Cell Analysis Resource Center
ThermoFisher Scientific – Resource for basic principles about cell analysis, applications, and techniques.
Fluorescent Probe Selection
Please review our equipment page to determine which fluorescent probes are compatible with the laser lines on our systems.
You may use the following spectral tools below, to help you with your selection. If you need further assistance, please contact Susan, email@example.com, 860-679-4686.
- ThermoFisher Scientific Fluorescence SpecrtraViewer
- BioLegend Fluorescence Spectra Analyzer
- FPbase Spectra Viewer
- AAT Bioquest Fluorescence Spectrum Viewer
- abcam Fluorochrome Chart
Cell Culture Media Considerations
Prokaryotic and eukaryotic naturally exhibit autofluorescence due to the cellular components and metabolites, such as flavins, NADH, lipofuscins, collagen, and elastin. Culture media can also interfere with fluorescent imaging due to phenol red and additional metabolites. Please review the following article as well as the two resources for media designed to aid with fluorescent imaging.
Bogdanov, A., Bogdanova, E., Chudakov, D. et al. Cell culture medium affects GFP photostability: a solution. Nat Methods 6, 859–860 (2009). https://doi.org/10.1038/nmeth1209-859
ThermoFisher Scientific – FluoroBrite DMEM Media
Millipore Sigma – BrightCell™ Photostable Cell Culture Media
Please note, the 780 and 880 Zeiss confocals are on inverted microscopes. Because of this, CCAM requires your samples to be sealed when mounted between a slide and cover glass. This will not only protect your sample but it will also protect our objectives and microscopes.
Widefield microscope sections typically range from 4-10 microns. Confocal sections often range from 10-40 microns.
Please use a #1.5, or 170-micron thick, glass coverslip. All the Zeiss objectives are corrected to be used with this material and thickness.
There are a variety of protocols to maintain cellular structures when preparing fluorescent samples. Please refer to this white paper for suggestions for preparing such samples.
Most commonly nail polish is used to seal slides, but it is toxic to live cells. Please refer to Coverslip Sealing for a variety of options.
CoverGrip is designed with ingredients that do not leach into aqueous mounting medium and affect specimen fluorescence.
Mounting media should have the same refractive index or be very close to Zeiss’ immersion oil which has a RI of 1.518.
It is highly recommended to use a mounting media that contains an anti-fade agent such as n-propyl gallate.
The type of mounting media you use will depend on which probes you are trying to visualize. Glycerol based mounts, ProLong Diamond, are better for preserving structural information while mounts that harden can disrupt cellular structure. You can seal these types of media before they harden and they will work equally as well and maintain the structures.
Mounting media can affect probes differently, so please make educated choices based on which probes you will be using. For example, Vectashield is not compatible with Alexa 647 and other far red dyes but works well with blue/green dyes. Likewise dated bottles of glycerol can result in unexpected staining results such as the nucleus appearing as background, as pictured below.
Jonkman, J., Brown, C.M., Wright, G.D. et al. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc 15, 1585–1611 (2020). https://doi.org/10.1038/s41596-020-0313-9
Homemade Mounting Media
Jackson ImmunoResearch Anti-fade Mounting Medium
Do not use mounting media that contains DAPI since premixed mounting solutions with DAPI tend to increase background fluorescence. It is best to first stain with DAPI and then mount your sample separately.
Live Cell Imaging Supplies
Cellvis – Glass Bottom Dishes
Fisher Scientific – Thermo Scientific Nunc Glass Bottom Dishes and Thermo Scientific Nunc Lab-Tek and Lab Tek II Chamber Slides
Ibidi – Chambered Coverslips
Mattek – Glass Bottom Dishes
Nunc Lab-Tek II Chambered Coverglass
Wilco – Glass Bottom Dishes
ThermoFisher Scientific – 5 Steps to Fixed-Cell Imaging