Label-free 3D imaging of weakly absorbing samples using spatially-incoherent annular illumination microscopy

Highlights

• The proposed technique can achieve label-free 3D images of weakly absorbing samples.

• The proposed technique employs spatially-incoherent annular illumination to enhance absorption contrast of in-focus images and reduce the phase contrast.

• A 3D gradient operation is employed to remove the background with defocused shadows caused by oblique illumination. A sequence of background-free sectioning images is acquired.

• The proposed technique is of simplicity, low cost, high performance and good compatibility. It can be implemented by simply adapting a standard bright-field microscope.

Abstract

In this paper, we present a simple but effective label-free three dimensional (3D) microscopy for weakly absorbing samples. The proposed technique employs spatially-incoherent annular illumination to enhance absorption contrast of in-focus images and reduce phase contrast. We also employ mechanical scanning along axial direction to acquire a volume of the sample images. A 3D gradient operation is further adopted to remove the background with defocused shadows caused by oblique illumination. As such, a sequence of background-free sectioning images is acquired. The 3D gradient operation results in that only the structural edges of the weakly absorbing sample are visible in the images. We can therefore reconstruct the 3D skeleton structure of the sample from the sectioning image sequence. A label-free diatom is used to verify our technique experimentally. The 3D skeleton structure of the diatom is reconstructed and presented. The proposed technique would find applications in various fields, such as life science, materials science, etc.

Introduction

Three-dimensional (3D) microscopy is a powerful tool for presenting 3D microstructures of samples. Most of 3D microscopies require fluorescence labeling [1], [2], [3], [4], [5], [6], [7], [8]. However, some structures in a sample cannot be labeled easily and the labeling process would potentially affect the biological sample. Actually, many biological samples are with weak absorption and appear transparent. There have been various phase-based microscopies proposed for 3D imaging of transparent samples [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. However, these phase-based techniques require a complex optical system and an extra computational reconstruction process, such as 3D deconvolution, etc. It is well known that the performance of the deconvolution heavily relies on how accurate the transfer function is modeled, but accurate modeling is challenging and difficult. Inaccuracy would introduce artifacts in the image.

Recently, we have reported the spatially-incoherent annular illumination microscopy (SAIM) for bright-field optical sectioning of strongly absorbing samples [21]. SAIM is a refinement of oblique illumination microscopy [22], [23]. For weakly absorbing samples, however, the defocused phase-contrast shadows due to oblique illumination cannot be ignored in the SAIM, making it difficult for 3D imaging. In this paper, we analyze the characteristics of imaging weakly absorbing samples by using SAIM in detail. Based on the theoretical analysis, we conclude that SAIM can enhance absorption contrast of in-focus images and reduce the phase contrast simultaneously. Aiming at label-free 3D imaging for weakly absorbing samples, we employ mechanical scanning along the axial direction to acquire a volume of the sample images, and use a 3D gradient operation to remove the background with blurred defocused shadows. A sequence of background-free sectioning images with high visual contrast can be acquired and the 3D skeleton structure of the sample can be reconstructed from the image sequence. A label-free diatom is used to verify the technique experimentally. The 3D skeleton structure of the diatom is reconstructed. The proposed technique would find applications in various fields, such as life science, materials science, etc.