Microscopy and Image Processing Resources
- Training Tutorials
- Opening Your Microscope Images
- Microscope objective and filter specification sheets
- Spectra and properties of fluorescent molecules
- Image processing and analysis software
- Data Management Tips
- Online microscopy references and tutorials
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 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.
Microscope Objective and Filter Specification Sheets
These sheets also list pixels sizes, which are necessary for creating scale bars and performing spatial measurements.
- Leica DM6b
- Nikon Eclipse
- Nikon TE2000 (widefield and confocal)
- Zeiss Axioskop
- Zeiss Axioscope.A1
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 Software section, below, to install)
- 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.
- Next steps with FIJI/ImageJ
- Excellent and highly recommended Introduction to BioImage Analysis (particularly with FIJI/ImageJ) by Peter Bankhead.
- Check-out the FIJI for Beginners Workshop from the University of Melbourne Advanced Microscopy Facility
- FIJI/ImageJ Reference and More Advance Topics
- Comprehensive FIJI/ImageJ reference at imagej.net
- Workshops for Quantification and Macro writing from the University of Melbourne Advanced Microscopy Facility
- Even more!
- @haesleinhuepf webinars on BioImage Analysis (mostly FIJI/ImageJ, but also CellProfiler, Python, Icy...)
- CellProfiler - program for interactively creating image analysis pipelines
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)
- Imaging methods are vastly underreported in biomedical research (Marques, Pengo, Sanders)
Microscopy data management resources
- Dr. Kubow's data management tips for students
- Dr. Kubow's tips for Data Management Plans involving microscopy data
- General data management tips from the JMU Library
- Even more tips from the Stanford Library
Online microscopy references and tutorials
- 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
- Microcourses YouTube channel
- iBiology microscopy lectures
- Microscopy educational websites
- Nikon’s MicroscopyU
- Olympus’s Microscopy Resource Center
- Zeiss Campus
- Excellent simulations from the PhET project at the University of Colorado Boulder