Determination of sub-diffraction-limited organelles and biomolecules using microsphere lenses in combination with fluorescence microscopy

Abstract

We propose the use of a high-refractive index glass microsphere in combination with a conventional fluorescence microscope for imaging sub-cellular organelles and biomolecules. The microsphere is placed on a sample that is immersed in water, collects the near-field nano-features of the sample and generates a magnified virtual image in the farfield, which is recorded through a conventional water immersion objective. We first investigate the imaging capability of the microspheres on size-calibrated fluorescent micro-/nano-particles. The experimental results obtained from a microsphere with 60 μm in diameter demonstrate imaging capability of features of ~λ/7-size (λ is the wavelength) with a magnification factor of 5.4. The position of the virtual image, the field-of-view (FOV) of the microsphere and the magnification factor are studied by using microspheres with different sizes. Finite Element Method (FEM) simulations are performed, providing key insight into the imaging effect of the microspheres with super-resolution capability. Moreover, the distribution and complex shape of different sub-cellular organelles, like centrioles, mitochondria and chromosomes in the AML12 cell line, are imaged with help of the glass microspheres. Thereafter, the subcellular location of mitochondrial encoded proteins could be studied.