O-MUG Introduction/Summaries

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)

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

Susan Newbigging (Director of Pathology at CMHD)

Abstract:

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)

Abstract:

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)

Abstract:

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)

References:
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.

References:
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:
Mueller-Klieser1993b.pdf
Mueller-Klieser2010.pdf

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

http://www.aperio.com/pathology-services/scanscope-FL-system-slides.asp

http://www.aperio.com/newsevents/press-release-090909-fluorescence-slide-imaging-biomarker.asp

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

www.bitplane.com

May 12, 2009 - Multispectral Imaging - top

April 6, 2009 - Stereology - top

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)
Schmitz C and Hof PR: Design-based stereology in neuroscience. Neurocience 2005; 130:813-831 (http://www.ncbi.nlm.nih.gov/pubmed/15652981)
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)
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

in vivo

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)
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)
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)
Rice B.W., et. al. In vivo imaging of light-emitting probes. Journal of Biomedical Optics 6(4), 432–440 (October 2001) (pdf)
Determining Luciferin Kinetic Curve for Your Model. Provided by Caliper Life Sciences. pdf

February 2, 2009 - Advanced Microscopy Techniques - an overview - top

June 2, 2008 - ImageJ/Colocalization - top

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)
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

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)
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)
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

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)
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)
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

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

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

Imaging Facility at The Hospital for Sick Children

Becker-Hickl

www.stke.org/cgi/content/full/sigtrans;2006/331/re2

November 5, 2007 - Beyond cellgazing: astronomy meets cell biology in advanced noise filtering and image restoration - top

WCIF (Wright Cell Imaging Facility)

http://micro.magnet.fsu.edu/primer/digitalimaging/russ/reducingnoise.html

http://micro.magnet.fsu.edu/primer/digitalimaging/concepts/ccdsnr.html

pdf

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

In this meeting, James Jonkman, along with a panel of users with related experience, will be discussing live cell imaging tips and techniques. Check out these references:
- http://micro.magnet.fsu.edu/primer/techniques/livecellimaging/index.html
- http://www.sciencemag.org/cgi/content/full/300/5616/82
- David J. Stephens and Victoria J. Allan. Light Microscopy Techniques for Live Cell Imaging. Science 300 (5616): 82-86.

Summary:

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.