Semi-automated 3D fluorescence speckle analyzer (3D-Speckler) for microscope calibration and nanoscale measurement.
Jonathan LoiXiaofei QuAussie SuzukiPublished in: The Journal of cell biology (2023)
The widespread use of fluorescence microscopy has prompted the ongoing development of tools aiming to improve resolution and quantification accuracy for study of biological questions. Current calibration and quantification tools for fluorescence images face issues with usability/user experience, lack of automation, and comprehensive multidimensional measurement/correction capabilities. Here, we developed 3D-Speckler, a versatile, and high-throughput image analysis software that can provide fluorescent puncta quantification measurements such as 2D/3D particle size, spatial location/orientation, and intensities through semi-automation in a single, user-friendly interface. Integrated analysis options such as 2D/3D local background correction, chromatic aberration correction, and particle matching/filtering are also encompassed for improved precision and accuracy. We demonstrate 3D-Speckler microscope calibration capabilities by determining the chromatic aberrations, field illumination uniformity, and response to nanometer-scale emitters above and below the diffraction limit of our imaging system using multispectral beads. Furthermore, we demonstrated 3D-Speckler quantitative capabilities for offering insight into protein architectures and composition in cells.
Keyphrases
- single molecule
- high throughput
- low cost
- high resolution
- atomic force microscopy
- living cells
- energy transfer
- deep learning
- induced apoptosis
- optical coherence tomography
- single cell
- fluorescence imaging
- machine learning
- cell cycle arrest
- electronic health record
- convolutional neural network
- oxidative stress
- gene expression
- high speed
- data analysis
- protein protein
- mass spectrometry
- cell proliferation