How can you improve the resolution of bioimaging techniques for more accurate images?

Begin with the end in mind. What do you need the assay to measure and how accurately do you need it to be measured? Sometimes researchers choose a technique that is overkill in terms of time, difficulty and cost for what they really need.

Fluorescence Microscopy: Ideal for visualizing specific components within cells, this technique uses fluorescent tags to illuminate structures. It's excellent for live-cell imaging and studying dynamic processes within cells. However, its resolution is limited by the diffraction limit of light Bioluminescence Imaging: Used primarily in vivo, this technique is great for non-invasive, longitudinal studies in animal models. It involves organisms or cells expressing a luminescent protein, like luciferase It's less suitable for high-resolution or single-cell analysis Common in ophthalmology and cardiology, Optical Coherence Tomography (OCT) provides realtime, crosssectional images of tissues. It's non-invasive and offers good depth penetration

I believe that all starts with having a good sample and continues with having a perfectly calibrated imaging system working in the best possible conditions

Yes. Sample preparation is a hugely critical component for fluorescent imaging. Growth of cells on appropriate substrate -- like a glass slide -- can change adhesion, for example, and morphology. Establishing proper growth is important for reproducibility. Fixation methods, permeabilization techniques, and handling of samples is important. The quality of the antibody is paramount to making conclusions between non-specific and specific staining. This cannot be overstated. Everything being equal, this is the single most important factor. The duration of staining can affect signal intensity -- 3D cells require longer staining to allow good penetration. A little time spent on getting these steps right can have a huge payoff.

For drug discovery, cells are often plated in microwell plates for imaging. The researcher may choose plate and well bottoms constructed of polystyrene, cycloolefin (and other glass-like polymers) or glass, typically #1.5 cover glass. Most microscope objective lenses are designed for imaging through #1.5 cover glass of 0.17 mm thickness. However, thick polystyrene is often chosen for microwell plates because of its low cost. Also, most cells are healthier when cultured on tissue-culture treated plastic than glass. Cycloolefin provides a better surface for cell health than glass with glass-like optical properties. Some cell types need additional surface treatments, e.g., poly-D-lysine or extracellular matrix proteins.

Post-processing techniques in bioimage analysis involve refining and enhancing images after initial acquisition. These methods address issues like noise reduction, contrast adjustment, and segmentation refinement to improve the accuracy and interpretability of results. They play a vital role in extracting quantitative data, identifying objects, and correcting artifacts. Post-processing also includes 3D reconstruction, image registration, and integration with machine learning for more precise and meaningful analyses, ultimately advancing our understanding of biological structures and processes.