Autofluorescent artifacts in grass imaging?

Damiano Marchiafava @Damiano-Marchiafava

20 June 2023 1 8K Report

I am trying to decellularize grass to be used as a myoscaffold for my muscle cells, but I continue to see these autofluorescent dots in my decellularized grass, making it hard to identify my cells. Does anyone know what these artifacts could be? Are they autofluorescent nuclei? I did not think that was possible. I see them in all channels and have not stained for anything yet in the photo.

Aman Prabhakar

The presence of autofluorescent dots in your decellularized grass samples can be challenging when trying to identify your cells. While it is difficult to provide a definitive answer without examining the samples directly, there are a few possibilities for these artifacts:

Chlorophyll remnants: Grass contains chlorophyll, which is responsible for its green color. Even after the decellularization process, small remnants of chlorophyll may persist and exhibit autofluorescence. These remnants can appear as autofluorescent dots and interfere with the identification of your cells.

Cellular debris: During the decellularization process, some cellular debris might not be completely removed, leading to residual autofluorescent components from the original grass cells. These remnants can contribute to the observed autofluorescent dots.

Contaminants: It's possible that there are contaminants present in your decellularized grass samples that exhibit autofluorescence. These contaminants could be from environmental sources, such as dust or other particles that have adhered to the grass.

Background fluorescence: Autofluorescence can sometimes arise from non-cellular components of the tissue itself, including extracellular matrix components, plant fibers, or other organic materials present in the grass. This background fluorescence can make it challenging to distinguish between true cell-specific fluorescence and nonspecific signals.

To overcome these challenges and improve the identification of your cells, you can consider the following strategies:

Use appropriate controls: Include appropriate controls in your experiments to differentiate between autofluorescence artifacts and specific cell fluorescence. These controls can help establish a baseline and distinguish between true cell-specific signals and nonspecific fluorescence.

Optimize decellularization protocols: Evaluate and optimize your decellularization protocols to ensure thorough removal of cellular debris and contaminants. You may need to modify the process or incorporate additional steps to minimize autofluorescence.

Consider different imaging techniques: Explore alternative imaging techniques that can minimize or differentiate autofluorescence from your target signals. For example, confocal microscopy with spectral detection can help separate autofluorescence from specific fluorescence by analyzing emission spectra.

Staining and labeling: Consider using specific stains or fluorescent markers to label your muscle cells. This can help distinguish them from autofluorescent artifacts and provide a clearer identification of your cells.

If the issue persists or you require more specific guidance, it is advisable to consult with experts in the field, such as researchers or technicians experienced in decellularization techniques or cell imaging, who can offer further insights based on their expertise.

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