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 and 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