Analysis of immunolabeled cells by atomic force microscopy, optical microscopy, and flow cytometry

Area of Science:

• Biophysics
• Cell Biology
• Immunology

Background:

• Atomic Force Microscopy (AFM) offers high-resolution imaging of biological samples.
• Immunogold labeling is a common technique for visualizing cellular structures.
• Combining these techniques presents challenges in label detection and resolution.

Purpose of the Study:

• To evaluate the applicability of silver-enhanced immunogold labeling for AFM.
• To correlate force and fluorescent imaging of labeled human lymphocytes.
• To determine optimal conditions for high-resolution cell surface antigen detection using AFM.

Main Methods:

• Human lymphocytes were labeled with anti-CD3 antibodies conjugated to fluorescein isothiocyanate.
• Secondary antibodies linked to 1-nm or 30-nm colloidal gold particles were used.
• Silver enhancement was applied to increase label size.
• Imaging was performed using a combined inverted optical and atomic force microscope setup.
• Flow cytometry was utilized for supplementary analysis.

Main Results:

• Direct correlation between force and fluorescent images was established.
• Silver enhancement significantly increased the size of the immunogold labels.
• Small labels (1 nm) combined with 30 minutes of silver enhancement yielded reliable results.
• High-resolution detection of cell surface antigens was achieved.

Conclusions:

• Silver-enhanced immunogold labeling is a reliable method for AFM.
• This technique enables high-resolution detection of cell surface antigens.
• Optimized labeling protocols are crucial for successful AFM-based cell surface analysis.