Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing

Area of Science:

• Biophysics
• Cell Biology
• Biomaterials Science

Background:

• Accurate live cell imaging is crucial for understanding cellular responses to mechanical forces.
• Photobleaching, a common issue in fluorescence microscopy, complicates reliable morphological studies.
• Dual photon excitation (DPE) microscopy is recognized for reducing photobleaching compared to one photon excitation (OPE).

Purpose of the Study:

• To investigate the impact of OPE and DPE photobleaching on articular cartilage chondrocyte morphology.
• To evaluate the efficacy of common image thresholding methods in correcting photobleaching effects.
• To assess these effects under both unloaded and compressed (loaded) tissue conditions.

Main Methods:

• Live imaging of in situ articular cartilage chondrocytes using OPE and DPE microscopy.
• Performing seven consecutive laser scans to induce and quantify photobleaching.
• Applying and comparing three standard image thresholding techniques.
• Analyzing cell volume changes with and without mechanical compression of the cartilage tissue.

Main Results:

• Photobleaching consistently led to apparent cell volume reduction in subsequent scans.
• DPE microscopy exhibited significantly less volume loss than OPE microscopy in unloaded cells.
• Image thresholding methods did not fully prevent photobleaching-induced volume loss, which varied across scans.
• Mechanical compression increased cell fluorescence, leading to varied volume change interpretations based on thresholding methods.

Conclusions:

• Photobleaching directly distorts cell morphology measurements, with DPE offering superior artifact reduction over OPE for uncompressed cells.
• The interplay between photobleaching and compression-induced fluorescence changes complicates the interpretation of mechanical stimuli responses.
• Microscopic techniques and image thresholding choices can lead to contradictory conclusions regarding chondrocyte volume changes during tissue loading.