High-resolution Hadamard transform microscope fluorescence imaging: quantifying the DNA content in single cells

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Conventional fluorescence microscopes lack advanced spatial resolution and multichannel detection capabilities.
• Accurate cellular quantitative measurements require high-resolution imaging and reliable standards.

Purpose of the Study:

• To develop a miniaturized Hadamard transform (HT) fluorescence imaging microscope for enhanced spatial resolution and multichannel detection.
• To apply the HT microscope for morphological analysis and quantitative DNA content measurements in various cell types.
• To evaluate eel erythrocytes as a potential standard for DNA content determination.

Main Methods:

• Integration of a conventional fluorescence microscope with Hadamard transform multiplexing encoding using a 1D movable mask.
• Utilizing a linear CCD for multichannel detection to achieve spatial resolution.
• Acquisition and analysis of HT images for morphological and quantitative cellular measurements.

Main Results:

• The HT microscope provides high-resolution, 0-255 gray level images for cell visualization and morphological analysis.
• Calibrated DNA content in chicken erythrocytes was 2.32 pg using human lymphocytes as a standard.
• Eel erythrocytes demonstrated a stable DNA value of 2.06 pg (CV 4.3%), suggesting their potential as a novel standard.

Conclusions:

• The developed miniaturized HT fluorescence imaging microscope enables high-resolution morphological and quantitative cellular analysis.
• Eel erythrocytes present a reliable and novel standard for DNA content determination across species.
• The HT imaging microscope holds significant value for applications in medicine and cell biology.