- Using Our Microscopes
- Opening Your Microscope Images
- Spectra and properties of fluorescent molecules
- Image processing and analysis software
- Microscopy data management and publishing
- Online microscopy references and tutorials
Using Our Microscopes
- Training tutorials
- "How to" videos and cheat sheets
- Instrument Technical Specifications (including objectives, resolution, pixel sizes, and fluorescence filters)
Opening Your Microscope Images
Unless you are collecting basic "screen-shot" quality images (jpgs), you will not be able to open your images using the basic software included in Windows and Mac OS. You have a few options:
- You just want to see your images, adjust the brightness, export them as JPGs for a presentation, etc.
- If you acquired them on one of the Nikon scopes, you can download a free viewing-only version of the Nikon Elements software.
- If you acquired them on one of the Leica scopes, you can download a free viewing-only version of the Leica LAS software (for Windows only).
- If you want to do image processing/analysis or the above options don't work for you, I recommend using ImageJ. The rest of this section explains how to use ImageJ to open images and later sections provide resources for learning how to use this powerful program for image processing and analysis.
ImageJ is a free, open-source image processing and analysis software and will work on nearly any operating system. You need to install the FIJI version of ImageJ, which comes pre-configured with lots of useful plugins. To install FIJI, click on the correct link for your operating system and save the file anywhere convenient (except in your Program Files folder). Uncompress the downloaded file and you're ready to go! Just double-click on the fiji.app file. If you already have ImageJ installed, you can install the Bio-Formats Package of plugins from the Open Microscopy Environment project to allow ImageJ to open most major proprietary image formats. Before you start using ImageJ, take a quick look through this webpage to orient yourself to the ImageJ program.
- Most image files can be opened by simply dragging and dropping the file onto the ImageJ toolbar. Alternatively, go to Plugins > Bio-Formats > Bio-Formats Importer and select the file to open.
- The Bio-Formats Import Options menu will display. There are many useful options in this menu, but unless you know what they do, I recommend using these basic options:
- View stack with: Hyperstack
- The Autoscale box should be checked
- No other boxes should be checked
- Some file formats may require additional steps...
- Leica (.lif) Project files: After you hit OK in the previous menu, the Bio-Formats Series Options window will display. Select the images you want to open and click OK. If a Console window also opens and lists warnings, ignore them.
- If you have a true-color image (NOT fluorescence) and it displays as three separate red, green, and blue images overlayed in a single window with a slider at the bottom, go to the Image menu, select Type, then RGB Color. Close the old image and work with the new one.
- Want to know more about how to work with your images in ImageJ? See the information, below, in Image Processing and Analysis.
Spectra and properties of fluorescent molecules and proteins
- FPBase Spectra Viewer
- FPBase Fluorescent Protein Database (compare properties, look at "evolutionary"/development lineages, ...)
- Nyquist Calculator: online calculator to estimate the optical slice thickness and optimal step size for your instrument; will also calculate the point spread function (PSF) for your microscope.
Image processing and analysis
Guides
- Beginners - start here!
- I recommend starting with FIJI/ImageJ (see the Opening Your Images section for installation instructions)
- This website gives a very brief introduction to the program
- My Basic Image Processing with ImageJ document gives some quick steps for how to do the most basic tasks
- Basic image analysis overview in the online Bioimaging Guide
- Basic overview of image analysis concepts (video) by Anne Carpenter
- Next steps with FIJI/ImageJ
- Excellent and highly recommended Introduction to BioImage Analysis (particularly with FIJI/ImageJ) by Peter Bankhead. Includes tutorials and worked examples in ImageJ and Python.
- Check-out the FIJI for Beginners Workshop from the University of Melbourne Advanced Microscopy Facility
- Comprehensive FIJI/ImageJ reference at imagej.net
- Workshops for Quantification and Macro writing (vidoes and tutorials) from the University of Melbourne Advanced Microscopy Facility
- More Analysis Tools and Resources
- @haesleinhuepf webinars on BioImage Analysis (mostly FIJI/ImageJ, but also CellProfiler, Python, Icy...)
- CellProfiler - program for interactively creating image analysis pipelines
- Ilastik - segmentation using machine learning, but without need for programming. Also see intro video by @haseleinhuepf.
Ethics (yes, it's something you must think about when you do any kind of image processing/analysis)
- Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images (D.W. Cromey)
- Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. (A.J. North)
Microscopy data management and publishing
- Data Management
- Dr. Kubow's data management tips for students
- Dr. Kubow's online tool for generating a metadata checklist
- General data management tips from the JMU Library
- Publishing Guidelines
- Publication checklist (see Fig. 2 for how to present your images; see Fig. 8 for how to report your image analyses) by QUAREP-LiMi
- Imaging methods are vastly underreported in biomedical research (Marques, Pengo, Sanders)
- Comparison table of different repositories for microscopy data by Beth Cimini
Online microscopy references and tutorials
- Online Bioimaging Guide: a great place to get started thinking and learning about all aspects of your experiment from planning and sample preparation to imaging and analysis.
- The Molecular Probes Handbook: An excellent, online, searchable reference for fluorescent probes for microscopy
- Excellent general textbook on light microscopy (available online if you're at JMU): Murphy, Douglas B. and Davidson, Michael W. (2012) Fundamentals of light microscopy and electronic imaging. Wiley-Blackwell (Library Call # QH205.2 .M87 2012)
- Microscopy instructional/educational videos
- Global BioImaging Training Platform
- Microcourses YouTube channel
- iBiology microscopy lectures
- Other microscopy educational websites
- Nikon’s MicroscopyU
- Olympus’s Microscopy Resource Center
- Zeiss Campus