A simple, inexpensive and multi-scale 3-D fluorescent test sample for optical sectioning microscopies.

Area of Science:

• Biophysics
• Microscopy
• Optical Imaging

Background:

• Quantitative fluorescence applications require reliable standards for quality control and inter-laboratory data comparison.
• Existing calibration samples are often expensive, technique-specific, and do not meet the growing demand for cross-instrument validation.
• Homogenous illumination and uniform detection sensitivity are crucial for advanced imaging analysis and quantitative measurements.

Purpose of the Study:

• To introduce and demonstrate the utility of a simple, versatile, and cost-effective 3D test sample for fluorescence microscopy.
• To showcase the application of fluorescent highlighter-labeled tissue paper as a complementary calibration standard.
• To validate its performance across a range of fluorescence microscopy techniques and scales.

Main Methods:

• Preparation of a 3D test sample by labeling commercial tissue paper with a fluorescent highlighter pen.
• Characterization of the sample's properties relevant for calibration.
• Acquisition of imaging data using epifluorescence, confocal, image scanning, two-photon (2P), and light-sheet microscopy.

Main Results:

• Demonstrated the versatility of the highlighter-labeled tissue paper across multiple fluorescence microscopy modalities.
• Showcased its applicability for calibration and quality control from sub-micrometer to centimeter scales.
• Provided examples of data acquired on various advanced microscopy systems.

Conclusions:

• Fluorescent highlighter-labeled tissue paper serves as a surprisingly versatile and simple 3D test sample.
• It can effectively complement existing, more expensive calibration standards for fluorescence microscopy.
• This approach facilitates improved data comparability and quality control in quantitative fluorescence imaging.