Methods

Users are encouraged to consult with Core staff in all microscopy methods.

Users are expected to be familiar with basic immunostaining and fixation techniques and slide preparation. A common method to prepare samples is available here.

The Fluorescent Protein database has several useful tools for choosing the right fluorophore for your experiment. It includes a spectra viewer, FRET efficiency calculators, and microscope fluorophore efficiency reports. 

Fluorophore DOs & DON'Ts

The following are general guidelines for new users. There are exceptions, caveats, and cases where each of these points DO NOT APPLY! Please consult with us if you are unsure.

  • DO use an antifade mounting media

    Why: Once excited, fluorophores can oxidize rather quickly. This also generates reactive oxygen species that destroy nearby fluorophores. Antifading agents let you image much, much longer.

    Caveat: Some microscopy methods like TIRF limit photobleaching. There are also live-cell antifading agents ike Trolox.

  • DO NOT use antifade/mounting media with built-in DAPI. Buy mountant without DAPI and do a separate DAPI stain and wash.

    Why: Antifade with built-in DAPI is meant for high-volume clinics. We recommend against using it for research. The DAPI goes bad very quickly, diffuses into tissues poorly, and always leaves a background signal. Instead, do a separate DAPI stain and wash out the excess before mounting. Why spend hours, days, or months on an experiment and ruin it at the last minute with a poor reagent when a 5-15 minute DAPI stain will work much, much better!

  • DO NOT use the following flow-cytometry dyes

    Why: Detection (flow) and imaging (microscopy) have different requirements. Some flow dyes and reagents do not work for imaging – especially with multi-band filters. Choose alternatives to these broad-spectrum and other problematic dyes

    • ❌❌ PE (Phycoerythrin) - Why: very broad spectrum
    • ❌❌ PerCP - Why: very broad spectrum
    • ❌ APC (Allophycocyanin) - Why: broad spectrum, Cy5 or AF647 are preferred
    • ❌❌ Quantum Dots - Why: they blink on/off. Caveat: They are appropriate for some experiments
    • ❌ Brilliant UV/Violet dyes other than BV421 - Why: most scopes are not setup for FRET dyes. Caveat: Spectral scanning scopes like the SP8 may work with these FRET dyes. Contact us for more information

  • DO NOT use primary labeled antibodies in the green channel (FITC, AF488, etc)

    Why: Tissue autofluorescence is strongest in the green channel. It can overwhelm weak primary-labeled antibody signals when imaging. Instead use a polyclonal fluorescent secondary antibody to amplify the signal. There is a similar but weaker autofluorescence in the orange channels (TRITC, AF548). Consider the no-labeled-primary rule for this color too. Remember, flow cytometry and microscopy are different beasts! What works for one doesn't always work for the other.

    Caveat: There are commercial autofluorescence quenching solutions that substantially reduce background and let you use these weak primary-labeled antibodies.

Additional methodology resources

Organizations

General protocols for microscopy

Microscopes

Lasers

Open Source Image processing

Fluorescent probes and antibodies

Fluorescence spectra viewer

Optical filters

Imaging chambers