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Sample preparation

• Make sure that the used antibodies are specific and do not cross-react.

• Choose fluorophores with well-separated excitation and emission spectra.

• Use the same mounting medium for the samples you will be comparing.

• Use cover glass of the same thickness for the samples you will be comparing.

• Try to avoid using anti-fading reagents, as they can increase background fluorescence.

• Use unstained control samples to check for autofluorescence.

Microscope set-up

• Use plan apochromatic lenses to reduce chromatic shift.

• Use optimized emission filters to maximize emission collection while avoiding bleedthrough.

• Use the same objective lens for the samples you will be comparing.

• Make sure the size of the microscope pinhole is properly set.

Image acquisition and handling

• Acquire images sequentially to minimize bleedthrough.

• Acquire images at maximum resolution to ensure reliability of analysis.

• Do not acquire too bright and too contrast images, as it will result in their saturation.

• Do not resave files in any other format than lossless TIFF. It may cause loss of the original image data needed to perform calculations.

• Do not manipulate images in graphics-editing programs, as it may ruin their suitability for quantification.

• By all means avoid using files in JPEG format.

• Always keep the original files. They may be needed to refer to the original software with which they were acquired to confirm metadata.

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