Seminars

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

• December 8, 2009
  • Localization based super-resolution: PALM - Sean McKinney
• November 3, 2009
  • Personalizing Imaris XT for Advanced 3D Image Analysis - Caleb Bailey
  • Cell Positional and Directional Relations to a Migratory Route: Computational Geometry Aspects - Boris Rubinstein
• October 6, 2009
  • Imaris: 3D Image Processing and Tracking - Katie Hollander
• June 23, 2009
• Multi position imaging: A simple tool that saves time and money - Joseph Steen
• Image Deconvolution: What it is all about? - Kasthuri Kannan
• May 19, 2009
• Higher Content Yeast Imaging and Data Processing - Richard Alexander
• Higher Content Yeast Imaging and Data Processing - Heather Cartwright
• April 7, 2009
• Fluorescence Cumulant Analysis of Molecular Stoichiometry In Vivo - Jay Unruh
• Moments, Cumulants and all that Stuff - Boris Rubinstein:
• February 3, 2009
• Strategies for fluorescent cell marking - Paul Kulesa
• Separating dye labels - Winfried Wiegraebe

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center
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Multi position imaging: A simple tool that saves time and money - Joseph Steen
Multi position imaging is a technique for imaging multiple samples in an automated fashion. Using existing software it can be used to accommodate other events in acquisition. This talk will go through the software and a few examples of how it can be used to acquire your data faster to shorten project duration.

Image Deconvolution: What it is all about? - Kasthuri Kannan
Deconvolution is a computationally intensive image processing technique that is being increasingly utilized for improving the contrast and resolution of digital images captured in the microscope. In this talk we will review the central ideas behind the deconvolution of images. The diversity of resulting algortihms reflects different ways of estimating the true signal under various idealizations of its properties. . .

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Higher Content Yeast Imaging and Data Processing - Richard Alexander
A specific example of how the Microscopy Center has helped solve imaging and data processing problems in yeast by combining multiple existing technologies available within the institute.
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Higher Content Yeast Imaging and Data Processing - Heather Cartwright
Examples of how microfabrication can help streamline data collection by combining custom devices with existing acquisition software.

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Fluorescence Cumulant Analysis of Molecular Stoichiometry In Vivo - Jay Unruh
For almost a decade, information about the confocal fluorescence intensity distribution has been used to measure the stoichiometry of molecular complexes in vivo. Fluorescence Cumulant Analysis, developed in the last few years, provides a simple approach to such analyses. I will discuss the theory, practical implications, and potential pitfalls of this analysis as well as new developments we have made extending this approach to camera based detection systems such as total internal reflectance fluorescence microscopy.

Moments, Cumulants and all that Stuff - Boris Rubinstein
Basic statistics knowledge about probability distribution functions and their discrete representations (regular, central, factorial moments and cumulants) is given. Methods of computation of k-statistics and its cumulants is discussed. Implementation of these method in Mathematica is presented.

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

Strategies for Fluorescent Cell Marking: Separating Individuals from the Heard - Paul Kulesa
I will focus on multicolor cell design.

Separating Dye Labels - Winfried Wiegeraebe
An introduction into ways to separate dye labels with spectral overlap in light microscopy experiments. I will focus on linear unmixing.

Stowers Links:
Stowers Institute Home Page
Stowers Core Facilities
Microscopy Center

• May 29, 2008
• Marco Blanchette:High Throughput RT-PCR for the Accurate Quantification of Alternatively Spliced mRNAs
• Brian Sanderson:Automated Analysis of DNA, RNA and proteins by eletrophoresis using the LabChip90
• January 31, 2008
• Karen Staehling-Hampton:Introduction to Next Generation Sequencing using the Illumina (Solexa) Genome Analyzer

Marco Blanchette: High Throughput RT-PCR for the Accurate Quantification of Alternatively Spliced mRNAs
In our efforts to design a bio-luminescent reporter to screen RNAi libraries for factors regulating alternative splicing, we have been developing a high-throughput RT-PCR assay using the LC90 capillary electrophoresis system. This procedure has the following characteristics: high throughput, sensitivity, reproducibility and accurate quantification of relative amount of PCR products. Using the LC90, we have been able to validate results from different sources. Results from gene expression analysis by next-generation sequencing and validation of bio-luminescent assays will be presented.

Brian Sanderson: Automated Analysis of DNA, RNA and proetins by Eletrophoresis using the LabChip90
The Caliper LabChip90 offers researchers an automated alternative for performing high-throughput electrophoretic separation and analysis of DNA, RNA and protein. Sampling from 96 and 384 well plates, the robot is able to reduce hands-on time and achieve digital results for sizing and quantitative examination. Electrophoresis is performed on a microfluidic chip loaded with reagents for staining samples prior to separating them using a sieving polymer and electrical current. In this presentation, I will detail this robot which is maintained by the Molecular Biology Facility and is a resource open to all Institute members.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Blanchette Lab
Molecular Biology Facility
Molecular Biology Internal Site

LabChip90 Presentation:
Powerpoint (5.25 MB)
PDF (1.17 MB)

Karen Staehling-Hampton: Introduction to next generation sequencing using the Illumina (solexa) Genome Analyzer
Next generation sequencing approaches offer tremendous excitement and will enable researchers to ask new questions that until now have been difficult or too costly to address. Along with the excitement come challenges in both the laboratory and in the data analysis. The enormous amount of data generated per run (>1TB) is particularly challenging, requiring large amounts of data storage and lengthy analysis times. I will provide an introduction to next generation sequencing and our new Illumina (Solexa) Genome Analyzer. I will discuss the technology, applications (expression profiling, ChIP sequencing, microRNA discovery, targeted re-sequencing and whole genome sequencing), our progress and current projects.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Molecular Biology Facility
Molecular Biology Internal Site

Presentation:
Powerpoint (17 MB)
PDF (2.47 MB)

• August 30, 2007
• Ho Yi Mak:The Double Life of Worms
• Winfried Wiegraebe:Image Processing in Light Microscopy
• June 28, 2007
• Heather Cartwright and Susan Abmayr:A Microfluidic Approach to Imaging
• April 26, 2007
• Rhonda Allen:Adventures in Electron Microscopy
• March 29, 2007
• Madelaine Gogol and Norman Pavelka : Tiling Arrays – probing genome and transcriptome structure
• February 22, 2007
• Danny Stark : An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo mode

Winfried Wiegraebe: Image Processing in Light Microscopy
Image processing is used to make features visible in light microscopic images and to quantify the information. In this presentation I will introduce standard image processing methods available at Stowers.

Ho Yi Mak : The Double Life of WormsThe locomotory behavior of C. elegans is subjected to changes in nutritional status. Using automated tracking, our goal is to characterize such changes in a quantitative manner both in wild-type and mutant animals that accumulate inappropriate amount of fat.

Stowers Links:
Stowers Institute Home Page
Mak Lab
Stowers Core Facilities
Stowers Microscopy Center

Image Processing Presentation:
Powerpoint (12.9 MB)
PDF (4 MB)
Powerpoint and movies (compressed) (218 MB)

C. elegans :
Powerpoint (6 MB)
PDF (1.8 MB)
Powerpoint and movies (compressed) (6.4 MB)

Web Links:
C. elegans WWW Server
Wikipedia Article
Image Processing Seminars

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities
Microscopy Center (Internal)

Web Links:
Wikipedia Microfluidics
Tutorial

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Presentation:
Powerpoint (34 MB)
PDF (26 MB)
Powerpoint and movies (compressed) (108 MB)

Web Links:
Scanning Electron Microscope
Transmission Electron Microscopy
Hitachi High-Technologies

Tiling Arrays offer high resolution views of genomic events, and are used to map transcriptome structure, protein/genome interactions, and examine loci or chromosomal copy number. Tiling arrays also present novel data analysis challenges. Design of an in-house full genome yeast array will be discussed, as well as design of custom Agilent arrays. Norman will discuss application of a high density Affymetrix array to study genomic changes that occur in yeast strains that have undergone experimental evolution over hundreds of generations.

Stowers Links:
Microarray
Microarray Internal Site
Rong Li Lab
Rong Li Research Website

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Web Links:
Affymetrix

• November 30, 2006
• Dan Bradford: Give the Pipette a Rest: Automated Services Provided by Molecular Biology
• October 26, 2006
• Gaye Hattem: Introduction to the Stowers Research Wiki: A documentation and collaboration tool
• September 28, 2006
• Les Choromanski: Laboratory Animal Husbandry - Government Regulation and Industry Standards
• Heather Marshall: LAS Technical Services
• Rachael Prater: Quarantine: The LASF's Dirty Secret
• Mike Elmore: Mouse Modeling and Expanded Services
• June 29, 2006
• Joel Schwartz: New TURF for TIRF
• May 25, 2006
• Arcady Mushegian: BLAST and other methods of probabilistic sequence comparison
• April 27, 2006
• Laurence Florens: Deeper into the MudPIT: Protein lists and beyond
• Philippe Prochasson: Analysis of the stoichiometry and modifications of the HIR complex using mass spectrometry
• March 30, 2006
• Winfried Wiegraebe: FCS - Measuring Molecular Interaction in Solution and Cells
• Charles Banks: Interactions between transcribing RNA Polymerase II and transcription elongation factors ELL and EAF1
• February 23, 2006
• Teri Johnson : Antigen Unmasking Methods in Immunohistochemistry: Strategies for Improving Immunoreactivity in Paraffin and Frozen Sectioned Material
• Angelo Iulianella : Microwave Immunohistochemistry: Probing Proteins On Frozen and Wax Sections
• January 26 , 2006
• Joel Schwartz: Measuring Intermolecular Distances with FRET
• Brian Slaughter: Analysis of Arp2/3 Conformation Using FRET

Photoactivatable fluorescent proteins (PFP) demonstrate altered photophysical properties after excitation with specific chromatic absorptions. We provide a comparison of four PFPs, including PAGFP, PSCFP, Kaede, and KikGr, within a chick embryo model. We found that In vivo response of the PFPs yielded results of rapid photoconversion and photobleaching, but also showed great potential of selectively marking individual or groups of cells.

Stowers Links:
Imaging Center
Imaging Center

Many routine laboratory tasks can be automated using liquid handling robotics. Laboratory automation also enables high throughput screens that otherwise would not be possible with manual pipettes. The molecular biology facility utilizes a Beckman-Coulter BiomekFX and Genetix QPix2 to provide services in robotic liquid handling and high throughput bacterial or yeast colony manipulations. In this presentation we will give an automation overview, describe the instrumentation available and give examples of services and projects we have automated.

Stowers Links:
Molecular Biology
Molecular Biology Facility

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What is a wiki? A brief introduction to using wiki technology as a simple way to create web pages that can be edited directly within a web browser. An overview of the resources currently available on the Stowers Research Wiki and examples of how a wiki can be used for sharing information among the research groups and support facilities.

Stowers Links:
Bioinformatics
Bioinformatics: Research Webpage
Stowers Wiki
Stowers Research Wiki
Bioinformatics: Wiki

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Web Links:
Wikipedia: Wiki
OpenWetWare: sharing of lab protocols
R Wiki: R software for statistical computation
AAA Wiki: Assembly, Alignment and Annotation of Drosophila genomes

Ever wondered why optimal laboratory animal husbandry is imperative in research facilities? Moreover, as rodent populations increase in research facilities as well as the desire to keep these animals healthy for longer periods of time, the LASF need to accommodate a current and more complex government and industry regulations and standards for the husbandry and care. The Animal Husbandry Section is responsible for maintaining the optimal macroenvironment and microenvironment in LASF, sanitation, nutrition, records keeping and transportation of animals into and out of facility.

Stowers Links:
Laboratory Animal Services
Laboratory Animal Services (Biospere)

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The LASF provides designated Research Technicians (RT) that perform a wide range of invasive and non-invasive procedures on the animals. These include injections, embryo harvests, blood draws, irradiation and tissue collection. Furthermore, the RTs perform much of the breeding colony management, enabling lab members to spend more time at the bench.

Stowers Links:
Laboratory Animal Services
Laboratory Animal Services (Biospere)

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Web Links:
Transnetyx: High Throughput Genotyping

With an ever-growing number of mouse models, both nationally and internationally, there is an increased interest among researches to incorporate them into specific projects and experiments. The Laboratory Animal Services Facility offers two quarantine areas to house these mice. Discussion topics will include: a general quarantine overview, how to submit a request, procedures for shipping mice out to collaborating institutions and utilizing the Strain Database.

Stowers Links:
Laboratory Animal Services
Laboratory Animal Services (Biosphere)
LASF: Strain Database
LASF: SARF (Shipping and Receiving Form)

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Through the LASF, the Stower's Institute for Medical Research provides it's members with a core facility for the production of Transgenic and Knockout mouse models. By using the techniques of DNA microinjection and gene targeting in mouse ES cells, the LASF is able to help our SIMR members create new and novel research models directed to their specific areas of interest. In addition, the core facility also provides a number of expanded services such as rederivation and cryopreservation for the maintenance and care of these novel animal models.

Stowers Links:
Laboratory Animal Services
Laboratory Animal Services (Biospere)

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Total internal reflection fluorescence (TIRF) microscopy is a well established technology that allows specific visualization of membrane localized structures. TIRF microscopy dramatically enhances z-axial resolution compared to confocal and widefield microscopies. We are currently developing some significant enhancements to incorporate polarization and image correlation with TIRF, these enhancements evaluate protein dynamics and protein interactions.

Stowers Links:
Imaging
Image Center

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I will review (with examples) some of the most useful methods of sequence comparison, will show how they can be used for inferring protein function, structure, and evolutionary history, and will also discuss what can go wrong in the process.

Stowers Links:
Mushegian Lab
BioInformatics Research Page

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Web Links:
King Jordan's class: a practical, hands-on introduction to the study of phylogenetics and computational genomics.
Sean Eddy's class: survey of algorithms and methods in computational molecular biology.
Steve Altschul's tutorial: The Statistics of Sequence Similarity Scores

Stowers Links:
Proteomics

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In chromatography-based shotgun proteomics analysis, spectral counts are good markers of protein abundance. Here, I will present how we used this approach to estimate the stoichiometry of the four HIR complex subunits by normalizing the SAF (spectral abundance factor) for each subunit against the total spectral count for the complex.The yeast HIR complex is involved in the cell cycle transcriptional regulation of the histone genes. Here, I will present how we are using Mass Spec. analysis to identify Post Translational Modifications (PTMs) of the subunits of the HIR complex during the cell cycle

Stowers Links:
Workman Lab
Workman Lab Research Web Page

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Fluorescence Correlation Spectroscopy (FCS) looks at diffusion properties, local concentration and interaction of molecules. It is a statistical method based on fluorescence fluctuations driven by Brownian Motion and electronic properties of dyes attached to molecules of interest. So far, most experiments where done in solution, but the technique is applied more and more to cell based systems.

Stowers Links:
Stowers Microscopy Center

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Transcription elongation factors affect the rate at which RNA polymerase II adds nucleotides to the growing RNA transcript. I am using FCS to investigate how the elongation factors ELL and EAF1 might interact with transcribing RNA Polymerase II.

Stowers Links:
Conaway Lab

Stowers Links:
Histology/Immunohistochemistry
Histology Facility

Go to link page

Immunohistochemistry is a powerful tool to get at the source of problems that arise during development of embryos and organs. But getting antibodies to work can be sometimes tricky and there are a number of ways to get beautiful fluorescent signals. One successful way for a number of diverse antibodies is Antigen Retrieval in Citrate buffer using a microwave oven. In this short talk, I will highlight some success I've had using the Antigen Retrieval Method for Immunohistochemistry to improve the specificity and intensity of antigen detection in tissues.

Stowers Links:
Trainor Lab

Go to link page

Foerster resonance energy transfer (FRET) is a valuable tool to measure protein proximity in living cells. Measuring the non-irradiative energy transfer from a donor molecule to an acceptor molecule establishes the distance by which the two molecules are separated. To-date, several different methods have been employed to measure FRET. I intend to highlight the various methods and provide a means to select the most appropriate method for individual experiments

Stowers Links:
Image Center
Image Facility internal
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Stowers Links:
Rong Li Lab
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• December 1st , 2005
• Brian Sanderson: Mapping Mouse Mutants
• Lisa Sadell: Trex mutation identified using microsatellites
• October 27, 2005
• Ron Yu: Targeting selective populations of neurons to silence
• Michael Elmore: Transgenic and Knockout Technologies in Biomedical Research
• September 29, 2005
• Paul Kulesa: In Vivo Imaging of Chick Development
• Paul Rupp: In vivo analysis of neural crest cell chain migration
• August 25, 2005
• Chris Seidel: Microarrays for detecting gene expression and the location of proteins bound to DNA in vivo
• Ray Camahort: Genome-wide localization Cse4 in S. cerevisiae
• July 28, 2005
• Jeff Haug: Cytometry and Fluorescent Activated Cell Sorting
• Linheng Li: The isolation of functional adult stem cells using FACS
• May 26 , 2005
• Winfried Wiegraebe: Imaging in 3D: Confocal vs. NLO Microscopy?
• Alexander Aulehla: Visualization of mouse embryos

The molecular biology facility has developed multiplexed panels of microsatellite markers to discern strains of mice. These markers, along with an ever-growing group of Single Nucleotide Polymorphisms allow the facility to quickly and easily scan the mouse genome for mutations.

Stowers Links:
Molecular Biology
Molecular Biology internal

A panel of microsatellite markers and SNP polymorphisms were used to map mutations from a screen for mouse developmental defects. With this strategy, a mutation called Trex, which produces craniofacial and limb abnormalities, was rapidly identified as a defect in a gene involved in retinoic acid metabolism.

Targeted expression of genes of interest in a specific population of cell is usually achieved by using tissue/cell type-specific gene promoters. However, quite often a specific promoter is not available for the cells one is interested in. We are developing a method to target specific populations of neurons in the nervous system to silence their functions using a combination of gene targeting and transgenic approaches.

Stowers Links:
Yu Lab
Yu Lab Research Web Page
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With the development of recombinant DNA technologies during the 1970's, scientist have been able to accelerate and refine the process of genetic manipulation in an animals genome that at the time could only be accomplished through  long,  labor intensive selective breeding programs.  From this, transgenics or the science of intentionally introducing a foreign gene or genetic construct into the genome of a target animal, was developed. Initial work involved techniques used to insert foreign genetic materials into mammalian cells maintained in culture. However, this in vitro work rapidly progressed into laboratory rodents, providing a more targeted and proactive approach for the establishment of new animal models for biomedical research. With the ongoing development of new techniques, current results have been very successful and provide a unique and precise mechanism for the study of a variety of specific conditions or diseases with a genetic basis or influence.

A major challenge to exploring in vivo cell lineages and testing the functional role of potential molecular mechanisms lies in designing novel embryo culture, cell labeling and cell tracking techniques within an advanced optical platform. Our expertise involves designing innovative intravital culture and imaging techniques to follow fluorescently marked cells using video, laser scanning and 2-photon, multispectral microscopy. We will describe basic principles for culture design, including techniques for cell tracking and 4D data analysis.

Stowers Links:
Kulesa Lab
Imaging Center
Image Facility internal

The cranial neural crest is a migratory, pluripotent population of cells required for normal craniofacial and cardiovascular development. Extensive information from transplants, fixed tissues and static imaging has shed light on how cells sort into and maintain the observed stereotypical patterns of cranial neural crest migration. Small Rho GTPases have been shown to be involved in the migration of a variety of cell types. Using in vivo labeling techniques, embryonic culturing systems and 4D-confocal time-lapse microscopy, we are now able to analyze, at high resolution, the dynamic behavior of individual neural crest cells migrating in r7 chains.

Stowers Links:
Kulesa Lab

Microarrays are complex probes allowing simultaneous detection or identification of labeled nucleic acids in complex samples. Thus they can be used to monitor gene expression, or for identifying DNA fragments which coprecipitate with a protein of choice. As such, they can be used to examine the cis-regulatory code, and to collect global views for unveiling the mechanisms controlling how proteins interact with DNA.

Stowers Links:
Microarray Facility
Microarray Resources
Chris Seidel Research Website
Madelaine Marchin Research Website

Many proteins intimately interact with DNA. Chromatin immunoprecipitation, coupled with DNA microarray analysis (ChIP-chip), has emerged as a powerful technique to study these protein/DNA interactions. Using Chip-chip I am studying the genome-wide localization of the S. cerevisiae cenH3 histone variant Cse4.

Stowers Links:
Gerton Lab
Gerton Research Website

Flow cytometry and fluorescent activated cell sorting (FACS) is frequently used as a tool in basic science research. Applications include the collection of cells for experiments involving, in vivo animal transplantation, in vitro culture maintenance, the harvest of nucleic acids, and the harvest of cellular proteins.

The process of FACS involves the movement of cells via a carrier fluid (i.e. sheath fluid) and the presentation of these cells for intersection with a finely focused laser beam. As the laser interacts with a cell, the light is scattered and is often emitted as fluorescence. The fluorescence/light is detected and organized in a fashion that allows for the characterization of cells. Microseconds after each cell is interrogated it is singly compartmentalized into electrically charged aerosolized sheath fluid. Sorting then occurs by the electromagnetic diversion of the fluid droplet containing a specified cell.

Stowers Links:
Cytometry Facility

Many adult tissues contain stem cells however; their location and phenotype are not yet defined. Cytometry and FACS are powerful tools that allow stem cell biologists to isolate and further study putative stem cell population and cells that occupy stem cell niches.

Stowers Links:
Linheng Li Lab

Recent development of calcium sensors has now been used in transgenic animals to monitor calcium activities. We have a number of probes and I will discuss the differences between these probes and our experience with them.

Stowers Links:
Yu Lab
Yu Lab Research Web Page

NLO (non-linear optics) microscopy allows to image deep within highly scattering tissue (up to 0.5 to 1 mm). Similar to a confocal microscope it eliminates out-of-focus light and creates 3D models with high resolution. In contrast to confocal microscopy NLO imaging excites dyes only at the focal plane, thus it does not need a pinhole, and avoids out-of-focus bleaching. The used near infrared light (NIR) allows higher penetration and is less toxic to most living species.

Stower's Links:
Advanced Instrumentation & Physics
Stowers Microscopy Center Research Website
Stowers Microscopy Center: Two-Photon Microscopy
Stowers Microscopy Center: Confocal Laser Scanning Microscopy

Web Links:
Optical Microscopy Primer: Laser Scanning Confocal Microscopy
Molecular Expressions: Multiphoton Fluorescence Microscopy

When imaging mouse embryos with NIR, they form somites more reliable compared to using shorter wavelengths.

Stowers Links:
Pourquié Lab

In this series of lectures we discuss fundamentals in microscopy and the related theoretical basics. It is intended for people who want to understand the inner workings of a microscope in order to improve their scientific results based on optical imaging or modify and built microscopes and related optical set-ups.

Please email Winfried Wiegraebe if you are interested in getting updated information about Foundations of Microscopy lectures.

• July 23, 2009: Christopher Wood - Image Processing
• July 16, 2009: Jay Unruh - Fluorescence Polarization Assays of Binding and FRET, Katie Perko - In vitro quantitative fluorescence measurement of brightness and binding
• July 9, 2009: Jay Unruh - Total Internal Reflection Fluorescence Microscopy (TIRF) and other Surface Imaging Technologies
• July 2, 2009: Jay Unruh - Fluorescence Correlation Spectroscopy for Measurements of In Vivo Molecular Interactions and Mobility
• June 18, 2009: Jay Unruh - Fluorescence Lifetime imaging of in vivo ionic concentrations and FRET
• June 11, 2009: Jay Unruh - Measuring Biological Interactions and Structures using FRET
• June 4, 2009: Jay Unruh - Two Photon and other Non-Linear Microscopy Techniques
• May 28, 2009: Winfried Wiegraebe - Basics of Microscopy: Digital Microscopy
• May 7, 2009: Jack Garner - Laser Capture Microscopy
• April 23, 2009: Peter J. Dwyer, Cri, Inc - Multispectral Imaging in Widefield Microscopy
• July 21, 2006 and February 19, 2009: Winfried Wiegraebe - Building Blocks of a Microscope
• August 4, 2006 and March 12, 2009: Winfried Wiegraebe - Image Formation
• August 18, 2006 and March 26,2009:: Winfried Wiegraebe - The Objective
• September 1, 2006: no lecture
• September 13-15, 2006: Advanced Microscopy Workshop (separate internal page)
• October 6, 2006: Amanda Combs - Illumination and Filters
• October 20, 2006: Winfried Wiegraebe - Non-Linear Optics
• November 3 , 2006: Winfried Wiegraebe - Fluorescence Lifetime Imaging Microscopy (FLIM)
• April 2, 2009: Winfried Wiegraebe - The Confocal Microscope

Stowers Links:
Stowers Microscopy Center: Microscopy
Image Center
Image Center (internal)
Advanced Microscopy Workshop 2006

Web Links:
Optical Microscopy Primer: Everything you want to know about microscopy.
Microscopy: Part of the Botany Online Internet Hypertextbook.

Literature:
M. Abramowitz: Microscope Basics and Beyond (Olympus)Download PDF
Microscopy from the very beginning (Carl Zeiss)Download PDF
M. Davidson, M. Abramowitz: Optical Microscopy Download PDF

This week will begin discussing digital image processing. We will talk about digital images, histograms, intensity transformations, and spatial filters such as smoothing and edge detection.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

TIRF is the method of choice for imaging of basal membranes in cell culture and has been a driving force in the elucidation of cell migration mechanisms. Nevertheless, recent developments in TIRF allow for observation of single molecule dynamics in living cell membranes as well as the capability to image apical membranes. In addition, other surface imaging modalities like surface plasmon enhanced imaging and single mode waveguides have been developed for membrane structural imaging as well as single molecule sequencing applications.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Much of cell and molecular biology hinges on homo and hetero protein interactions. This week we will discuss fluctuation methods for measuring such interactions for diffusing molecules. These methods overcome the distance limitations of FRET, but have some limitations of their own that must be accounted for. In addition, advanced Image Correlation measurements allow for measurement of binding and diffusion components of molecular diffusion.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

We will discuss requirements to combine a digital camera with an optical microscope. We will talk about resolution, magnification and signal-to-noise. Different options for digital cameras for optical microscopy will be introduced.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Jack Garner from Carl Zeiss will give an introduction into Laser Capture Microscopy. This method allows to cut single cells out of tissue or cell cultures and capture them for further analysis. Such a system is available at the Stowers Microscopy Center.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Web Links:
Carl Zeiss

Peter Dwyer will present the Nuance multispectral imaging systems.
For details see http://cri-inc.com/products/nuance.asp

We are testing one of these cameras at the Stowers Microscopy Center during the next days. If you would like to have a look at this camera, please contact Richard Alexander.

These systems enable users to quantitate molecular markers even when they are co-localized in a single tissue section, producing clear and accurate images of each individual label on a multi-label tissue section. These systems also offer the powerful capability to unmix and remove autofluorescence.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

We will discuss the different components of a wide-field light microscope, their locations and functions. To understand their optical properties we will review basic ray optics. We will look at the beam-path of a infinity corrected microscope for bright-field and fluorescence imaging and the role of image-forming and pupil beam path.

Stowers Links:
View Presentation
Download Presentation

Web Links:
Optical Microscopy Primer: Microscope Optical Components.
Optical Microscopy Primer: Lenses and Geometrical Optics.
Wikipedia: Optics.

Literature:
Light and Matter: Optics Online introductory physics textbooks by Ben Cromwell Download PDF pp. 1-50 Download PDF pp. 11-100 Download PDF pp. 101-end

Geometric optics can help to understand the magnification of a light microscope, but not its limit in resolution. In this lecture we will introduce the concept of wave optics. This will help to understand the formation of a diffraction limit image in a light microscope. Point spread function and contrast transfer function will be discussed.

Stowers Links:
View Presentation
Download Presentation

Web Links:
Optical Microscopy Primer: Image Formation.
Optical Microscopy Primer: Modulation Transfer Function

The objective is the most important part of any microscope. We will discuss its parameters like resolution, magnification, correction and transmission. We will look at the importance of cover-slips, immersion- and mounting media for optimal imaging.

Stowers Links:
Stowers Microscopy Center: Objectives
Image Center: Objectives

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Web Links:
Optical Microscopy Primer: Objectives
Carl Zeiss: Objective Database
Leica: Microscope Objectives

Literature:
Carl Zeiss: LSM Objectives Download PDF

For bright-field as well as for fluorescence microscopy we use specially designed light sources. We will discuss their characteristics and the influence on image formation. Especially in fluorescence microscopy the proper selection of filters is crucial. The main parameters of these filters are discussed. A short refresher on basics of fluorescence will be given.

Stowers Links:
Stowers Microscopy Center: Fluorescence

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Web Links:
Optical Microscopy Primer: Microscope Illumination
EXFO: Manufacturer of X-Cite fluorescence lamp.
Optical Microscopy Primer: Light Filters
Optical Microscopy Primer: Introduction to Fluorescence
Carl Zeiss: Fluorescence Dye, Filter and Objective Databases
Chroma: Manufacturer of fluorescence filter sets.
Omega: Manufacturer of fluorescence filter sets.
Semrock: Manufacturer of fluorescence filter sets.

Literature:
Lichtman, J. W. and J.-A. Conchello (2005). "Fluorescence microscopy." 2(12): 910-919. Download PDF
Chroma: Handbook of Optical Filters for Fluorescence Microscopy Download PDF

Non-Linear Optics (NLO) is the basis of two- and multi-photon microscopy. The optical setup is similar to a confocal microscope. While in confocal epi-fluorescence microscopy one photon at a time excites a fluorescence dye, in two-photon microscopy two photons are needed. Thus the excitation wavelength is longer than the wavelength of the emitted light. Because the probability of two photons reaching the fluorophore at the same time is close to zero outside the focal spot, only in-focus fluorophores are excited. Thus a two-photon microscope provides intrinsic optical sectioning.

An other non-linear optical phenomenon used in microscopy is second-harmonic generation. This method can be used to image non-stained biological material and to increase the signal-to-noise of voltage sensitive dyes.

Stowers Links:
Stowers Microscopy Center: Two-Photon Microscopy
Image Center: LSM-510-NLO
Image Center: LSM-510-DEV

Web Links:
Molecular Expressions: Multiphoton Fluorescence Microscopy
User group: Multi-Photon Laser Scanning Microscopy
W.W.Webb Group at Cornell University

Literature:
Molecular Expressions: Multiphoton Fluorescence Microscopy - Selected Literature
Denk, W., J. H. Strickler, et al. (1990). "Two-Photon Laser Scanning Fluorescence Microscopy." Science 248(4951): 73-76.Download PDF
M. Dickinson: Multiphoton Laser Scanning Microscopy: Introduction into Theory and Operation
Koenig, K. (2000). "Multiphoton microscopy in life sciences." Journal of Microscopy, 200(2): 83-104Download PDF
Mertz, J. (2004). "Nonlinear microscopy: new techniques and applications." Curr Opin Neurobiol 14: 610-6.Download PDF

Like absorption and fluorescence spectra, fluorescence lifetime can be used to identify a specific type of dye molecules. The fluorescence lifetime is the time it takes an electron in an excited energy level of the dye molecule to return to its ground level while emitting fluorescence light.
The fluorescence lifetime of a dye depends on its environment. If there are alternative routes to fluorescence to depopulate the excited energy state, the measured lifetime is reduced. One important way to depopulate an energy state is Fluorescence (Förster)-Resonance-Energy-Transfer (FRET) or quenching.
In this lecture we will focus on the implementation of FLIM in the time-correlated single photon counting mode (TCSPC) as implemented at our system.

Stowers Links:
Stowers Microscopy Center: FLIM
Image Center: LSM-510-DEV

Web Links:
Becker & Hickl: Applications

Literature:
Wolfgang Becker: TCSPC Handbooks
TCSP Handbook
FLIM-LSM 510 Handbook

We will introduce the main components of a confocal microscope. We will discuss the importance of the pinhole and the fact that most confocal microscopes are laser scanning microscopes.

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Image processing is becoming a major part of microscopy. In this series of lectures we discuss theory and implementation of image processing algorithms. It is intended for users of image processing software who want to ensure optimal results while avoiding artifacts. We will discuss different software implementations and how to develop new algorithms.

Please email Winfried Wiegraebe if you are interested in getting updated information about Foundations of Image Processing lectures.

• August 25 , 2006: Winfried Wiegraebe - Convolution
• September 29 , 2006: Earl Glynn - Fourier Transformation
• October 27 , 2006: Christopher Wood - De-Convolution
• November 24 , 2006: no lecture (Thanksgiving)

Stowers Links:
Stowers Microscopy Center: Software
Bioinformatics
Image Center: Services

Web Links:
Optical Microscopy Primer: Basic Concepts in Digital Image Processing
HyperMedia Image Processing Reference: by R. Fisher, S. Perkins, A. Walker and Erik Wolfart
Image Processing Fundamentals: by I.T. Young, J.J. Gerbrands, and L.J. van Vliet

Software filters based on convolution kernels are among the most widely used image processing tools. The value of each image pixel in real space is recalculated taking its and its neighbors values into account. Convolution filters are used for smoothing, contrast enhancement and edge detection operations. Convolution as image processing tool is related to the convolution of sample features by the point-spread-function of the microscope when creating an image.

Almost all image processing packages used at the Stowers Institute use convolution kernels. The AIM software for our Carl Zeiss confocal microscopes, Axiovision, Image-Pro Plus, NIH ImageJ and IDL support user-defined kernels.

Stowers Links:
Stowers Microscopy Center: AIM software
Adv. Instr. & Physic: NIH ImageJ
Adv. Instr. & Physic: IDL
Image Center: Services

View Presentation
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Web Links:
Optical Microscopy Primer: Convolution Kernel Mask Operation
Optical Microscopy Primer: Convolution Kernels
Image Processing Fundamentals: Convolution-based Operations
Wolfram MathWorld : Convolution
NIH ImageJ: Process Menu

Literature:
Image Processing in IDL (see p.191ff)

Images can be described in real as well as in frequency space. The representation in frequency space is the Fourier transformation of the real image. In a microscope the Fourier transformation of the image of the sample is found at the back-focal-plane.

In image processing Fourier transformations are used as a fast implementation of some convolution operations. Working in the frequency domain allows effective filtering (enhancing and suppression) and detection of periodic features.

Frequency domain filtering is implemented in Axiovision, NIH ImageJ and IDL.

Stowers Links:
BioInformatics: Earl Glynn
Adv. Instr. & Physic: NIH ImageJ
Adv. Instr. & Physic: IDL
Image Center: Services

View Presentation
Download Presentation

Web Links:
Image Processing Fundamentals: Fourier Transforms
NIH ImageJ: FFT
ImageJ: Documentation Portal
Optical Microscopy Primer: Fourier Transform Filtering Techniques
HyperVis: Teaching Scientific Visualization Using Hypermedia
Reindeer graphics : Image Analysis Cookbook 6.0 - Part 7: 2.F.2. Deconvolution of blurred focus

Wolfram MathWorld: Fourier Series
Wikipedia: Fourier transform
Wikipedia: Continuous Fourier transform
Wikipedia: Fast Fourier transform
Wikipedia: Nyquist–Shannon sampling theorem
EFG's Computer Lab: Complex Numbers
California State University: Complex Analysis Project for Undergraduate Students
FFTW: C subroutine library for computing the discrete Fourier transform

University of Texas : Jean Baptiste Joseph Fourier

Literature:
Image Processing in IDL (see p.128ff)

In optical microscopy the image of the sample is blurred by light diffraction at the lens and optical imperfections like aberration due to refractive index mismatch. In mathematical terms this process can be described as a convolution of the sample with the point-spread-function of the optical system. De-convolution tries to reverse these effects.

While de-convolution can not re-create lost information (e.g. it can not break the diffraction limit), it can improve signal-to-noise and reassign light diffracted into out-of-focus image planes. This results typically in an amplification of high spatial frequencies which can result in an increase in resolution.

De-convolution is implemented in Axiovision, the Carl Zeiss AIM software and NIH ImageJ.

Stowers Links:
Stowers Microscopy Center: AIM software
Adv. Instr. & Physic: NIH ImageJ
Image Center: Services

Web Links:
Optical Microscopy Primer: Deconvolution in Optical Microscopy
SVI-wiki: Doing Deconvolution

Literature:
Schaefer LH, Schuster D, Herz H. 2001. Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy. Journal of Microscopy 204(2):99-107. Download PDF
Rooms F, Philips W, Lidke DS. 2005. Simultaneous degradation estimation and restoration of confocal images and performance evaluation by colocalization analysis. Journal of Microscopy 218(1):22-36.Download PDF

At the FCS User Club we are discussing classic papers about FCS and related techniques or results we got during our work with these methods. The FCS User Club is a forum for current and potential users of fluorescence correlation spectroscopy.

Each paper will be introduced by a volunteer, but we assume that everyone has carefully read it. We want to focus on the discussion of methods and findings within the paper.

Please email Winfried Wiegraebe if you are interested in getting updated information about the FCS User Club.

Copies of papers can be found at Literature Fluorescence Correlation Spectroscopy (FCS) (pdf for internal use only).

Time and location: every 2^nd Friday, 1:00 - 2:00 pm, room 421

• Friday, February ^9th, 2006, 1:00-2:00 pm, room 421
  Brain Slaughter: Association and oligomerization of MAPK proteins in live yeast examined by fluorescence correlation methods
• Friday, November 10^th, 2006, 1:00 -2:00 pm, room 421
  Joseph Huff: Characterization of Fluorescent Proteins by FCS.
• Friday, October 13^th, 2006: no seminar
• Friday September 8^th, 2006, 1:00 -2:00 pm, room 421
  Schwille, P., F. Meyer-Almes and R. Rigler (1997). "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophys. J. 72(4): 1878-1886.
• August 11 , 2006: Rigler, R., U. Mets, J. Widengren and P. Kask (1993). "Fluorescence Correlation Spectroscopy with High Count Rate and Low Background: Analysis of Translational Diffusion." European Biophysics Journal 22(3): 169-175.

Stowers Links:
Stowers Microscopy Center: FCS
Image Center: LSM-510-DEV

Literature:
Literature Fluorescence Correlation Spectroscopy (FCS)

Using FCS, Fluorescence cross-correlation, and the photon counting histogram, the mobility, association, and oligomerization of MAPK signaling proteins were analyzed in live yeast. Data are interpreted to yeild insights into how specificity is maintained between two overlapping signaling pathways, yeast invasive growth and mating response. In addition, challenges for implementing fluorescence correlation methods in live cells will be discussed.

Stowers Links:
Rong Li Lab
Li Lab: Research Web page
Li Lab: Brian Slaughter

At this seminar we will discuss recent results.

Fluorescence Proteins are powerful tool to label proteins for FCS investigations in the test tube as well as living cells. Most of these fluorescence proteins exhibit complex photo-physical behavior. We examine these behaviors and discuss the implications for FCS measurements.

Stowers Links:
Stowers Microscopy Center: FCS
Stowers Microscopy Center: Joseph Huff
Stowers Microscopy Center: Wiki
Image Center: LSM-510-DEV

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.

Presented by Joel Schwarz

External Literature Link Download PDF

Stowers Links:
Stowers Microscopy Center: FCS
Image Center: LSM-510-DEV

View Presentation
Download Presentation

Literature:
Literature Fluorescence Correlation Spectroscopy (FCS): S-V
Schwille, P., F. Meyer-Almes and R. Rigler (1997). "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophys. J. 72(4): 1878-1886.Download PDF

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Paper will be presented by Winfried Wiegraebe.

External Literature Link Download PDF

Stowers Links:
Stowers Microscopy Center: FCS
Image Center: LSM-510-DEV

View Presentation
Download Presentation

Literature:
Literature Fluorescence Correlation Spectroscopy (FCS): O-R
Rigler, R., U. Mets, et al. (1993). "Fluorescence correlation spectroscopy with high count rate and low background: Analysis of translational diffusion." European Biophysics Journal 22(3): 169-175.Download PDF

Stowers Links:
Stowers Institute Home Page
Stowers Institute Core Facilities

Web Links:
Carl Zeiss

Literature:
LinkstoLiterature, Download PDF