survey & X-ray (Chandra) Spectral analysis of Fermi LAT gamma pulsarsSaurabh Bondarde
The document describes a thesis project analyzing archival X-ray data from the Chandra observatory of gamma-ray pulsars detected by the Fermi Gamma-ray Space Telescope. It provides background on electromagnetic spectra, high-energy astrophysics missions including Fermi and Chandra, X-ray telescopes and imaging capabilities. It also summarizes literature on pulsar basics, types of pulsars, research status on magnetars, and prior multi-wavelength studies of gamma-ray pulsars. The analysis will include source detection, spectrum extraction and spectral fitting of selected pulsars in the Chandra data to study their multi-wavelength high energy characteristics.
Microscope part 2 BY DR. C. P. ARYA (B.Sc. B.D.S.; M.D.S.; P.M.S.; R.N.T.C.P.)DR. C. P. ARYA
The document discusses the components and operation of optical microscopes. It describes the body tube, stages, illumination methods including Kohler and Nelson techniques, objectives, eyepieces, and other parts. It also covers magnification calculation, microscope setup, cleaning, measurement using micrometers, and different microscope types such as fluorescence, polarization, and electron microscopes. The document provides detailed information on the structure and use of optical microscopes.
This document discusses biometry techniques used to measure eye dimensions needed for intraocular lens (IOL) power calculations during cataract surgery. It describes keratometry to measure corneal curvature, A-scan ultrasound to measure axial length, and various IOL formulas used to calculate the needed IOL power based on the measured parameters. Key biometry techniques discussed include keratometry, A-scan ultrasound, optical biometers like the IOL Master and Lens Star, and common IOL formulas like SRK/T.
NG3D902 - Basic Ray Optics Experiments - 2016Chris Francis
This document describes 6 experiments conducted on the fundamentals of ray optics, including the laws of reflection, refraction, total internal reflection, dispersion, and the properties of convex/concave lenses and the Lensmaker's equation. The experiments were led by Christopher Francis and aimed to demonstrate how light behaves at the boundaries between transparent media based on these optical principles. Key findings included verifying Snell's law, identifying the critical angle for total internal reflection, showing dispersion's effect on the index of refraction, and using the Lensmaker's equation to calculate a lens's focal length.
Microscopy - Magnification, Resolving power, Principles, Types and ApplicationsNethravathi Siri
Magnification, Resolving power, Principles and Applications of Simple, Compound, Stereozoom, Phase contrast, Fluorescent and Electron microscopes (TEM & SEM).
Microscopy is the technical field that uses microscopes to observe samples which are not in the resolution range of the normal-unaided eye.
Introduction to microscopy
Different parts of a microscope & their function
Different types of microscopy
Different types of optical microscopy
Different types of electron microscopy
Different terms used in microscopy
Staining- Simple, Differential, Special
Gram Staining
This document provides an overview of microscopy used in diagnostic microbiology. It discusses the history and types of microscopes including bright field, dark field, phase contrast, fluorescence, transmission electron, and scanning electron microscopes. It describes how each microscope works and its applications. Key aspects covered include the use of microscopy to identify microorganisms, detect viruses, and examine cellular structures in detail not visible to the naked eye. Microscopy is an important tool in diagnostic microbiology.
This document summarizes an interdisciplinary master's project that evaluates machine learning algorithms for tracking neuronal signals recorded by electronic depth control probes. The project aims to identify recording channels and track neural activity over time by classifying features extracted from neuronal signal measurements. Different supervised learning algorithms are tested and evaluated to determine the most appropriate method. Preliminary results show it is possible to track neural activity between recording sessions, and shifts in probe position between sessions may be detectable from the classification results.
Introduction
Definition
Basic mechanism
Prerequisite of flow cytometer
Components of flow cytometry
Flow system
Optics system
Concept of scattering
Advantage
Limitation
Application
Conclusion
References
Microscopy is the technical field of using microscopes to view objects that cannot be seen with the naked eye. There are three main types of microscopy - light microscopy, which uses visible light; electron microscopy, which uses electrons; and scanning probe microscopy, which uses a physical probe. Light microscopes like brightfield, darkfield, phase contrast, and fluorescence microscopes are commonly used to view living and stained specimens. Electron microscopes have much higher resolving power than light microscopes and are able to view much smaller structures. Transmission electron microscopes form images using electrons transmitted through thin specimens while scanning electron microscopes form images from electrons emitted from surfaces.
Microscopy is the technical field of using microscopes to view objects that cannot be seen with the naked eye. There are three main types of microscopy - light microscopy, which uses visible light; electron microscopy, which uses electrons; and scanning probe microscopy, which uses a physical probe. Light microscopes like brightfield, darkfield, phase contrast, and fluorescence microscopes are commonly used to view living and stained specimens. Electron microscopes have much higher resolving power than light microscopes and are able to view much smaller structures. Transmission electron microscopes form images using electrons transmitted through thin specimens while scanning electron microscopes form images from electrons emitted from surfaces.
Principles of optical coherence tomographyJagdish Dukre
OCT uses interferometry to perform non-invasive imaging of biological tissues. The first OCT images of the retina were obtained in 1990. Time domain OCT works by scanning a reference mirror to measure echo time delays, while Fourier domain OCT measures spectral interference patterns without scanning. Fourier domain OCT allows for much faster acquisition speeds compared to time domain OCT. Integrating OCT with scanning laser ophthalmoscopy enables localization of OCT scans on fundus images.
This document discusses laser medicine and medical imaging projects at RLE including:
1) Developing an ultrahigh resolution OCT system using a microstructured fiber for continuum generation, achieving 2.5 μm resolution for in vivo imaging.
2) Demonstrating spectroscopic OCT of water absorption using a 200 nm bandwidth light source centered at 1400 nm.
3) Designing OCT imaging devices like a colposcope that integrates OCT with standard clinical imaging to enable early disease detection.
SEM, AFM, and 3D Optical Profiler IntroductionKristina Boberg
This document provides an introduction to various types of microscopy used in nanotechnology. It explains that light microscopes have resolution limitations and then describes scanning electron microscopes which use electron beams rather than light to achieve higher resolution down to 1nm. Atomic force microscopes are also discussed, which use a physical probe to scan samples with nanoscale resolution. Finally, 3D optical profilers are introduced, which use light waves to generate 3D renderings of micrometer-scale surface topography. Overall, the document outlines different microscopy techniques and their applications from the micrometer to nanometer scale.
This document provides information about retinal recognition as a biometric identification method. It discusses the anatomy of the retina and how retinal scanning works. Retinal recognition analyzes the unique blood vessel patterns in a person's retina. While very reliable, retinal recognition is also considered invasive and expensive. The document reviews the history and development of retinal scanning technology over time. It examines the strengths of retinal recognition in providing a large number of identification points and its stability over a person's lifetime. However, weaknesses include the need for user cooperation, discomfort of the scanning process, and the high cost of retinal scanning devices.
Microscopy is used to study microorganisms that are too small to be seen with the naked eye. Bacteria are typically 2-5 μm in size, below the resolution of the human eye, so microscopy is needed. There are different types of microscopes that provide magnification and resolution, including brightfield, darkfield, phase contrast, fluorescent, and electron microscopes. Each type has a specific working principle and applications - for example, phase contrast microscopy can be used to study microbial motility while living cells are visible. Microscopy allows rapid identification and detection of organisms in patient specimens and provides diagnostic information in microbiology.
1. April 8th
2014
Maria Helene Kalkvik
Cell Biology BI2012
N o r w e g i a n U n i v e r s i t y o f S c i e n c e a n d T e c h n o l o g y
Live cell imaging and advanced image analysis
of the Golgi apparatus in plants
2. 2
Abstract
This experiment was conducted in order to obtain a better understanding of laser scanning
confocal microscopy, and also study the movement and stucture of the Golgi apparatus and
endoplasmatic reticulum in plant cells. The chosen organells were labled with fluorescent
proteins in order to observe them in the microscope. Arabidopsis thaliana was the chosen
organism for the experiment. A Leica SP5 microscope was used along with computer software.
Image processing softwares Amira and ImageJ were used to analyse the images of the specimen.
Overview images of both the ER and Golgi apparatus was captures, and measurements of the
Golgi apparatus’ size were contucted. Point spread function of fluorescent beads was determined
in order to perform deconvolution.
Abbreviations
GFP: Green fluorescent protein
PSF: Point spread function
FWHM: Full with half maximum
ROI: Region of interest
ER: Endoplasmatic reticulum
LUT: Look up table
NA: Numerical aperture
3. 3
Table of contents
ABSTRACT....................................................................................................................................... 2
ABBREVIATIONS........................................................................................................................... 2
1 INTRODUCTION ........................................................................................................................ 4
1.1 CONFOCAL MICROSCOPY ................................................................................................................................ 4
1.2 FLUORESCENSE................................................................................................................................................. 5
1.2.1 Green Fluorescent Protein.............................................................................................................................. 5
1.3 POINT SPREAD FUNCTION .............................................................................................................................. 6
1.4 DECONVOLUTION............................................................................................................................................ 7
1.5 RESOLUTION ..................................................................................................................................................... 9
DYNAMIC RANGE..................................................................................................................................................11
1.6 BIOLOGICAL SAMPLES.......................................................................................................... 11
1.6.1 ARABIDOPSIS THALIANA ............................................................................................................................11
1.6.2 THE PLANT CELL .........................................................................................................................................12
1.6.3 ENDOPLASMATIC RETICULUM ..................................................................................................................12
1.6.4 THE GOLGI APPARATUS .............................................................................................................................12
1.6.5 ACTIN.............................................................................................................................................................12
2 MATERIALS AND METHODS..................................................................................................13
2.1 SPECIMEN PREPARATION..............................................................................................................................13
2.2 BEAD PREPARATION ......................................................................................................................................13
2.3 MICROSCOPE ...................................................................................................................................................13
2.4 IMAGE PROCESSING AND SOFTWARE .........................................................................................................13
2.5 OPTIMAL RESOLUTION..................................................................................................................................14
2.6 PSF ANALYSIS..................................................................................................................................................15
2.7 DECONVOLUTION..........................................................................................................................................15
3 RESULTS......................................................................................................................................15
3.1 IMAGE ANALYSIS OF THE GOLGI APPARATUS ...........................................................................................15
3.2 IMAGE ANALYSIS OF THE ENDOPLASMATIC RETICULUM........................................................................16
3.3 DECONVOLUTION OF THE GOLGI Z-STACK.............................................................................................17
3.4 SIZE ANALYSIS OF THE GOLGI Z-STACKS ..................................................................................................17
4 DISCUSSION ...............................................................................................................................18
4.1 IMAGE ANALYSIS OF THE GOLGI APPARATUS AND ER...........................................................................18
4.2 OPTIMAL RESOLUTION..................................................................................................................................18
4.3 PSF ANALYSIS..................................................................................................................................................18
4.4 DECONVOLUTION OF THE GOLGI Z-STACK..............................................................................................19
4.5 SIZE ANALYSIS OF THE GOLGI STACKS ......................................................................................................19
5 LITTERATURE ...........................................................................................................................21
5.1 ILLUSTRATIONS...............................................................................................................................................22
7 APPENDIX...................................................................................................................................23
A CALCULATIONS .................................................................................................................................................23
A.1 Resolution limit, pixel size and number of pixels needed.................................................................................23
B METROLO J ANALYSIS ......................................................................................................................................24
B.1 PSF analysis of fluorescent beads....................................................................................................................24
B.2 Size analysis of the Golgi stacks .....................................................................................................................24
4. 4
1 Introduction
1.1 Confocal microscopy
Laser scanning confocal microscopy has become an important tool for studying
and observing biological cells. Through this technique one can obtain knowledge
on how the organells in the cells move through the cytosol, and gain insight in
cellular functions. It also gives the opportunity to perform optical cross sections of
transparent sampels, withouth physically slicing them into sections. The ability to
remove glare from out of focus layers has been an important improvent for studies
involving biological imaging. [1]
In a laser scanning confocal microscope (LSCM) a laser is used as a light source.
The light is then filtered by an acousto-optical tunable filter (AOTF), which allows
for regulation of both the wavelength of emitted light from the laser as well as
exitation intensity. These microscopes makes it possible for examination of
fluorescence emisson from 400 to 750 nanometers. [2]
After the light is filtered by an AOTF, the light is reflected by a dicromatic mirror,
which only reflects certain wavelengths and let others pass through. The Leica SP5
uses an acousto-optical beam splitter (AOBS) insted of a dicromatic mirror. Here,
the crystalline materials only reflect certain wavelengths of light by the interaction
of acoustic waves. This is done by manipulates the refractive index of the crystal.
[3]
The specimen is scanned by the laser, while two high speed oscillating mirrors
direct the beam in a certain pattern across a chosen area of the specimen. One
mirror controls the light along the x-axis, and the other along the y-axis. When a
region of interest is found (ROI), the light moves along the x-axis from a starting
point, and then returns to the starting point to scan in the y-dimension. [4]
Before the emission light reaches the detectors, it passes through a pinhole
aperture. The pinhole’s function is to reduce light disturbance, like blur, from
planes above and below the plane of focus. Only a small part of the light from
other planes in the specimen will pass through the pinhole (Figure 1). [4]
Figure 1: Illustration of the pin hole’s function in a confocal microscope 2
5. 5
1.2 Fluorescense
Fluorescense is the ability certain compounds have to emit visible light. When the
compund is exited by a photon, electrons move to a higher state of energy. This
state is highly unstable which results in the electrons moving back to their original
state, and when they do, energy is released. Energy in this case is in the form of
light. Different compounds have different electron configuration and orientation of
chemical bonds. This leads to emission of different wavelengts as the exited state
for the electron may be in an higher or lower state of energy, depending on the
compund. Since light can be viewd as a form of energy, differences in energy
because of electron configuration, the electrons exited state and its original state,
leads to emission of different light.
The wavelenth of light required to exite a compound is designated as 𝜆!"#
, where
the unit is nanometers (nm). Some of the energy is lost as it forms heat and
vibration, not only photons (light). Therefore the photons emitted from the
compund has less energy, and thus a longer wavelength than the light used to exite
the compound. This difference between the exitation wavelength and emission
wavelength is called Stokes shift. The emission wavelength is given by 𝜆!"
, and is
also in nanometers.
1.2.1 Green Fluorescent Protein
Green fluorescense protein (GFP) was isolated from the jellyfish Aequorea victoria.
Research shoes that the molecule is able to fluoresce when it is expressed by itself
in any organism, thus making it a well suited marker for biological studies and cell
imaging [5]. GFP consists of eleven 𝛽-barrels and one 𝛼-helix, surrounding the
cromophore in the core (Figure 2). The cromophore is the part of the molecule
that makes fluorescense possible. It has been modified in order to create a more
stable and effective version of the fluorescent protein, compared to the wild type
found in A. victoria. One of the modified versions are called enhanced yellow
fluorescent protein (EYFP). It has a 𝜆!"#
of 514 nm and a 𝜆!"
of 527 nm. [6]
6. 6
Figure 2: A model of Green Fluorescent Protein (GFP). The cromophore is not
depicted in the model. The model was created in the software Amira, by using the
protein data bank, 1EMA.
When studies involving fluorescent proteins are contucted, the protein is fused to
proteins who binds to, or target the chosen organelle. When studying ER, the
protein is fused to a signal peptide called Arabidopsis thaliana wall associated
kinase 2 (AtWAK2 ). The fusion happens at the N-terminus of the peptide, and the
ER recognises the signal sequence His-Asp-Glu-Leu at the C-terminus. When
studying the Golgi apparatus the fluorescent protein is fused to a cytoplasmatic and
transmembrane domain of a protein calles soybean 𝛼-1,2-mannosidase I.[6]
1.3 Point spread function
A point spread function is a result of diffraction of light in a specimen. When light
travels through the microscope it is defracted as a result of interactions with
materials it passes through, lenses. The consequence of light diffraction is that a
given point in the specimen will seem larger than it actually is. It can be said that
the PSF is a measurement of how many neighboring pixels are affected when one
pixel is fully enlightened. The given point will often be surrounded by alternating
dark and light rings, as a result of light diffraction. This patterns is refered to as an
Airy pattern, and was first discovered by George Biddel Airy. [7] The central disc is
called an Airy disc. This pattern in three dimentions is what creates what is called a
point spread function, since it describes how light spread out from a single point.
[2]
7. 7
Figure 3: The corrolation between an airy pattern and a point spread function. The graph (red)
shows the PSF as light intensity, with the airy pattern in the background. 3
1.4 Deconvolution
Blur is created as a result of the diffraction of light when it interacts with lenses in
the microscope. As blur is an undesirable factor when working with image analysis,
it can be corrected for by using the PSF. Every point, or pixel, in the image is
essensially a point spread function with a corresponding airy pattern. The out of
focus blur is a result of the alternating dark and light rings of the airy pattern. The
goal is to remove these rings, and obtain a single point in focus. In order to do so,
one must apply the point spread function to every point of the object. [8]
Deconvolution is a mathematical process based on algoriths, that determines the
most likely estimation to reassigh out of focus blur back to its point source. To
determine the point spread function, beads with a known size can be used to
calculate it. A 3D image of the beads can be analysed with image processing
software (Figure 4).
8. 8
Figure 4: A 3D visualization of beads in the image processing software Amira.
As the PSF shows spreading of light from the point source, it can be used to
reverse the blurring effect of convolution. The PSF can be applied to chosen
images by using image processing software, and thereby reducing the blur in the
image. This creates a more realistic depiction of the specimen.
9. 9
Figure 5: The raw image (left) and the deconvolved image (right). The grana in the chloroplasts
was almost impossible to observe before deconvolution.
1.5 Resolution
Several factors affect resolution of an image taken with a confocal microscope. The
numerical aperture (NA) of the objective plays a big role in the resolution of an
image. NA is defined by [10]:
𝑁𝐴 = sin 𝛼 𝑥 𝜂 (1)
Where 𝛼 is defined as half the angle of the cone of ligth the objective captures
from the focal point, and 𝜂 is the refractive index of the immersion medium used
[10]. As a result, when the objective size increases or the refractive index increases,
so does the NA. A high NA value gives a higher resolution.
The Rayleigh criterion is an optical unit that describes the minimal distance
between two point sources at which they are distinguishable from each other. It is
given by the formula [9]:
𝐷!! =
!,!"!!"#
!"
(2)
Where 𝐷!" is the Rayleigh criterion, 𝜆!"#
is the wavelength of the excitation light,
and NA is the numerical aperture of the objective. In the axial dimension, the
Rayleigh criterian can be calculated using the following equation [9]:
10. 10
𝐷! =
!!!"#!
(!")!
(3)
Where 𝜂 is the refractive index of the immersion medium. The most common
immersion mediums are water, air, oil and glass. Their refractive indices are
presented (Table 1).
Table 1: Refractive indices of the immersion mediums water, air and oil/glass [12]
Immersion medium Refractive index
Air 1.00
Water 1.33
Oil/Glass 1.52
To illustrate how the NA affects PSF, one can generate different PSFs with varying
NA values (Figure 6).
Figure 6: How NA affects PSF. The NA varies from 1.0 (left), 1.2 (middle) and 1.4
(left). The 𝜆!"#
was set to 514 for all PSFs. The model was created using the Amira
software.
The sampling rate is also an influencial factor to resolution. The Nyquist theorem
states that it should be two samples per resolvable element to obtain an accurate
image.[9] The size of the pixel determines the sampling rate, as one pixel only can
have one light intensity value. Therefore the the sampling rate directly correlates to
11. 11
the pixel size. The following equation must be used to calculate pixel size in lateral
dimentions, in order to fulfill the Nyquist theorem:
𝑃𝑠!" =
!!"
!
(4)
Where 𝑃𝑠!" is the pixel size.
A voxel is relevant when the axial dimension is under study. While a pixel only has
two dimensions, a voxel has three, making it a volume element rather than just an
area. The voxel size is determined by the pixel size, in the x- and y-dimensions, and
also the distance between scans in the z-dimension. The Nyquist theorem can also
be applied here. The depth of a voxel should not exceed the value given by the
following equation:
𝑃𝑠! =
!!
!
(5)
Where 𝑃𝑠! is the voxel depth.
The number of pixels needed in an image can be calculated using the following
equation, by taking the Nyquist theorem in account:
#𝑝𝑥 =
!
!!"
(6)
Where the number of pixels needed in both the x and y dimensions are denoted
#𝑝𝑥, and 𝑠 represents the size of the ROI.
Dynamic range
To ensure good image quality, the microscope and the software needs to be set up
and adjusted correctly. The bit depth is an important aspect to consider. A high bit
depth will be more likely to produce an accurate depiction of the object under
study. A low bit depth results in a larger variance in light intensity [12]
The software needs to be adjusted so that the image is shown within the detectors
dynamix range. A LUT is usually used, as it gives different colours to different light
intensities.[12] One should make sure that no pixels are completely saturated, in
order to achieve a better understanding of the pixels intensity relative to each other.
1.6 Biological samples
1.6.1 Arabidopsis thaliana
Arabidopsis thaliana is one of the most commonly used species in plant studies. It
is a plant with simple growth requirements and a relativly short life cycle of eight
weeks, which makes concucting studies on the plant quite easy. Also, A. Thaliana’s
12. 12
genome has been fully mapped, and it is therefore often used in genetic
experiments. The plant is also susceptible to genetic transformations, only by
spraying the plant with a bacterium which holds the gene of interest.
1.6.2 The plant cell
The plant cells contain an endomembrane system that incorporates all membrane
bound organelles in the cell. These organelles are the endoplasmatic reticulum and
golgi apparatus, which will be discussed in the report, and also the nuclear
envelope, the cell membrane, vacuoles, lysosomes and transport vesicles. The
vaculoles controlles turgor pressure in the cell, as well as it works as a storage space
for water and inorganic ions among other things. As a result, the plant vacuole is
quite large, which only makes it possible for the other organelles to occupy the
small cytoplasmic space between the central vacuole and the cell membrane.[13]
1.6.3 Endoplasmatic reticulum
The endoplasmati reticulum (ER) is an organelle consisting of both the rough and
smooth ER, which have specialized functions in the cell. It is a continuous network
of tubules and sacs. The rough ER plays a prominent role in protein synthesis, as
well as protein modification and marking. It is called the rough ER because of
ribosomes sitting on its membrane, giving it an uneven surface. The smooth ER is
active in lipid synthesis, detoxification and calcium storage, among other things.
Overal the ER has a wide range of functions including biosyntesis, metabolism and
storage. Like several other organelles the ER is connected to actin filaments which
makes movement of the ER possible. [13,14]
1.6.4 The Golgi apparatus
The Golgi apparatus consists of several sacks, each enclosed by a membrane. Its
functions include modification of protein marking, transport and secretion of
proteins and other molecules. Vesicles from the ER fuse together with the Golgi
apparatus, where the cargo’s final destination is decided by the Golgi. Either it is
sent to the area in the cell where it is required or secreted through exocytosis. The
reciving side of the Golgi is called the cis-golgi network, and is the side closest to
the ER. The opposite side is the trans-golgi network, where the proteins or
molecules are secreted in a vesicle formed by the Golgi membrane. [13,14]
1.6.5 Actin
Actin is a part of the cells cytoskeleton. It is necessary for the cell to have a
cytoskeleton to obtain its structure, and also for transporting organelles or vesicles
containing cargo molecules. Actin contributes to shaping the cell surface, as well as
cell movement. Actin makes movement of the Golgi and ER possible. The
transport process is mediated by the acto-myosin system, where actin filaments
13. 13
represent the road or track the organelles move on, and myosin functions as a
motor protein, moving the organelles in a direction. [14]
Actin filaments are created by polymerization of actin monomers. Latrunculin B
forms complexes with actin monomers, resulting in inhibition of the
polymerization process. If the cell is exposed to Latrunculin B, movement of the
organelles will be inhibited. [15]
2 Materials and methods
2.1 Specimen preparation
An Arabidopsis thaliana plant was marked with fluorochrome YFP or GFP, in
order to visualize the golgi apparatus or the endoplasmatic reticulum.
A small leaf was removed from a young A. Thaliana plant. The leaf was placed on
an object slide with a drop of MQ-water. Any dirt og particles, as well as air
bubbles was removed from the object slide, and the specimen was sealed using
wax.
2.2 Bead preparation
A solution of TetraSpeckTM
microspheres was vortexted for two minutes. Three
different samples were created by diluting the solution to the factors 1:100, 1:1000
and 1:10 000. The purpose of diluting the solution was to avoid cluttering of the
beads, in order for visualization of a single bead to be possible.
2.3 Microscope
The type of microscope used was a Leica SP5, along with the Leica Application
Suite Advanced Fluorescence (LAS AF) software. An argon laser was used, with an
intensity of 30 %. The chosen wavelength of 514 nm light was set to 15 %. The bit
depth used was 12 bits, and the pinholde aperture was set to 1 airy unit (AU). One
photo-multiplier tube (PMT) detector was set to detec light with wavelengths in the
range between 520 and 570 nm.
The overview images and the RGB images of the ER and Golgi were taken with a
10x/0.40 air-immersion objective.
2.4 Image processing and software
The images of the specimens containing golgi and ER-marked cells were scanned.
The images were then analysed and applied a LUT of chosen colour in the
computer software ImageJ. The gain and offset values of the images was adjusted
to make the structures of interest clear. Scalebars and LUT colour bar, indicating
light intensity was added to the overview images.
Different ROIs were selected for the movies, resulting in the RGB images, of the
ER and Golgi apparatus. The acquisition mode of the microscope software was set
to xyt, with a frame rate of 1 frame per second.
14. 14
Frame number 1, 6 and 11 were chosen in both movies to illustrate movement of
the organelles in the RGB images. The RGB images were processed in the software
ImageJ. The first frame is red, the second green and the third blue. Merging the
pictures of the three frames create an illustratin of organelle movement in the cells.
Since the colours red, green and blue create white when they overlap, white areas
represent those areas where no movement was detected. The organelles that moved
will have either a red, blue or green colour, or a mixture of two of the colours.
2.5 Optimal resolution
The z-stacks of the Golgi apparatus and the fluorescent beads was visualized using
a 63x/1.20 water immersion objective. A PMT detector was used for the z-stacks
of the golgi apparatuses. For the imaging of the beads a hybrid detector (HYD) in
photon counting mode was used. Acquisition mode was set to xyz.
Latrunculin B was used on the specimens containing the Golgi apparatuses to
inhibit movement. To be sure that there would be no movement in the specimen,
one should wait one hour to let the inhibitor work. After one hour the specimen
was scanned for ROI.
The Rayleigh criterion was applied. The Dxy and Dy was found, and the Nyquist
theorem was applied with oversamling by a factor of three. The optimal pixel size
was set by adjusting the zoom factor and the number of pixels in the image
(Equation 6). The LUT was adjusted so that the pixels of interest were neither fully
saturated or at zero intensity.
When performing optical cross sections in the z-direction, the start point was set to
a plane above the chosen Golgi apparatus, where there was zero light intensity. The
same was done with the end point; it was set to a plane well below the Golgi
apparatus where the structure was not visible and there was zero light intensity.
This is done to ensure that the whole golgi stack was depicted in the z-region. If
the regions above or below the Golgi was too large, one can crop the image using
an image processing software. The distance between the scans in the z-dimensions
was set so that it corresponded with the optimal voxel depth calculated. The stack
was visualized using image processing software.
The settings listed above was also used for the fluorescent beads, except for the
zoom factor, which was set to a higher value. This resulted in greater oversampling
in this stack, compared to the stack of the Golgi apparatus.
A ROI was chosen based on the requirement of the visualization of three beads to
be possible in the chosen aera. The beads also had to be distinguishable from one
another. Here, the start and end point was also chosen were there was zero
intensity above and below the chosen beads.
15. 15
2.6 PSF analysis
The z-stacks of the fluorescent beads were analysed in the software Amira. The
three chosen beads were marked, and a BeadExtract module was attached to the z-
stack module. The BeadExtract module estimates the PSF size based on the
average of the three marked beads. The PSF was then visualized using a Projection
View module.
The PSF was opened in ImageJ, where the MetroloJ plugin was used to calculate
size at FWHM of the PSF.
2.7 Deconvolution
The computer software Amira was used for the analysis of the z-stacks of the golgi
apparatuses. The PSF module from the beads’ PSF analysis were resampled in
order to have an sampling rate equal to the z-stack of the Golgi apparatuses. This
was done by connecting the two modules to a resample module (Figure 7). A
Deconvolution module was connected to the Golgi z-stack module and the
resampled PSF module. The correct NA and 𝜆!"#
values, as well as refraction
index, was entered in the deconvolution module. Deconvolution by maximum
likelihood estimation was startet, and 20 iterations were run.
Figure 7: Module window in Amira. The modules containing data is marked green,
the operation modules are coloured red and the yellow boxes show visualisation
modules. The operation modules process the raw data in order to produce
resampled and deconvolved images.
3 Results
3.1 Image analysis of the golgi apparatus
An image of a cell marked with EYFP of the golgi apparatus is presented as well as
a RGB-image to illustrate movement of the organelles (Figure 8). The RGB-images
were created by filming the movement of the organelles and choosing three
different frames, numer 1, 6 and 11, where number one is red, 6 is green and 11 is
blue. The three frames were overlayed, creating the images shown below. Frame
16. 16
number 1, 6 and 11 were used both for the RGB-image of the Golgi apparatuses
and the ER.
Figure 8. (To the left) An overview of the Golgi Apparatus marked with EYFP in
plant cells of A. Thaliana. The image was taken using a 10x/ 0,4 air immersion
objective. The colour scale of the LUT to the right indicates the light intensity
raging from zero, black, to fullt saturated pixels, shown in white. (To the right). A
RGB-image to illustrate movement of the golgi apparatuses in the cell.
3.2 Image analysis of the endoplasmatic reticulum
The images presented are an overview image of cells with an EYFP-marked ER,
next to a RGB-image illustrating the movement of the organelle within the cell
(Figure 6). The procedure for creating the RGB-image was the same as the one for
the Golgi apparatus.
17. 17
Figure 9: (To the left) An overview of the ER marked with EYFP in plant cells of
A. Thaliana. The image was taken using a 10x/ 0,4 air immersion objective. The
colour scale of the LUT to the right indicates the light intensity raging from zero,
black, to fullt saturated pixels, shown in white. (To the right). A RGB-image to
illustrate movement of the ER in the cell.
3.3 Deconvolution of the Golgi z-stack
The software Amira was used to obtain a deconvolved image of the golgi stacks.
This was done to remove blur from the image, in order to observe the golgi stacks
in 3D with better resolution.
Figure 10. The golgi z-stacks before deconvolution (left) and after (right). The
images were created in the Amira image processing software.
3.4 Size analysis of the Golgi z-stacks
The deconvolved z-stacks of the golgi apparatus were analysed so that the size
measurements of a single stack could be contucted. The software ImageJ was used
with the MetroloJ plugin. This software interprets the raw data and produces a
gaussian line fit based on the data. The line fit makes it possible to measure
FWHM. Three different golgi stacks were analysed, and the average size was
calculated (Table 2)
Table 2: FWHM size of the three Golgi stacks, and average size based on these
data. The asterisk denotes a possibly inaccurate value, as the fittet curve did not
incorporate all the data points.
Dimension Golgi 1 [nm] Golgi 2 [nm] Golgi 3[nm] Average size [nm]
x 260 447 266 324,33
y 302 502 250 351,33
z 595 617 809* 673,67
18. 18
4 Discussion
4.1 Image analysis of the Golgi apparatus and ER
The images clearly illustrated the cells structure and organization of the organelles
under study. The cell membrane is clearly visible, making it possible to distinguish
between to different cells. This is probably a consequence of the vacuole pushing
the organelles towards the membrane. Individual organelles can be observed in the
cell, and their shape and size can be used for further analysis.
In the overview image of the Golgi apparatuses, the stomata is clearly visible. This
shows that the cell is one of the cells at the top layers of the leaf. The Golgi
apparatuses are clustered together in the stomata guard cells. The overview image
also shows that the Golgi apparatus is quite abuntant in cells. Golgi is a very
important organelle in the cell because of its role in sorting of proteins, among
other things.
The overview image of the ER shows a clear network structure. Its long tubular
structures creates a large surface area, suggesting that a large surface area is an
important tool to secure effiency of the processes occuring in the organelle. The
ER seems to be tightly packed near the cell membrane, as the light intensity here is
quite high. The underlying network is shown in blue.
The RGB images is able illustrate movement of the organelles in the cells quite
well. In both the RGB images of the ER and the Golgi apparatuses it shows
significant movement. Also, it shows areas where there was not detected any
movement, coloured white. It is possible to observe certain areas that are
particularly active in both the ER- and the Golgi image. These active areas can be
seen as multicoloured strands in the cell. In other areas the movement of the
organelles seem more random than directional.
4.2 Optimal resolution
The calculated values for the resolution limit, pixel and voxel size were enterted in
the Leica softwar for optimal sampling rate. The software automaticly rounded of
the numbers, creating a possible source of error. Because of this, the calculations
were performed with oversampling.
4.3 PSF analysis
The PSF from the bead extract could be seen as a dot surrounded by alternating
light and dark rings. This fits well with the theoretical knowlegde of the PSF. This
suggests that the z-stacks of the beads were accurate.
19. 19
A 3D model of the PSF was generated using computer software, making it possible
to view the intensity of the PSF in the z-dimension. Through this model the airy
pattern was clearly visible (Figure 8).
Figure 11: Three different cross sections in the z-dimension of the PSF, showing
different light intensities through as a pillar in the chosen plane. The airy patterns,
or rings, are clearly visible in the PSF. The PSF was created by an NA value of 1.4
and the 𝜆!"#
value was created by Amira. The values used to create this model was
not from the results.
4.4 Deconvolution of the golgi z-stack
The deconvolved image was clearly an improvement from the raw image, as the
organelles are easier to see and most of the blur was removed from the image.
Deconvolution has shown to be a useful tool when it comes to live cell imaging.
However, the process did not work perfectly. This could be due to a deviation
between the PSF of the golgi z-stacks and the PSF from the beads. Nontheless, the
abberation seems to not have been very significant, as the deconvolved image does
not show any obvious flaws and is not missing any crucial information. It seems as
though the utmost of blur has been removed, and thus creating a better depiction
of the specimen.
4.5 Size analysis of the Golgi stacks
The FWHM size for three different golgi stacks were calculated using the MetroloJ
plugin. Gaussian line curves where fitted to the light intensity values of the z-
stacks. In the analysis of Golgi 3 the gaussin fit did not include all the data points in
the z dimension, resulting in a possibly inaccurate R2
-value. The R2
value is a
measure on how well the fitted line corresponds with the data points. A perfect line
will have a R2
value of 1.
20. 20
There were differences between the three chosen Golgi apparatuses in size
dimensions, suggesting that the organelle is not symetrical in shape. This may be
due to the fact that the Golgi apparatus consists of several stacks, or cisternae,
which can have different shapes and sizes. The data suggests that all three Golgi
apparatuses are largest in the z-dimension. The minimum resolution was larger in
the z-dimension, resulting in inaccurate values as the smallest unit will become
larger.
The data collected from the experiment regarding size of the Golgi apparatus is not
representable. For one thing, only one cell type was analysed, and the sample size
was also quite low. To achieve a more representable measurement of the Golgi
more cell types should be analysed, to remove any inaccuracies.
21. 21
5 Litterature
1. Corle, Gordon. Confocal Scanning Optical Microscopy and Related Imaging
Systems [0-12-408750-7;9786611046699] (1996).
2. Nathan S. Claxton, Thomas J. Fellers, and Michael W. Davidson. Laser
Scanning Confocal Microscopy.
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22. 22
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5.1 Illustrations
1. Cover photo: Plant seed. URL: http://wodumedia.com/wp-content/uploads/Plant-seed-from-
freshwater-pond-near-Moscow-Russia.-Photographed-with-fluorescence-10x-objective.--
960x1531.jpg
[Access date: 01.04.2014]
2. Illustration of the pin hole’s function in a confocal microscope
URL: http://www.photonic-lattice.com/en/technology/polarization-longitudinal-
slit-technology/
[Access date: 27.03.2014]
3. The corrolation between an airy pattern and a point spread function. The graph (red) shows the
PSF as light intensity, with the airy pattern in the background. URL:
http://www.asinen.org/
[Access date: 27.03.2014]
23. 23
7 Appendix
A Calculations
A.1 Resolution limit, pixel size and number of pixels needed
Calculations of the resolution limit was performed using equations (2) and (3). The objective used
was a 63x/1.2 water immersion objective, which gives an NA value of 1.2 and 𝜂 = 1.33. The
excitation wavelength used was 514 nm.
Lateral resolution limit:
𝐷!" =
0.61 𝑥 514 𝑛𝑚
1.2
= 261,3 𝑛𝑚
Axial resolution limit:
𝐷! =
2(514 𝑛𝑚 𝑥 1.33)
(1.2)!
= 951,6 𝑛𝑚
Optimal pixel- and voxel size was calculated using equations (4) and (5):
Lateral pixel size:
𝑃𝑠!" =
261,3 𝑛𝑚
3
= 87,1 𝑛𝑚
Axial voxel depth:
𝑃𝑠! =
951,6 𝑛𝑚
3
= 317,2 𝑛𝑚
24. 24
B Metrolo J analysis
B.1 PSF analysis of fluorescent beads
The gaussian line fit graphs for the PSF analysis of the fluorescent beads are shown (Figure B1).
(a) (b) (c)
Figure B1: Gaussian line fit in the x (a), y (b) and z (c) dimensions.
The R2
value is presented (Table B1).
Table B1. R2
values
Gaussian curve R2
a 0.99
b 0.99
c 0.98
B.2 Size analysis of the Golgi stacks
Gaussian line fits for Golgi apparatus number 1,2 and 3 are presented (Figure B2, B3, B4)
(a) (b) (c)
Figure B2: Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 1.
The R2
value is presented (Table B2).
Table B2. R2
values
Gaussian curve R2
a 0.99
b 0.97
c 0.99
25. 25
(a) (b) (c)
Figure B3. Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 2.
R2
values are presented (Tabel B3)
Table B3. R2
values
Gaussian curve R2
a 0.98
b 0.98
c 0.99
(a) (b) (c)
Figure B4. Gaussian line fits in the x (a), y (b) and z (c) dimensions, of Golgi apparatus 3.
R2
values are presented (Tabel B4)
Table B4. R2
values
Gaussian curve R2
a 0.99
b 0.99
c 0.99