Development of a digital holographic microscopy system integrated with atomic force microscope

Atomic Force Microscope (AFM) imaging, due to the scanning method of recording, requires significant recording time for examination of wide sample area. In contrast, digital holographic microscopy (DHM), owing to the wide-field method, allows recording of the hologram in very fast rate which could be numerically analyzed to reveal surface of the sample with axial resolution at the nanometer scale. However, DHM yields quantitative phase properties of the sample, and therefore sensitive to changes in refractive index along with physical thickness. Therefore, to accurately determine the refractive index map, it is imperative to estimate the physical thickness map of the sample. This was achieved by AFM imaging. Further, since the transverse resolution of DHM is limited by diffraction limit, co-registration of AFM image provided higher transverse resolution at nanometer scale. The interference of the AFM probe was observed to be minimal during simultaneous AFM and DHM recording due to the transparent nature and bent configuration of the optical fiber based AFM cantilever. Integration of DHM and AFM led to realization of a powerful platform for nanoscale imaging. The integrated AFM-DHM system was built on an inverted fluorescence microscope to enable fluorescence imaging of the sample. The integrated system was employed to analyze fluorescent polystyrene microspheres, two-photon polymerized microstructures and red blood cells.