In widefield microscopy the excitation wavelengths which illuminate the sample, and the emission wavelengths which reach the CCD camera are selected throughout a filter cube. A filter cube consists of three elements: an excitation filter, an emission (or barrier) filter, and a dichromatic beamsplitter (or dichroic mirror). The filters and the mirror are designed such to avoid overlaps between their spectral profiles. It is particularly important to avoid crosstalk between the excitation filter and the dichroic mirror. In this case some excitation light could reach the sensor reducing the contrast of the resulting image. Residual leakage through filters is also termed bleed-through.
More diffusely, bleed-through refers to the partial overlapping of the excitation and/or emission spectra of two fluorophores, in multi-channels acquisitions. We distinguish three cases: cross-emission cross-excitation cross-emission AND cross-excitation Bleed-through artefacts complicate the interpretation of the acquired image and particularly can lead to false results in case of colocalization analysis and fluorescence quantification. In widefield microscopy bleed-through is intrinsically minimized, as the different channels images are usually acquired sequentially (in contrast with confocal microscopy), even if simultaneous acquisition is also possible using triple filters. In any case a careful design of your experiment in terms of colours separation is important to get high quality images. The case in which both excitation and emission spectra of two fluorophores overlap remains critical.
Cross-emission The emission spectra of two fluorophores overlap. Cross-emission tipically happens toward the longer wavelength, as emission spectra have often long tiles toward the right. DAPI ex DAPI em Alexa488 ex Alexa488 em Λ (nm) Suggestion: to reduce cross-emission in multichannels acquisition, the reddest dyes should be imaged first Note: concerning crosstalk, DRAQ5 can be a good alternative to DAPI
Cross-excitation The emission spectrum of the bluest fluorophore overlaps with the excitation spectrum of the reddest fluorophore. FITC em Texas Red ex FITC ex Texas Red em Λ (nm)
Cross-excitation and cross-emission The emission spectrum of the bluest fluorophore overlaps with the excitation spectrum of the reddest fluorophore. Moreover the emission spectra of the two fluorophores partially overlap. Alexa488 em Cy3 em Alexa488 ex Cy3 ex Λ (nm) Suggestion: to reduce recording of reddest fluorophore emission in the bluest channel, use narrow band pass filter to isolate the bluest fluorophore emission.
Strategies for bleed-through minimization and correct results interpretation Experimental design: choose carefully a correct combination of fluorophores and filters (if possible, prefer band-pass filters to long-pass filter). Concerning the choice of the fluorescent dyes, other factors are however to be considered, as target specificity and quantum yield. Note also that the more the emission spectra are separate, the higher the probability of facing significant chromatic aberrations, colour shift and variation in resolution between the different channels. Acquisition: image before the reddest fluorophores. Controls: the analysis of controls which are labeled with only a single fluorophore helps in individuating bleed-through artefacts. Moreover it is possible to estimate background in the single channels (for example due to tissues autofluorescence). In critical situations linear spectral unmixing can be a solution.
Strategies for bleed-through minimization and correct results interpretation Specimen labeling: the degree of labeling and the intensity of fluorescence emission from the different fluorophores should be equally balanced. A good hint is for example to use the brightest and most photostable fluorophore for the least abundant cellular target. For increasing concentration of fluorescein respect to rhodamine concentration, the degree of cross-emission quickly increase (areas [1+2] -> 3 -> 4).