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Comprehensive Course on Fluorescence Microscopy

Meetings Intro/Summaries

14 November 2017 (Second Harmonic Generation Microscopy)
10 October 2017 (Super Resolution Microscopy)
12 September 2017 (Fundamentals of Fluorescence Microscopy)
2 May 2017 (Live-cell microscopy and Zeiss' Cell Discoverer system)
11 April 2017 (A new web tool for finding high-quality antibodies from publications)
14 February 2017 (Zoom, Zoom!  Stereomicroscopes and Macroscopes for large specimens)
10 January 2017 (Whole-slide scanning and analysis – brightfield and fluorescence)
6 December 2016 (Why Your Microscopy Data Likely Isn’t Quantitative, And How To Fix It)
8 November 2016 (Sensitivity, Resolution, and Speed:  What you really need in a microscope camera)
11 October 2016 (Tissue Clearing Techniques)
13 September 2016 (Any Way You Slice It - A comparison of optical sectioning techniques)
9 February 2016 (Structured Illumination Microscopy: SIMply the Perfect Focus for a Talk, Benefits of Resolution in the 100 nm Range)
8 December 2015 (Automated live-cell imaging with the IncuCyte ZOOM)
8 September 2015 (Pinhole Oversampling and Superresolution Techniques)
10 March 2015 (Multiphoton Imaging in the Life Sciences)
10 February 2015 (Fast 3D imaging using new Phaseview technologies)
13 January 2015 (Fundamentals of Fluorescence Microscopy)
2 December 2013 (Analyze Like a Pro: Ten things that you can't do with free analysis software)
7 October 2013 (Closing the Gap - Standardizing the Wounding Healing Assay for Measuring Cell Migration Rate)
6 May 2013 (White Light Laser - Opening the Light Gate in Confocal Microscopy)
2 April 2013 (Need for Speed)
3 December 2012 (You Call That Quantitative!?  PART 2:  A TUTORIAL)
5 November 2012 (3D/4D Visualization and Analysis with IMARIS)
1 October 2012 (Labeling and Detection Strategies in Fluorescence Microscopy)
7 May 2012 (Any Way You Slice It - A comparison of optical sectioning techniques)
2 April 2012 (Top Ten Tools for Optical Microscopy)
6 February 2012 (Pathology of Genetically Engineered Mice: Understanding Sample Preparation)
7 November 2011 (You call that quantitative!?)
3 October 2011 (What the FRAP?)
6 June 2011 (Advances in Super-Resolution Microscopy)
2 May 2011 (Focus on Microscopy 2011: latest developments in optical microscopy)
4 April 2011 (Laser Microdissection)
7 March 2011 (A bioluminescence microscope for metabolic imaging of tumors)
7 February 2011 (Tweaking the Raleigh Limit - Improvements in conventional imaging modes)
6 October 2010 (Fluorescence Molecular Tomography (FMT): Imaging of Disease Biology and Therapeutic Response In Vivo)
8 June 2010 (Super-Resolution and High-Speed Imaging with DeltaVision|OMX)
3 May 2010 (Invitrogen Molecular Probes Labeling and Detection Technologies)
9 April 2010 (Aperio's New Fluorescence Scanner and AQUA analysis software)
1 March 2010 (IVIS Spectrum: Small Animal Imaging of Fluorescent and Bioluminescent Probes)
1 February 2010 (Scratching the Surface with TIRF)
7 December 2009 (New Instruments for Automated Long Term Time Lapse Imaging)
2 November 2009 (3D and 4D Image Visualization and Analysis)
12 May 2009 (Multispectral Imaging)
6 April 2009 (Stereology)
2 March 2009 (Invivo Imaging with the IVIS system)
2 February 2009 (Advanced Microscopy Techniques)
2 June 2008 (ImageJ/Colocalization)
5 May 2008 (High Content Screening)
3 March 2008 (Raman Microscopy)
4 February 2008 (Two-photon Microscopy)
3 December 2007 (FLIM - Fluorescence Lifetime Imaging Microscopy)
5 November 2007 (Beyond Cellgazing)
1 October 2007 (Fast Confocal Imaging)
10 September 2007 (Live Cell Imaging)
4 June 2007 (Automated analysis using macros)
7 May 2007 (Intravital Microscopy)
2 April 2007 (Brightfield Whole Slide Scanning)
5 March 2007 (Deconvolution)
5 February 2007 (FRET Microscopy)
4 December 2006 (Frontiers in Microscopy)

November 14, 2017 - Second Harmonic Generation Microscopy - top

James Jonkman

Summary coming soon.

October 10, 2017 - Super Resolution Imaging - top

Lianne Dale (Leica Microsystems)

How super is YOUR resolution?  A good confocal microscope can resolve details down to about 200nm.  Until recently, this was the best you could do without moving to electron microscopy.  But as commercial super-resolution microscopes have matured, it is now quite straight-forward to get down to 160nm, 120nm, or even 50nm depending on the technique.  The AOMF has recently installed STED and Localization (GSD/STORM) microscopes, and there are also several Structured Illumination microscopes around Toronto.  But which super-resolution technique should you try? Lianne Dale from Leica Microsystems will explain the more popular techniques and what resolution you can expect from them.  She will also describe important aspects of sample prep that are required for all high- and super-resolution imaging (such as coverslip thickness, choice of fluorophores, etc.). 

September 12, 2017 - Fundamentals of Fluorescence Microscopy - top

James Jonkman (AOMF)

Fluorescence microscopy has become one of the most important tools in the biologist's toolbox. This presentation will provide a solid foundation for the beginner and a helpful review for more experienced microscope users by discussing:
  • The advantages of fluorescence microsocpy
  • When to use confocal microscopy
  • Choosing fluorophores
  • What resolution you can expect to achieve (and what about super-resolution!?)
  • Four key microscope elements that require optimization
Here is a pdf of the presentation.

May 2, 2017 - Live-cell microscopy and Zeiss' Cell Discoverer system - top

James Jonkman (AOMF) & Dan Stevens (Zeiss)

April 11, 2017 - A new web tool for finding high-quality antibodies from publications - top

Maurice Shen(BenchSci)

You might be a skilled microscopist and have access to the best fluorescence microscopes around, but the results of your immunofluorescence imaging will only be as good as the antibodies you use when preparing your samples.  For this month’s O-MUG meeting, we invite Dr. Maurice Shen to present a new UofT start-up company called BenchSci.  Looking for antibodies for your experiments?  BenchSci is a free, web-based, search platform that uses machine learning technology to decode primary research papers and extract antibody usage data from peer-reviewed publications. With BenchSci, you can examine individual published figures on a single site and identify which antibody was used in each figure.  The figures can be filtered by specific experimental contexts including techniques, tissues, cell lines, and more, in order to match your specific experimental interest.  Forget googling for antibodies:  BenchSci adds a wealth of context to your antibody search!

Here is a pdf of the presentation.

February 14, 2017 - Zoom, Zoom!  Stereomicroscopes and Macroscopes for large specimens - top

Chris Souwand (Carl Zeiss Canada)

There are several ways you can image a large specimen.  1. Cut it up into smaller specimens.  In particular, if you section it nice and thin, you can run it through a brightfield or fluorescence slide scanner as we talked about last month.  2.  Put it on a microscope and just look at a tiny part of the specimen.  If the microscope is motorized, you can tile-and-stitch to give yourself a bigger field of view.  3.  Use a Stereomicroscope or Macroscope to image a large specimen in one shot.  These instruments usually allow you to zoom in or out as required for your application.  Stereomicroscopes (sometimes called “Dissecting scopes”) are helpful when you need to perceive the depth of the specimen, as they give you a 3D view of the specimen.  But Macroscopes have a tremendous advantage for capturing fluorescent images:  our new Axiozoom Macroscope has nearly 3 times the Numerical Aperture of our old Stereomicroscope, which means we get 3 times the resolution and 9 times the sensitivity of the Stereomicroscope.  By adding a fast, high-resolution sCMOS camera we can capture large specimens with unprecedented sensitivity at speeds of 100 images/second.  Chris Souwand  from Carl Zeiss is a bit of an expert on the topic, and will discuss with us the advantages and applications of stereomicroscopes and macroscopes.

January 10, 2017 - Whole-slide scanning and analysis – brightfield and fluorescence - top

James Jonkman (AOMF) and Anne Hellebust (Indica Labs)

More often than not, a few snap shots under the microscope are not terribly representative of an entire tissue section (consider tumour heterogeneity, for example).  For nearly 10 years now, the AOMF has been encouraging users to scan their entire brightfield histology slides; and with the installation of a fluorescence slide scanner last month we’re now able to offer the same service for fluorescently labeled tissue sections.  I will start with a brief introduction to the capabilities of our new fluorescence slide scanner; and then Anne Hellebust from Indica labs will talk about how our new Halo analysis software will enable users to extract meaningful data from whole-slide images.  Halo uses pattern recognition (like our old Genie software) to first classify tissue areas (eg: tumour versus stroma).  Users then follow up with general analysis modules such as Nuclear, Membrane, and Cytoplasmic quantification, or more specific ones such as TMA analysis, Immune cell quantitation, and more. 

December 6, 2016 - Why Your Microscopy Data Likely Isn’t Quantitative, And How To Fix It - top

James Jonkman (AOMF)

Any reasonably modern fluorescence microscope can produce beautiful images of your cells and tissues without too much effort. Users are probably also diligent about using the same microscope settings (exposure time, laser power, etc.) for control and experimental conditions.  But would you believe me if I told you that up to half of the images taken on our microscopes are essentially unquantifiable!? Lurking behind those colourful facades are:
  • Lamp/ laser intensity fluctuations (I’ve seen it as bad as 20% in a 3-hr session!).
  • Non-uniform illumination across the field-of view (15–30% variations!).
  • Uncorrected background and bleedthrough.
  • Poor shutter control, leading to varying degrees of photobleaching.
Despite the fact that these (and other) issues are surprisingly widespread, users of these instruments are blissfully unaware of any problems and will likely go on to analyze the resulting images. I’ll present several case studies that illustrate the extent of these problems, and then I’ll suggest 7 specific steps that you can follow to ensure you get quantitative data from your microscope images.

November 8, 2016 - Sensitivity, Resolution, and Speed:  What you really need in a microscope camera - top

James Jonkman (AOMF)

Most smart phones and tablets have integrated cameras with at least 5 Megapixel (MP) resolution; so why are we still stuck using 1.4MP cameras or even 0.25MP cameras on many of our microscopes?  New scientific CMOS cameras can boost your imaging performance with reasonably high resolution (4.2MP), superb sensitivity (> 80% QE), and blazing fast frame rates (100 images per second!).  Explore the trade-offs between these parameters as we walk through some of the advantages and disadvantages of new CCD and sCMOS microscopy cameras.

October 11, 2016 - Tissue Clearing Techniques - top

Judy Cathcart (AOMF)


The scattering of light inhibits our ability to image deep into thick tissues in order to ascertain its inherent 3-dimensional structure. Tissue clearing is a term used to describe a diverse collection of techniques aimed at producing transparent tissues more amenable to volumetric fluorescence imaging. This month, we’ll discuss the nature of a cleared tissue, survey some different clearing methods and outline a few challenges in preparing cleared tissues.


Douglas S. Richardson and Jeff W. Lichtman. "Clarifying Tissue Clearing", in Cell, 162, July 16, 2015. (pdf)

Click here to download a pdf version of Judy's talk.

September 13, 2016 - Any Way You Slice It - A comparison of optical sectioning techniques - top

James Jonkman (AOMF)


Confocal microscopes are now a standard tool for the life sciences researcher; but which kind of confocal microscope is BEST for your particular application?  For this month’s O-MUG meeting, James will compare the classic laser-scanning confocal, the speedy spinning-disk confocal, and their younger cousin the grid-based optical sectioning microscope (sometimes called “the poor-man’s confocal”).  He will briefly describe how each of these instruments works and what its advantages SHOULD be, and then he will attempt both qualitative and quantitative comparisons on thin samples (cultured cells), thicker samples (15um-thick tissues), and very thick samples (70um-thick embryos).


José-Angel Conchello & Jeff W Lichtman. "Optical Sectioning Microscopy", in Nature Methods, Vol.2 No.12, December 2005. (pdf)

Click here to download a pdf of James' presentation.

February 9th, 2016 - Structured Illumination Microscopy - top

Kevin Conway (Nikon Canada)

Using the best objective lenses on our high-end confocal microscopes, we’re able to resolve structures down to about 200nm; but several targets of biological interest tempt us just below this resolution limit.  By projecting grid patterns into the sample at various angles, Structured Illumination Microscopy (SIM) promises resolutions down to about 100nm.  SIM is relatively fast (>1.5 fps)  and compatible with live-cell imaging.  We currently have ~ $1M in funding to purchase a confocal microscope with super-resolution capabilities, and one of the technologies that we’ll be evaluating is the Nikon A1R with SIM.  Come out and discuss whether your applications would benefit from SIM! br>
December 8th, 2015 - Automated live-cell imaging with the IncuCyte ZOOM - top

Kurt Harris (Essen BioScience)

Please join us on Tuesday for a seminar provided by Kurt Harris from Essen BioScience on the IncuCyte ZOOM live-cell imaging system. The system allows researchers to perform kinetic assays and observations on living cells inside a cell culture incubator over long periods of time; from hours to weeks. The system accommodates a variety of standard tissue culture vessels, including microplates, dishes, flasks and slides for simultaneous experiments and multiple user accessibility. The automated imaging allows walk-away data acquisition over time without the need to remove the cells from the incubator.

The IncuCyte ZOOM software provides a remote interface for setup, viewing and exporting images, time-lapse movies and data from remote users computers. The software provides a quantitative panel of metrics for scoring and graphing the kinetic response of various phenotypic cellular events, including:

  • Labeled and Unlabeled Proliferation
  • Cytotoxicity & Apoptosis
  • 3D Spheroids
  • Stem Cell Biology
  • Migration & Invasion & Chemotaxis
  • Angiogenesis & Co-culture Tube Formation
  • Cell-cell Interactions (Cancer Immunotherapy)
  • Reporter Gene Expression
  • Neurite Outgrowth & Toxicity
  • Phagocytosis
You can view a list of assays and peer-reviewed publications on the IncuCyte technology and its live-cell assays in the links below:

IncuCyte ZOOM Live-Cell Assays

IncuCyte ZOOM Publications by Research Area

September 8th, 2015 - Pinhole Oversampling and Superresolution Techniques - top

Dan Stevens (Zeiss Canada)

New confocal detectors for increased resolution and sensitivity the laser-scanning confocal has been the fluorescence microscopy workhorse of many research labs for more than a decade. Rather than this technology stagnating, however, it has become increasingly more sophisticated with new kinds of lasers, scanners, detectors, and software. One particularly exciting development is the replacement of the traditional confocal pinhole with the Airyscan detector array: the resulting Pinhole Oversampling provides improved lateral resolution and sensitivity without any modification of the sample preparation. Several other advances in confocal imaging technology will also be discussed, along with a brief introduction to some competing superresolution techniques.

March 10th, 2015 - Multiphoton Imaging in the Life Sciences - top

Louisa Mafeld (Scientifica)

Further to James Jonkman’s talk on the Fundamentals of Fluorescence, this presentation will provide an introduction to multiphoton imaging. Unlike single photon methods of microscopy which are limited to the observation of surface tissue, multiphoton imaging offers the user deeper penetration, reduced photoxicity and a favourable signal to noise ratio- this is particularly relevant when observing changes in fluorescence. Multiphoton imaging has been ground-breaking in in-vivo applications where tissue can not only remain intact but also be paired with genetically encoded indicators of neural activity (GINAs) to obtain large-scale recordings in a variety of domains.
The presentation will give an overview of multiphoton imaging by covering the following main areas:
  • What is multiphoton imaging?
  • Examples of typical research applications
  • What to consider when purchasing a multiphoton system
If you have any questions or require any specific information prior to the presentation then please feel free to contact Louisa Mafeld.

February 10th, 2015 - Fast 3D imaging using new Phaseview technologies - top

Presenter: Dr. Igor Lyuboshenko, Lead Scientist, PhaseView (France)
Sponsor: Bernd Michaelis, CPX-Solutions, Canada

Fast 3D imaging using new PhaseView technology Biological specimens are typically three-dimensional, and viewing them in 3D is usually preferred.  In addition to the well-known confocal microscopes, there are several lesser known 3D imaging techniques that have various advantages and disadvantages.  The company PhaseView will present fast 3D widefield imaging using their patented acquisition technology, which is available as an add-on to most optical microscopes.  Instead of using stepper motors or piezo stages, Phaseview uses digitally controlled tunable lenses for vibration-free focusing without moving the specimen or objective lens. Applications have included:
· in-vivo imaging of C. elegans, Drosophilia and Zebrafish
· 3D functional imaging of neuronal activity
· high-speed CA²++ imaging
· live imaging of cellular dynamics in three dimensions
· and more!
They will also be announcing a new Phaseview Light Sheet microscope, available as an add-on to existing research microscopes.

January 13th, 2015 - Fundamentals of Fluorescence Microscopy - top

James Jonkman (AOMF)

Fluorescence microscopy has become one of the most important tools in the biologist's toolbox.  In this presentation I hope to provide a solid foundation for the beginner and a helpful review for more experienced microscope users by discussing: . When to use fluorescence . How fluorescence microscopes work . Choosing fluorophores . What resolution you can expect to achieve . When to use confocal microscopy . Four key microscope elements that require optimization

Click here for a summary of James' presentation.

December 2th, 2013 - Analyze Like a Pro: Ten things that you can't do with free analysis software - top

James Jonkman (AOMF) & Nick Beavers (Media Cybernetics)

You've worked hard to prepare samples, then spent countless hours snapping pictures of them on the microscopes. Now it's time to extract meaningful data from these images. Many people use the free ImageJ software, and it is a decent start; but I'll go through 10 things that the new Image Pro Premier 9 software lets you do that you simply can't do with free software. Things like:
- Smart Segmentation, which helps you select objects based on texture rather than just intensity;
- Automated and semi-automated tracking of moving objects
- One-click output of your images to Powerpoint slides
- Apps, including the newly-released Wound-Healing app

Nick Beavers from Media Cybernetics will follow with a short demo of Image Pro Premier 9.1.

October 7th, 2013 - Closing the Gap - Standardizing the Wounding Healing Assay for Measuring Cell Migration Rate - top

Judy Cathcart (AOMF)

Wound healing assays are typically performed to measure cell migration, proliferation and interactions for biomedical applications such as embryogenesis, tissue regeneration and cancer metastasis. A gap is created in a monolayer of confluent cells, stimulating the inflammatory, proliferative and regenerative phases of wound healing for which various measurements and analyses can then be made. The development of different types of wound healing/migration assay has been directed toward improving both accuracy and repeatability of assay results, as well as overcoming some of the limitations posed by traditional, manual scratch techniques.
Several post-acquisition, image processing steps can help improve and standardize the quantification of either mechanical or barrier type wound healing assays. Our experiment compared a mechanical (scratch) method based on an established protocol and barrier type (Ibidi chamber) assays with two cell lines having different migration rates. Image analysis procedures such as contrast enhancement, smoothing, edge detection and setting thresholds are applied to the collected data in order to illustrate how such tools can be used to standardize the results of this and other types of experimental image data.

May 6th, 2013 - White Light Laser - Opening the Light Gate in Confocal Microscopy - top

Lianne Dale (Leica Microsystems)

Conventional laser scanning imaging systems are equipped with a set of gas or solid state lasers that have a limited number of fixed laser lines available for use. As a result, the dyes and labels available to use are dictated by the imaging system. However, think about all of the possibilities available if you could adapt your imaging system to your sample. You would be able to optimally excite the dye and in turn decrease the damage to the cell, reduce unwanted cross-excitation, potentially minimize nagging autofluorescence, and use new combinations of dyes and labels. This is all possible with one of the latest advancements in laser scanning confocal microscopy; Leica's White Light Laser (WLL). The WLL has 200 laser lines to select from and providing over a trillion combinations of laser lines. This adds a whole new dimension to spectral imaging and opens the light gate in confocal microscopy.

Apr 2nd, 2013 - Need for Speed (How resonant-scanning confocals compare) - top

James Jonkman (AOMF) and Kevin Conway (Nikon Canada)

The classic laser-scanning confocal microscope gives you unmatched resolution and sensitivity, but it generally tops out at about 1 or 2 frames per second (fps). The Yokogawa spinning-disk confocal can give you 10 fps or more, but only for a single colour (usually) and with a compromise on resolution and depth performance. If your application demands blazing fast speed, a resonant-scanning confocal can give you full-frame scans at 30 fps, or a thinner strip of your image at up to 420fps! Come out and discuss what speedy resonant scanning confocals can do for your imaging applications.

Dec 3rd, 2012 - You Call That Quantitative!?  PART 2:  A TUTORIAL - top

James Jonkman, Manager of AOMF

Most researchers are aiming to get quantitative data when they sit down at the microscope.  Indeed, any reasonably modern fluorescence microscope is capable of generating beautiful images - but as we spoke about last year, fluorescence microscopes all have hidden issues that can easily render the data rather non-quantitative:
  • Lamp/ laser intensity fluctuations (I've seen it as bad as 20% in a 3-hr session!)
  • Non-uniform illumination across the field-of view (15-30% variations!)
  • Uncorrected background and bleedthrough
  • Dark craters on camera surfaces
  • Poor shutter control, leading to varying degrees of photobleaching
In last year's session, I mostly dwelt on the problems, but this month I'm presenting solutions: a tutorial on quantitative fluorescence microscopy. I'll suggest specific steps that users can follow, and we'll discuss what role vendors and facility managers should play in ensuring quantitative results.

Here's a good related article to check out: Cromey DW.  “Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images”. Sci Eng Ethics. 2010 Dec;16(4):639-67. (pdf)

Sponsoring us this month, will be Lumen Dynamics (makers of X-Cite fluorescence illumination systems) and they are giving away an IPad! Fill out a survery here for your chance to win!

Click here for a copy of James' presentation.

Nov 5th, 2012 - 3D/4D Visualization and Analysis with IMARIS - top

Wendy Allan, Bitplane

This month, the AOMF welcomes Dr. Wendy Allan from Bitplane, who will tell us how their Imaris software enables rapid and accurate data visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets.  Confocal microscopes are well-equipped for 3D (X, Y, Z) or even 4D (X, Y, Z, time) image acquisition; and yet probably 90% of the images taken on our confocals are just 2D slices.  It's true that in many cases, a single confocal slice may be representative of the entire specimen (much like we take images of 100 cells on a coverslip to be representative of all of the cells).  However, there are some compelling reasons why you should consider extending your acquisition to 3D:

1. Some types of analysis (particularly tracking and co-localization) are more accurate in 3D.  It used to be nearly inconceivable to try doing these tasks in 3D, but Imaris makes it pretty easy.

2. 3D renderings (particularly with Imaris!) make your presentations sexier: you may not need to collect all the data in 3D, but try a few representative ones in 3D and wow your supervisor, colleagues, and reviewers.

3. In the last couple of years, 64-bit computers with multiple CPUs, gobs of RAM, and terabyte harddrives have made 3D analysis nearly painless.

4. A generous benefactor has contributed the latest version of Imaris to the AOMF, so you can access what is arguably the best 3D visualization and analysis tools on the market for just $10/hr.

Oct 1st, 2012 - Labeling and Detection Strategies in Fluorescence Microscopy - top

Nick J. Dolman Ph.D, Sr. R&D Scientist from Molecular Probes, part of Life Technologies


Fluorescence microscopy offers the benefit of spatially resolved and multi-parametric analysis of cells in heterogenous populations. Life Technologies offers three foundational approaches to fluorescent probes for microscopy; small-molecule organic dyes, Qdot®nanocrystals, and fluorescent protein biosensors.This comprehensive solution to fluorescence labeling and detection enables unmatched flexibility and ease of use, translating readily into a powerful toolbox of probes and applications for cell biology. 10:00a.m–Advancements in probing cell structure & function by fluorescence microscopy The first seminar will provide an overview of new and existing cellular assays for traditional fluorescence microscopy. Click-iT®chemistry, pH sensor dye technologies, and BacMam gene delivery will be presented as particular examples of platforms which enable robust imaging of cell structure as well as various aspects of cell function such as cell cycle, internalization, and autophagy. New MolecularProbes® imaging assays for apoptosis and oxidative stress will also be discussed. 11:00a.m-General considerations for labeling & detection in fluorescence microscopy The second seminar will provide an introduction to fluorescence as well as basic considerations for performing immunolabeling and microscopy. Probe selection tools, tips for sample processing, and reagents for workflow simplification will also be discussed.

Here are pdfs of the presentations:
Introduction to Imaging
Advanced Imaging

May 7, 2012 - Any Way You Slice It - A comparison of optical sectioning techniques - top

James Jonkman (AOMF) & Dan Stevens (Carl Zeiss Canada)


Confocal microscopes are now a standard tool for the life sciences researcher; but which kind of confocal microscope is BEST for your particular application?  For this month’s O-MUG meeting, the AOMF has teamed up with Carl Zeiss Canada to compare the classic laser-scanning confocal, the speedy spinning-disk confocal, and their younger cousin the grid-based optical sectioning microscope (sometimes called “the poor-man’s confocal”).  We will briefly describe how each of these instruments works and what its advantages SHOULD be, and then we will attempt both qualitative and quantitative comparisons on thin samples (cultured cells), thicker samples (15um-thick tissues), and very thick samples (70um-thick embryos).


José-Angel Conchello & Jeff W Lichtman. "Optical Sectioning Microscopy", in Nature Methods, Vol.2 No.12, December 2005. (pdf)

April 2, 2012 - Top Ten Tools for Optical Microscopy - top

James Jonkman

Abstract: Over the years, I've enjoyed interacting regularly with other microscopists from Toronto (O-MUG meetings), from across Canada (such as the recent CCA meeting) and from around the globe (international meetings such as the Focus on Microscopy). One of the great things that happens when we get together is a kind of cross-pollination: I'm eager to learn of new tools and resources for optical microscopy, and I'm also happy to share the ones that I've found useful. For this month's O-MUG meeting I will present a list of tools for Optical Microscopy that I've compiled from fellow core facility staff and others.

February 6, 2012 - Pathology of Genetically Engineered Mice: Understanding Sample Preparation - top

Susan Newbigging (Director of Pathology at CMHD)


The mouse is by far the most frequently used and most sophisticated organism available to model complex human disease processes. Immense resources are consumed to develop transgenic and knock-out mice, and furthermore, to subsequently fully characterize them. In order to maximize morphological data output, GEMs must undergo careful anatomical and histopathological analyses, even after extensive in-life characterization and imaging techniques. Inadequate tissue preparation can result in the loss of relevant histological architecture from these precious, sometimes irreplaceable, samples. Moreover, routine tissue preparation as performed in diagnostic labs, is often not sufficient for the type of analyses required in GEM pathology investigations. The importance of expertise in mouse necropsy techniques, tissue fixation, embedding modalities, histochemical and immunohistochemical techniques for both embryonic, post-natal and adult will be discussed in the context of preparing these samples for optimal gross morphology and histopathology interpretation as well as for superlative image acquisition and analysis.

November 3, 2011 - You call that quantitative!? - top

James Jonkman, Manager (AOMF)


Any reasonably modern fluorescence microscope can produce beautiful images of your cells and tissues without too much effort.  But would you believe me if I told you that up to half of the images taken on our microscopes are completely unquantifiable!?  Lurking behind those colourful facades are:
  • laser power fluctuations as great as 20% over three hours
  • 30% variations in illumination intensity across a slide
  • dozens of dark craters on camera surfaces
  • uncorrected background and bleedthrough

Despite the fact that these (and other) issues are widespread, users of these instruments are blissfully unaware of any problems and will likely go on to analyze the resulting images.  I'll present three Case Studies that illustrate the problems, and I'll describe some ways of correcting for these issues when possible.  I hope that you will discuss with me the responsibilities of the various parties (microscope users, facility staff, and manufacturers) in ensuring fluorescence microscopy results in quantitative data.

October 3, 2011 - What the FRAP - top

James Jonkman, Manager (AOMF)


Fluorescence Recovery After Photobleaching (FRAP) is a relatively well-known technique for measuring protein mobility in living cells. Nevertheless, I'm quite surprised by how few people are making use of this tool!  After tagging the protein of interest with a fluorescent protein (such as GFP), many confocal microscopes will facilitate photobleaching a small region in the cell and monitoring the recovery of fluorescence in this region as bleached molecules exchange with unbleached ones from elsewhere in the cell. A qualitative assessment can already tell you whether the mobility is fast or slow; but a more quantitative analysis can help to determine whether binding interactions are present, the number of binding states, and whether there is a proportion of immobilized protein that doesn't participate in the exchange. I'll show you some FRAP data taken on a spinning-disk confocal which has been optimized for straight-forward (yet powerful) acquisition and for simple (yet rigorous) processing and analysis. Then, I hope we can discuss together whether You (my fellow O-MUG participants) are currently "FRAPing" (qualitatively or quantitatively), or whether you you're using other techniques to measure protein mobility. I've selected a couple of excellent FRAP papers that you may wish to look through to prepare you for the talk (but you can still come if you haven't read them!). The first one is a nice review of FRAP, and the second shows a nice application of FRAP (as well as photoactivation and photoswitching) while doing in vivo imaging with a window chamber.

If you missed James' presentation, you can download a pdf version of it here: (pdf)

Brian L. Sprague, James G. McNally. "FRAP analysis of binding: proper and fitting", in Trends in Cell Biology, Vol 15:  pp 84-91. (pdf)

Marta Canel, Alan Serrels, Derek Miller, Paul Timpson, Bryan Serrels, Margaret C. Frame, and Valerie G. Brunton. "Quantitative In vivo Imaging of the Effects of Inhibiting Integrin Signaling via Src and FAK on Cancer Cell Movement: Effects on E-cadherin Dynamics", in Cancer Res, Vol 70: pp 9413-9422. (pdf)

Jamie White and Ernst Stelzer. "Photobleaching GFP reveals protein dynamics inside live cells", in Trends in Cell Biology, Volume 9, Issue 2, 61-65, 1 February 1999 (pdf)

Zobeck KL, Buckley MS, Zipfel WR, Lis JT. "Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells", in Mol Cell. 2010 Dec 22;40(6):965-75. (pdf)

Lippincott-Schwartz J, Snapp E, Kenworthy A. "Studying protein dynamics in living cells", in Nat Rev Mol Cell Biol. 2001 Jun;2(6):444-56. (pdf)

Eric A.J. Reits and Jacques J. Neefjes. "From fixed to FRAP: measuring protein mobility and activity in living cells", in Nature Cell Biology 3, E145 - E147 (2001). (pdf)

June 6, 2011 - Advances in Super-Resolution Microscopy - top

Thoma Kareco, PhD    Senior Biosystems Applications Specialist (Nikon Canada)
Kevin Conway, PhD     Advanced Imaging Specialist (Nikon Canada)

Super-resolution microscopy is a family of techniques designed to address resolution limits of the light microscope. Here we discuss new super-resolution systems, N-SIM (Stuctured Illumination Microscopy) and N-STORM (STochastic Optical Reconstruction Microscopy), the hardware and software requirements for super-resolution work, and experimental considerations.

In Structured Illumination Microscopy (N-SIM), the cellular ultrastructure is elucidated by analyzing the moiré pattern produced when illuminating the specimen with a known high-frequency patterned illumination. Capturing super-resolution images at over 1 frame per second enables the study of dynamic interactions in living cells.

STORM reconstructs a super-resolution image by combining the high-accuracy localization information of each fluorophore in 3 spatial dimensions and multiple colours. Stochastic activation of small numbers of fluorophores using low-intensity light enables high-precision Gaussian fitting of each molecule in 2-space. Use of Nikon’s 3D-STORM optics also permits axial localization, producing an unprecedented increase in 2D and 3D resolution.

May 2, 2011 - Focus on Microscopy 2011: latest developments in optical microscopy - top

As many of you know, James Jonkman has been away in Cambridge accompanying his wife on sabbatical the past few months. While there, he had the great opportunity to scoot over to Germany for the Focus on Microscopy conference. For this month's O-MUG he's going to tell us all about it via Skype.

The Focus On Microscopy 2011 conference was held in Konstanz, Germany from April 17 - 21. The FOM is truly a 'meeting of the microscopy minds', with many of the founding fathers in the field of optical microscopy participating. The AOMF's James Jonkman reports back on some of the exciting new developments including: superresolution, superresolution, more superresolution (do you detect a recurring theme?), Second and Third Harmonic Generation, Light-Sheet Based Fluorescence Microscopy (particularly for developmental biology), automated image analysis, and more.

Click here for a page of references from James' talk.

April 4, 2011 - Laser Microdissection (LCM) - top

Laser microdissection is used to isolate specific cells or tissue sections of interest from tissue samples including blood smears, cytologic preparations, cell cultures and aliquots of solid tissue. Frozen and paraffin embedded tissue may also be used . LCM is a useful method of collecting selected cells for DNA, RNA and/or protein analyses, because it doesn't alter the morphology or chemistry of the sample collected , nor does it disturb the surrounding cells.

The AOMF manages an MMI (Molecular Machines & Industries) laser microdissection system at Max Bell and it's been gaining much use over the past year. Jeff Butler from Quorumm Technologies, which distributes the MMI laser microdissection system, will be giving an overview of the technique and some of its applications. Following his talk, we will have two of our current LCM users talk about their applications for the system.

March 7, 2011 - A bioluminescence microscope for metabolic imaging of tumors - top

Understanding the metabolic differences between healthy and cancerous cells has become an exciting and productive area of research in the past decade. As early as 1930 and prior to the discovery of the genetic contribution to tumourigenesis, Otto Warburg determined that cancer cells meet their massive energy requirements by altering the pathways that metabolize glucose and produce ATP. Since then, our knowledge about tumour metabolism has grown substantially. The ability to identify and understand the altered metabolism and microenvironment of solid tumours will ultimately help improve diagnoses, treatments and patient survival.

Our OMUG meeting this month will focus on an experimental bioluminescence technique devised in the early 1990's by a group of German researchers and further developed here at OCI by Dr. Eduardo Moriyama, who will discuss the technique and its advantages in further detail. The instrument and technique developed by Dr. Moriyama will be moved to the AOMF this month and we expect to begin acquiring data for the Hypoxia and Microenvironment Program headed by Dr. Brad Wouters beginning in April. We are scheduled to offer this technique as full service imaging to other researchers early in June 2011.

YouTube links to some talks on tumour metabolism by Brad Wouters and Mike Milosevic:
Brad Wouters: Part 1
Brad Wouters: Part 2
Mike Milosevic: Part 1
Mike Milosevic: Part 2  

Also, you can check out the Mueller-Klieser and Walenta articles below:

Summary of talk: A bioluminescence microscope for metabolic imaging of tumors

Eduardo Moriyama, PhD
We have developed a novel bioluminescence imaging system to assess the distribution of metabolites, such as lactate, glucose and ATP in tumor cryosections. The technique involves the application of specific metabolic enzymes involved in bioluminescence reactions in tumor cryosections. The emitted photons are subsequently detected, enabling high-resolution imaging of the distribution of metabolites within the area of interest.            Bioluminescence offers several advantages over other techniques for evaluating metabolism, including ease of translation to the clinic, microscopic spatial localization of metabolites, quantitation of metabolites and spatial co-registration of metabolic maps with tumor histology and/or immunohistochemical biomarkers.

February 7, 2011 - Tweaking the Raleigh Limit - Improvements to conventional methods for live cell and three dimensional imaging - top

Jeff Butler and Paul Constantinou, Quorum Technologies

Quorum technologies will be presenting two short talks discussing  improvements to current technologies - showing them to be powerful,  often underutilized techniques.
The first talk will cover the Riveal Imaging system - utilizing oblique illumination to show a degree of detail that many other  techniques can miss. Moreover, it is a technique requiring no staining and is ideal for live cell applications.
The second talk will discuss conventional structured illumination. Many researchers have utilized this technique, but it has yet to  become a mainstay in many facilities. We will discuss some of the  historic challenges these techniques have faced, and the solutions  currently available.

October 6, 2010 - Fluorescence Molecular Tomography (FMT): Imaging of Disease Biology and Therapeutic Response In Vivo - top

Gilberto Prudencio, Perkin Elmer

The FMT 2500 LX quantitative tomography system with Perkin Elmer’s suite of fluorescent agents provides the leading fluorescence tomographic imaging solution for true quantification of deep tissue targets in vivo. Using one or multiple of Perkin Elmer’s targeted, activatable and vascular agents and labels (for multiplexed results), a researcher can quantify a broad range of biologic targets, pathways and processes in vivo. In addition, the FMT is specifically developed for robust multi-modality imaging, allowing for the easy fusion of FMT with PET, MRI, CT and SPECT data sets. Whether you are imaging a particular biomarker, disease pathway or monitoring therapeutic efficacy, the FMT is easy to learn, fast to incorporate into a high-throughput workflow and quickly produces unmatched results.

June 8, 2010 - Super-Resolution and High-Speed Imaging with DeltaVision|OMX - top

Paul Goodwin, Director of Advanced Development, API
Laurence Pelletier, Principle Investigator, SLRI

This presentation will be given in two parts. First, Paul Goodwin from Applied Precision will present “Super Resolution Microscopy with 3D SIM”. In this talk, Mr. Goodwin will present a tutorial on Three Dimensional Structured Illumination Microscopy (3D SIM). He will cover the mechanisms that limit resolution with conventional microscopes and then move into a description of the mechanisms and methods for creating 3D SIM images and finish with a few examples of the data that has been obtained using the technology. Dr. Pelletier will present the second portion of this talk. He will show examples of how his laboratory at the Samuel Lunenfeld Research Institute has used microscopy based screening, high resolution, time resolved microscopy, and 3D SIM to identify proteins involved in the assembly and maintenance of the mitotic spindle in mammalian cells. He will show specific examples of results obtained with the DeltaVision|OMX system located at the SLRI. For more on Dr. Pelletier's research, click here.

May 3, 2010 - Invitrogen Molecular Probes Labeling and Detection Technologies - top

Kary Oakleaf, from Invitrogen, will be coming to talk to us about new technologies for structural and functional analysis of live and fixed cells by fluorescence microscopy. Fluorescence microscopy offers the benefit of spatially resolved and multi-parametric interrogation of cells in heterogenous populations. Molecular Probes, part of Life Technologies, offers three foundational approaches to fluorescent probes for microscopy; organic dyes, Qdot® nanocrystals, and fluorescent proteins. This comprehensive approach to fluorescence labeling and detection enables unmatched flexibility and translates into a powerful toolbox of probes and applications for cellular imaging. This seminar will provide an overview of fluorescent probes and assays for live and fixed cells in traditional fluorescence microscopy. The Click-iT® and BacMam technologies will be presented as particular examples of platforms which enable robust imaging of cell structure as well as various aspects of cell function such as proliferation, endocytosis, autophagy and cytotoxicity.
Download a pdf copy of the presentation here

April 9, 2010 - Aperio's New ScanScope FL Fluorescence Scanner and HistoRX AQUA analysis software - top



March 1, 2010 - IVIS Spectrum: Small Animal Imaging of Fluorescent and Bioluminescent Probes - top

In Vivo bioluminescence imaging has become a very important tool for our researchers, as evidenced by the increased usage of our Xenogen instrument and the fact that last year’s O-MUG meeting on this topic garnered a record attendance. Now in vivo imaging has received another boost: two new IVIS Spectrum instruments have recently been added to the AOMF’s roster (one at our sister facility STTARR in the MaRS building, and another at PMH). In addition to the highly sensitive 2D bioluminescence in vivo imaging, these new instruments feature 3D Bioluminescence Imaging, 2D and 3D Fluorescence imaging, and more! Come out to this special 2-hr O-MUG session to find out how you can take advantage of these advanced in vivo imaging modalities.

Caliper Life Sciences, who sells the IVIS instruments, is a leader in the field of biophotonic imaging in small animals and has developed a technology which allows biological processes to be non-invasively monitored, both three dimensionally and in real-time. Genes encoding luciferase proteins are engineered into cells (e.g., cancer cell lines and infectious disease agents) and/or animals (transgenic mice and rats) to enable them to produce light that can be visualized through the tissues of a live animal using specialized imaging equipment (the IVIS Spectrum) and software (Living Image 4.0) designed and built by the company. Furthermore, this technique is equally applicable to fluorescent imaging and can be used to monitor a wide range of fluorophores in vivo (e.g., fluorescent proteins, dyes and quantum dots), allowing fluorescently tagged biological events to be three dimensionally visualized both independently and in combination with bioluminescently tagged events. To date, Caliper’s technology has been used predominantly to facilitate drug discovery and innovative biological research in areas including but not limited to oncology, infectious disease, gene therapy, stem cell biology, and transplantation. Caliper's Advanced Imaging Training Specialist, Brad Taylor will be providing an overview of basic and advanced imaging concepts as well as information on the proper use and function of the IVIS Spectrum hardware and the Living Image 4.0 software.

Here is the schedule for the talk:
10:00am – 10:45am: The New IVIS Spectrum: Bioluminescence and basic fluorescence acquisition (plus tips and tricks).
10:45am – 11:00am: Short coffee break (bring your own mug!)
11:00am – 12 noon: Advanced acquisition: DLIT, FLIT, Spectral Unmixing

February 1, 2010 - Scratching the Surface with TIRF - top

Kevin Conway, from Nikon, will be providing an overview of total internal reflection fluorescence microscopy (TIRFM), including some of its applications in cell biology as well as recent advances. An introduction to the Nikon TIRF system at AOMF will also be given.

Some reference material: December 7, 2009 - New Instruments for Automated Long Term Time Lapse Imaging - top

George Sakellaropoulos, from Olympus Canada will be talking about some of their new microscope in a box systems, including the FV10i and the Viva View, for automated time lapse imaging.
  • FV10i is the world's first self-contained Confocal Laser Scanning Microscope.
    The unique advantage of the FV10i is its self-contained design. We have completely re-engineered the design of the confocal laser scanning microscope into a self-contained package integrated with a variety of functions including an incubator and a laser combiner. You can install the FV10i easily in a laboratory without having to prepare a dedicated room. The FV10i also has unique features like a vibration isolation function, and light-tight cover, eliminating the need for a dark room. The FV10i has the same functionality of a high end confocal laser scanning microscope with easy-to-use software delivering it in a compact design.
  • Viva View Incubator Microscope
    The new VivaView FL incubator microscope from Olympus incorporates optimal cell growing conditions with proven imaging optics. Maintaining all the benefits associated with a dedicated incubator, the VivaView FL provides a fully integrated and motorized inverted microscope to allow high quality, long-term time-lapse imaging in a constant and optimized environment. Multiple locations in up to 8 samples can be imaged simultaneously with fluorescence or Differential Interference Contrast. Simple, intuitive computer operation removes all the difficulty previously associated with configuring a live cell imaging system. The VivaView FL is the next generation in live-cell imaging providing researchers the ability to image cells for significant longer than has previously been possible with absolute control of the surrounding environment.

    We've arranged for a demo of these instruments in the afternoon at the AOMF. If you are interested in participating in the demo, please contact Chantal Blanchard (chantal.blanchard@olympus.com or 416-476-3050).

November 2, 2009 - 3D and 4D Image Visualization and Analysis - top
    Cory Glowinski from Bitplane (www.bitplane.com), will be presenting their IMARIS software for 3D and 4D visualization and analysis. We currently have a license for IMARIS at the AOMF, so we thought this could be a great opportunity to spread the word! After the talk, Cory will be running a workshop over at PMH (7-319). For anyone interested in participating in the workshop, please contact Cory at cory@bitplane.com. If you can’t make it, but would still like to know more about IMARIS, you can always setup a time with one of the AOMF staff members.

May 12, 2009 - Multispectral Imaging - top
    Ruba Sarris from CRi will be talking about multispectral imaging using their Nuance system. As biological imaging becomes more complex with multiple labels used in a single sample, it becomes more difficult to accurately separate the signals from each other and from background autofluorescence. The Nuance system allows for imaging of multi-labeled samples and has powerful capabilities for separating out autofluorescence, which dramatically improves signal-to-noise and thus accuracy of quanitificaton.

April 6, 2009 - Stereology - top
    Stereology is defined as the science of estimating higher dimensional information from lower dimensional samples such as serial histological sections. It consists of a collection of methods quantifying 2D and 3D structures using estimation methods based on fundamental principles of geometry and statistics. We use the term "design-based stereology" to describe a subset of these methods whose probes and sampling strategies are "designed", i.e. defined a priori and independent of the size, shape and spatial orientation and distribution of the objects to be studied. A key component of unbiased estimation is the requirement for each object to be counted once and only once. The principle of systematic random sampling ensures just that. Correctly applied stereological methods are accurate, more efficient and reliable than other ad hoc quantitative analyses.

    1. Dockery P and Fraher J: The quantification of vascular beds: A stereological approach. Exp. Mol. Pathol. 2007; 82:110-120 (http://www.ncbi.nlm.nih.gov/pubmed/17320863)
    2. Schmitz C and Hof PR: Design-based stereology in neuroscience. Neurocience 2005; 130:813-831 (http://www.ncbi.nlm.nih.gov/pubmed/15652981)
    3. Howell K et al: Combined confocal microscopy and stereology: a highly efficient and unbiased approach to quantitative structural measurement in tissues. Exp. Physiol. 2002 87:747-756 (http://www.ncbi.nlm.nih.gov/pubmed/12530405)
    4. West MJ: Design-based stereological methods for counting neurons. Prog. Brain Res. 2002; 135:43-51 (http://www.ncbi.nlm.nih.gov/pubmed/12143362)
March 2, 2009 - In vivo Imaging on the IVIS system - top
    Bioluminescence imaging is commonly used to monitor tumor growth and treatment responses in vivo. When luciferin, a chemical found in bioluminescent organisms such as the firefly, is oxidized under the catalytic effects of luciferase and ATP, a bluish-green light is produced. This constitutes the bioluminescent signal. Because the reaction is also dependent on ATP, it allows one to determine the presence of energy or life. It seems that more and more users are interested in doing in vivo bioluminescence imaging experiments here in our facility using the IVIS system these days. And many of them have questions about animal preparation, how to optimize the imaging and analysis. So we thought it would be a good idea to bring all the users together to share what they are doing and tips for other users as well as to discuss any issues that they've encountered and possible solutions. There will be a brief introduction followed by a few examples from a couple of our current users and then a word from our sponsor, Caliper Life Sciences, who sells IVIS systems.

    1. Moriyama EH, Niedre MJ, Jarvi MT, Mocanu JD, Moriyama Y, Subarsky P, Li B, Lilge LD, Wilson BC. The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro. Photochem Photobiol Sci. 2008 Jun;7(6):675-80.(pdf)
    2. Eduardo H. Moriyama, Stuart K. Bisland, Lothar Lilge and Brian C. Wilson. Bioluminescence Imaging of the Response of Rat Gliosarcoma to ALA-PpIX–mediated Photodynamic Therapy. Photochemistry and Photobiology, 2004, 80: 242–249.(pdf)
    3. Akens, M. K., A. J. Yee, B. C. Wilson, S. Burch, C. L. Johnson, L. Lilge and S. K. Bisland (2007). "Photodynamic Therapy of Vertebral Metastases: Evaluating Tumor-to-Neural Tissue Uptake of BPD-MA and ALA-PpIX in a Murine Model of Metastatic Human Breast Carcinoma." Photochem Photobiol 83(5): 1034-9. (pdf)
    4. Rice B.W., et. al. In vivo imaging of light-emitting probes. Journal of Biomedical Optics 6(4), 432–440 (October 2001) (pdf)
    5. Determining Luciferin Kinetic Curve for Your Model. Provided by Caliper Life Sciences. pdf
February 2, 2009 - Advanced Microscopy Techniques - an overview - top
    James Jonkman will give a brief introduction and then John Dow from Agilent will present an overview of some advanced techniques such as FRAP, FRET, TIRF, and structured illumination.

June 2, 2008 - ImageJ/Colocalization - top
    One of the most useful aspects of immunocytochemistry is the ability to compare the location of two or more proteins tagged with different fluorophores between or within cells. The routine method for doing this is to stain (or pseudostain) one fluorescent image red, the other green and look for yellow in the superimposed image. While this is an invaluable screening tool there are major limitations that are often not considered. Further, the method is often applied without thought as to whether it will answer the critical question. Thus, if one is simply interested in the question 'are the two proteins are located in the same region of the cell or the same cell' this approach may be sufficient. However, if the question is 'are these two proteins parts of a common complex' this method can be woefully insufficient and even totally misleading. These concepts will be introduced and debated and some methods that have been devised that may improve on the simple dye-overlay will be discussed.

    1. Bolte, S. and Cordelieres, F. P. A guided tour of subcellular colocalization analysis in lilght microscopy. Journal of Microscopy, Vol. 224, Pt3 December 2006, pp.213-232. (pdf)
    2. Li, Q., Lau, A., Morris, T. J., Guo, L., Fordyce, C. B., and Stanley, E. F. (2004). A Syntaxin 1, Galphao, and N-Type Calcium Channel Complex at a Presynaptic Nerve Terminal: Analysis by Quantitative Immunocolocalization. J. Neurosci. 24, 4070-4081. (pdf)
May 5, 2008 - High Content Screening - top
    High Content Screening encompasses a range of techniques that enable similar experiments to be multiplexed to examine thousands of experimental conditions and allow the measurement of more than one single experimental value in an individual experiment. In cell biology experiments this takes the form of the phenotypic screening of culture conditions, chemical and RNAi libraries using cells in multi-well plates.
    High Content Microscopy is the application of automated (primarily) confocal and epi-fluorescence microscopy and image analysis in order to quantify and analyze the results of High Content Screening experiments. In this meeting we will discuss some applications of High Content Microscopy and image analysis and look at the advantages and limitations of this technique.

    1. Das AK, Sato M, Story MD, Peyton M, Graves R, Redpath S, Girard L, Gazdar AF, Shay JW, Minna JD, Nirodi CS. Non-small-cell lung cancers with kinase domain mutations in the epidermal growth factor receptor are sensitive to ionizing radiation. Cancer Res. 2006 Oct 1;66(19):9601-8.(pdf)
    2. Garippa RJ, Hoffman AF, Gradl G, Kirsch A. High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera. Methods Enzymol. 2006;414:99-120. (pdf)
    3. Kolas NK, Chapman JR , Nakada S, Ylanko Y, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science. 2007 Dec 7;318(5856):1637-40. Epub 2007 Nov 15. (pdf)
March 3, 2008 - Raman Microscopy - top
    Raman microscopy offers a unique combination of spatial resolution (~ 1um) and chemical/physical characterization. Although its spatial resolution is worse than that of electron microscopy, it enables measurements of a host of chemical and physical properties, including chemical composition, molecular orientation, conformation, crystallinity, strain, temperature and so on. In this meeting we will discuss some applications of Raman spectroscopy and microscopy for in vivo imaging. We will also look at the advantages and drawbacks of this technique.
    1. Evans C, Potma E, Puoris’haag M, Cote D, Lin C, Xie X (2005), “Chemical imaging of tissue in vivo with video-rate CARS”, PNAS November 15 pp. 16807-16812 (pdf)
    2. Uzunbajacava N, Lenferink A, Kraan Y, Willekens B, Vrensen G, Greve J, Otto C (2003) “Nonresonant Raman Imaging of Protein Distribution in Single Human Cells”, Biopolymers (Biospectroscopy) v.72 pp.1-9(pdf)
    3. Cheng J, Jia Y, Zheng G, Xie X (2002), “Laser scanning CARS Microscopy and applications to Cell Biology” Biophysical Journal v.83 pp 502-509(pdf)
February 4, 2008 - Two-Photon Microscopy - top
    There are some clear applications of two-photon microscopy, like imaging thick samples, but there are also many misconceptions such as that two-photon is better for live cell imaging or that it gives higher resolution than confocal. In this meeting, we will discuss some real applications of two-photon microscopy and debunk some of these myths.

    Advantages of two-photon microscopy:
    • Good for imaging thick samples (tissues, embryos, scaffolds, etc – plated cells do not constitute thick samples!). Because it uses longer wavelengths, there is less scattering of light in the sample, allowing for deeper penetration into thick samples and better depth resolution.
    • Excitation occurs mainly in the focal plane, so there is a reduction of photobleaching and phototoxicity above and below the focal plane
    • Absorption spectra is broader so simultaneous excitation of multiple fluorophores is possible
    • Useful in applications such as uncaging, PDT, and fluorescence cross correlation spectroscopy (FCS), because the signal is confined to focal plane/volume
    Disadvantages of two-photon microscopy:
    • Lateral resolution is reduced, because it depends on wavelength (the longer the wavelength, the worse the resolution).
    • Within the focal plane, photobleaching may actually be worse, because the pulsed laser yields high peak powers (can be close to 1MW)
    • Need detectors close to objectives to collect the most signal, otherwise some signal will be lost in going through the optical path.
    • Absorption spectra are broader, so selective excitation becomes difficult
    • Specialized applications may require rebuilding of microscope setup for optimization
    • Fluorophore excitation sometimes is not exactly a factor of 2
    • More expensive
    • Denk, W., J. H. Strickler and W. W. Webb (1990). "Two-photon laser scanning fluorescence microscopy." Science 248(4951): 73-6 (pdf).
    • Helmchen, F. and W. Denk (2005). "Deep tissue two-photon microscopy." Nat Methods 2(12): 932-40 (pdf).
    • Zipfel, W. R., R. M. Williams and W. W. Webb (2003). "Nonlinear magic: multiphoton microscopy in the biosciences." Nat Biotechnol 21(11): 1369-77 (pdf).
December 3, 2007 - FLIM (Fluorescence Lifetime Imaging Microscopy) - top
    Mike Woodside from the Imaging Facility at The Hospital for Sick Children will be talking about time correlated single photon counting and how it can be used for FLIM and how FLIM can be used to get FRET measurements. He will cover the types of equipment needed for these types of experiments and some of the problems encountered.
    Check out the following references for some background information on the topic:
November 5, 2007 - Beyond cellgazing: astronomy meets cell biology in advanced noise filtering and image restoration - top
October 1, 2007 - Fast Confocal Imaging - top
  • In this meeting, Farid Jalali from Robert Bristow's lab will be presenting fast confocal imaging using Spinning Disk Confocal Microscopy. Confocal microscopy provides great resolution, but imaging tends to be too slow for most live cell imaging experiments and photobleaching is a concern. A good compromise to this is disk scanning confocal microscopy. This imaging modality uses a disk with multiple pinholes to achieve acceptable resolution, fast imaging speeds, with less photobleaching, without the need of lasers and at a moderate cost.
    Check out the following reference for more of a background on the topic:
    • Chapter 10 (pdf) from the Handbook of Confocal Microscopy (Third Edition) by James B. Pawley.
September 10, 2007 - Live Cell Imaging - top
  • James' introduction:
    • need to know biology
    • sample preparation
    • microscope optimization (what microscope will you use)
    • other hardware (eg. Incubators, such as the Chamlide TC)
    • software
  • Noah Fine's presentation:
    • need to keep cells at ~37 degrees Celsius
    • pH most be kept between 7.3-7.4 (5% CO2 or add 10-20mM HEPES buffer)
    • if using a water lens, there’s the problem of the water evaporating over time and then the focus will change
    • focus can drift over long periods of imaging, need to correct for this
    • if imaging over short term (30min-1hr), usually worries
    • if imaging for longer (several hours), need to use HEPES buffer plus stage heater or return plates to incubator
    • if imaging over long term, need a full incubator (CO2 chamber/humidifier/stage heater)
  • Discussion: - there are certain fluorescent dyes that have been successful for use in long live cell experiments (up to 48 hours), some suggestions were: DRAQ5 and cFNA?
  • Presentation by Christ Cathcart from Nikon Canada
    • Biostation IM/CT – provides a controlled environment, essentially a microscope in a box
    • LiveScan – high speed swept field spinning disk confocal (sweeps pinhole array across), up to 1000 frames/second
    • C1Si Spectral confocl
    • TE2000E Perfect focus system – integrated system which provides continuous monitoring of focus 20 times/second
June 4, 2007 - Automated image analysis using macros - top
  • Even the simplest macro can prove to be very powerful for image analysis. They save you tremendous amounts time. And although, they may sound complicated, in fact, writing a macro can be much simpler than you think. In this meeting, we will have a few of our users tell us about how they used a macro to help simplify and automate the steps involved in analyzing their images. We will also demonstrate how to create a simple macro.
May 7, 2007 - Intravital Microscopy - top
  • There’s nothing quite like imaging living cells in their natural environment - the body of an animal. Trevor McKee is one of the few users who have begun doing intravital microscopy (microscopy in live animals) here in the AOMF. Although the definitions are often interchanged, I would distinguish the term “intravital microscopy” from “in vivo imaging” by their resolutions: “intravital microscopy” is microscopic imaging in live animals, giving cellular detail and often including timelapse imaging to follow dynamic processes; while “in vivo imaging” generally refers to whole-body imaging of mice or rats, producing low-resolution macroscopic images often of clusters of cells (such as a tumour).Trevor will show us some of his recent intravital imaging results from our Two-Photon microscope, and describe some of the challenges he’s faced in the process. (written by James Jonkman)

    Introduction/Summary (from Trevor McKee):
  • In this O-MUG session I will talk about some principles behind intravital microscopy, and then some of the work I have ongoing as part of my research program in this area. As this topic covers a wide array of techniques, I will do my best to summarize these techniques and the various advantages and disadvantages associated, along with a discussion of which techniques are best for which application. The focus will be on microscopic methods, although some intravital imaging applications will also be covered, however we have had a number of recent talks on intravital imaging, so I will try not to re-cover too much ground.
    Intravital microscopy involves the application of microscopic methods and analytic techniques to the study of cells and tissues within living animals. There are a range of preparations that can be acheived using a variety of techniques - from relatively non-invasive to more invasive surgical preparations. Both acute, as well as chronic methods can be utilized to monitor physiological processes within mice over varying periods of time. As an example, I will show recent data I have produced monitoring tumor associated fibroblast remodeling of fibrillar collagen within tumors growing in mice using the dorsal skinfold chamber preparation. Other examples of cellular processes, methods to monitor drug delivery and penetration within tumors growing in vivo, and the formation and remodeling of collagen will be presented. Finally, I will discuss analytical methods that can be used to assist in both data acquisition and in the quantitation and interpretation of images post-acquisition, in order to extract meaningful results.

    Check out this website on intravital imaging: http://www.aecom.yu.edu/aif/intravital_imaging/introduction.htm
    Also check out these reference papers:
    Rakesh K. Jain, Lance L. Munn, Dai Fukumura. Dissecting tumor pathophysiology using intravital microscopy. (pdf) (Nature Reviews Cancer 2, 266 - 276, 2002)
    John Condeelis and Jeffrey E. Segall.Intravital Imaging of Cell Movement in Tumours (pdf)
    Ronald N. Germain, Mark J. Miller, Michael L. Dustin and Michael C. Nussenzweig. Dynamic Imaging of the Immune System: Progress, pitfalls and promise (pdf)
    Thomas Misgeld and Martin Kerschensteiner.Invivo Imaging of the Diseased Nervous System (pdf)
  • O-MUG survey - click here to see results from the survey. If you didn't take part in the survey, you can download it here and drop it off or email it to us. We appreciate your opinions.
April 2, 2007 - Clinical and educational applications of brightfield whole slide scanning - top
  • Whole slide scanning is changing the way people do microscopy these days. With the current advances in automated microscopes, computer power, and software, scanning is becoming faster and easier than ever before. So people don’t have to settle for small fields of view anymore, they can digitize entire slides in a matter of minutes, analyze and even share them over the internet with others. Slide scanning is a powerful tool in both research and clinical settings.
    Doug Tkachuk is a pathologist here at PMH and he has been instrumental in introducing the technology to us, which culminated in the purchase of our Aperio scanner. He has also started a company called Objective Pathology, which provides digital pathology imaging services to individuals or institutions. In his talk, he will speak about clinical and educational applications of slide scanning, which will also raise discussion of applications in research. Presentation by Doug Tkachuk:
    Brightfield imaging is of great importance in cancer diagnosis. Whole slide brightfield scanning combined with fluorescent biomarker data will make routine testing easier as well as easing the data flow between hospitals and research institutions.
    Word from sponsor:
    George Sakellaropoulos and Chantal Blanchard from Olympus Canada presented the new Olympus Nanzoomer whole slide scanner.
March 5, 2007 - Deconvolution - top
  • There are various phenomena responsible for image degradation in microscopy. One such phenomenon is blurring, which is caused by the spreading of light as it passes through microscope optics. Since blurring is a non random effect, it can be predicted with various optics models and if you can predict the effect, you may be able to reverse it to a certain extent. This is the basis of deconvolution. Deconvolution is a computational technique used to reduce blurring and haze in 3D fluorescence images. There are many different algorithms that are used to do this and they are discussed in the review paper by Wallace et al. cited below. With our computing power these days, running a deconvolution operation is easy and it's a reasonable way to sharpen up your images. James Pawley, author of the "Handbook of Biological Confocal Microscopy", recommends that you should always deconvolve your images whether their acquired in widefield or confocal mode. Here in the AOMF, we now have access to a spinning disk confocal microscope with deconvolution software so it would be great to learn more about deconvolution and try it out using this system.

    Check out these references for more details:
    • Wes Wallace, Lutz H. Schaefer, and Jason R. Swedlow."A Workingperson's Guide to Deconvolution in Light Microscopy." BioTechniques Vol.31: 1076-1097 (November 2001) (pdf).
    • Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. (Chapters 23, 24, and 25 pp. 453-500).
  • Summary of presentation by Robin Battye
    • Convolution in microscope – The very process of using a lens to resolve a specimen, leads to diffraction and creates blur/haze in images. The amount of distortion can be described by the PSF (Point Spread Function) of the system
    • Solutions – Some solutions for reducing this blur/haze include: using confocal, deconvolution, or structured illumination (grid confocal)
    • Deconvolution - Calculate or measure the PSF and with the known image, you can estimate what the object was.
      • Classifications
        • No neighbour/nearest neighbour – filter like an unsharp mask, resulting data is not quantifiable
        • Inverse filter/Wiener filtering – doesn’t work too well with noisy data
        • Constrained Iterative – most common method, which leads to quantifiable data
      • Limitations: The technique breaks down with really thick samples because noise increases and you must also be aware of artifacts that may arise due to the computation process.
  • Take home message: Deconvolution can be a very powerful technique to sharpen images and in principal you should always deconvolve, but it’s not always practical since the best algorithms are still very expensive. It is important that you know what the biological question that you are trying to solve is so that you can decide if the results from the deconvolved data are valid, because artifacts may arise.
February 5, 2007 - FRET Microscopy - top
  • FRET(Fluorescence Resonance Energy Transfer) microscopy is a technique for measuring the interaction between molecules in a cell.
    Check out these review papers:
    • Irina Majoul, Yiwei Jia, and Rainer Duden. "Practical fluorescence resonance energy transfer or molecular nanobioscopy of living cells." Handbook of Biological Confocal Microscopy, Third Edition 45:788-808.
    • Ammasi Periasamy. "Fluorescence resonance energy transfer microscopy: a mini review." Journal Of Biomedical Optics 6(3): 287-291 (July 2001).
    • Peter van Roessel and Andrea H. Brand. "Imaging into the future: visualizing gene expression and protein interations with fluorescent proteins." Nature Cell Biology Vol.4: E15-E20 (January 2002).
    • Fred S. Wouters, Peter J. Verveer and Philippe I.H. Bastiaens."Imaging biochemistry inside cells." TRENDS in Cell Biology Vol.1 No.5: 203-211 (May 2001).
    • Elizabeth A Jares-Erijman and Thomas M Jovin."FRET imaging." Nature Biotechnology Vol.21 No.11: 1387-1395 (November 2003).
    • Zongping Xia and Yuechueng Liu."Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microsopes." Biophysical Journal Vol.81: 2395-2402 (October 2001).
  • Presentation by Sarang Kulkarni - "FRET ... or ... Don't FRET"
    “WM Keck Center for Imaging” website for FRET (www.cci.virginia.edu)
    Molecular Imaging: FRET Microscopy and Spectroscopy. Oxford University Press. Periasamy and Day, 2005. Chapter 7.
    Mochizuki et al., 2001, Nature411:1065. (Raichu probes for intramolecular FRET)
    Shyu, Y., Liu, H., Deng, X., and Hu, C.-D. Identification of new fluorescent fragments for BiFC analysis under physiological conditions. BioTechniques, 40:61-66 (2006). (Original report and detailed experimental procedures of the improved BiFC assay)
    Hu, C-D. and Kerppola TK. Direct visualization of protein interactions in living cells using bimolecular fluorescence complementation analysis. Protein-Protein Interactions (ed. P. Adams and E. Golemis), Cold Spring Harbor Laboratory Press, 2005. (Detailed design and experimental procedures of multicolor BiFC assays)
    Hu, C.-D. and Kerppola, T. Simultaneous visualization of interactions between multiple proteins in living cells using multicolor bimolecular fluorescence complementation analysis. Nat. Biotechnol. 21, 539-545 (2003). (Original report of multicolor BiFC assay and its applications)
  • Word from our sponsor: Olympus Canada. There will be a demo of Hamamatsu's Nanozoomer digital pathology system on February 12th and 13th. If you're interested in attending, please contact Chantal Blanchard.
  • Discussion about issues with FRET (including: BiFC, different methods for measuring protein-protein interactions)
December 4, 2006 - top
  • Presentation by James Jonkman on Frontiers in Microscopy
    • Introduction to O-MUG
    • Discussion of possible future topics (some ideas included: FRET/FLIM, probe development, Raman spectroscopy, hyperspectral imaging, sample preparation, deconvolution, LCM, analysis)
    • Extended Resolution Microscopy
      • Structured Illumination
        Gustafsson, M. G. L. (2005). "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution." PNAS 102(37): 13081-13086.
        Hell, S. W. (2003). "Toward fluorescence nanoscopy." Nature Biotechnology 21(11): 1347-1355.)
        Gustafsson, M. G. L. (2000). "Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy." Journal of Microscopy-Oxford 198: 82-87.
        Gustafsson, M. G. L. (1999). "Extended resolution fluorescence microscopy." Current Opinion in Structural Biology 9(5): 627-634.
      • 4Pi Microscopy
        Bewersdorf, J., B. T. Bennett and K. L. Knight (2006). "H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy." Proc Natl Acad Sci U S A 103(48): 18137-42.
        Medda, R., S. Jakobs, S. W. Hell and J. Bewersdorf (2006). "4Pi microscopy of quantum dot-labeled cellular structures." J Struct Biol 156(3): 517-23.
        Bewersdorf, J., R. Schmidt and S. W. Hell (2006). "Comparison of I5M and 4Pi-microscopy." J Microsc 222(Pt 2): 105-17.
        Gugel, H., J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz and S. W. Hell (2004). "Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy." Biophys J 87(6): 4146-52.
  • Word from our sponsor:Biomedical Photometrics Inc. (BPI)
    We will be demoing their TISSUEscope instrument until the end of December.

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