Fluorescent Probe Guide
Don't know which probes to use for your experiment at the CMCI? Let this guide help you! The good people at the Harvard Medical School Imaging Centre have developed fpbase.org, a website that allows you to input the specifications of your microscope, and see which fluorescent probes you can best use with it. Below are links to each of our microscopes on FPBase.
To use these links, click on the microscope that you are interested in using, and you will be taken to the FPBase.org website. Then select the Fluorophore you want to visualise with the microscope (e.g. EGFP, Alexa488, FM 4-64), and select a configuration (e.g. "Green", "GFP", "Far Red"). The website will account for the excitation level of the fluorescent protein by the selected configuration, and the amount of emitted signal captured by the system to give you a "brightness" value. Brightness levels close to 40 are very high, close to 2 are negligible. Note that some fluorescent proteins (e.g. mNeonGreen) are extremely bright, and so will give much higher brightness values than dimmer proteins (e.g. mCherry). A fluorescent protein repository is coming soon to the CMCI, so we may be able to provide you with plasmids encoding the current generation of fluorescent proteins, which are generally the brightest. Synthetic dyes do not provide a Brightness value, but they are generally significantly brighter than fluorescent proteins - if a significant proportion of their emission is detectable and they are undergoing a significant excitation in a configuration then they will likely be visible in that configuration - you can use this to check for bleedthrough!