Transport-of-intensity phase imaging using commercially available confocal microscope.

Abstract

Significance: Confocal microscopy is an indispensable tool for biologists to observe samples and is useful for fluorescence imaging of living cells with high spatial resolution. Recently, phase information induced by the sample has been attracting attention because of its applicability such as the measurability of physical parameters and wavefront compensation. However, commercially available confocal microscopy has no phase imaging function.

Aim: We reborn an off-the-shelf confocal microscope as a phase measurement microscope. This is a milestone in changing the perspective of researchers in this field. We would meet the demand of biologists if only they had measured the phase with their handheld microscopes.

Approach: We proposed phase imaging based on the transport of intensity equation (TIE) in commercially available confocal microscopy. The proposed method requires no modification using a bright field imaging module of a commercially available confocal microscope.

Results: The feasibility of the proposed method is confirmed by evaluating the phase difference of a microlens array and living cells of the moss Physcomitrium patens and living mammalian cultured cells. In addition, multi-modal imaging of fluorescence and phase information is demonstrated.

Conclusions: TIE-based quantitative phase imaging (QPI) using commercially available confocal microscopy is proposed. We evaluated the feasibility of the proposed method by measuring the microlens array, plant, and mammalian cultured cells. The experimental result indicates that QPI can be realized in commercially available confocal microscopy using the TIE technique. This method will be useful for measuring dry mass, viscosity, and temperature of cells and for correcting phase fluctuation to cancel aberration and scattering caused by an object in the future.