Three-dimensional fluorescence imaging using the transport of intensity equation.

Sudheesh K Rajput,Yasuhiro Awatsuji,Osamu Matoba,Manoj Kumar,Mitsuhiro Morita,Tomoyuki Furuyashiki,Xiangyu Quan,Enrique Tajahuerce

doi:10.1117/1.jbo.25.3.032004

Abstract

.We propose a nonscanning three-dimensional (3-D) fluorescence imaging technique using the transport of intensity equation (TIE) and free-space Fresnel propagation. In this imaging technique, a phase distribution corresponding to defocused fluorescence images with a point-light-source-like shape is retrieved by a TIE-based phase retrieval algorithm. From the obtained phase distribution, and its corresponding amplitude distribution, of the defocused fluorescence image, various images at different distances can be reconstructed at the desired plane after Fresnel propagation of the complex wave function. Through the proposed imaging approach, the 3-D fluorescence imaging can be performed in multiple planes. The fluorescence intensity images are captured with the help of an electrically tunable lens; hence, the imaging technique is free from motion artifacts. We present experimental results corresponding to microbeads and a biological sample to demonstrate the proposed 3-D fluorescence imaging technique.

Highlights

IntroductionFluorescence imaging is an important technique to get the functional information of a biological sample for cellular and microbiological investigations, as has been proved by several studies.[1,2,3,4,5,6,7,8,9,10,11,12,13] Most of the reported fluorescence imaging techniques are either two-dimensional in nature or involve sectioning to get three-dimensional (3-D) information, such as laser scanning confocal microscopy and other related techniques.[1,2,3,4,5,6,7,8,9,10,11] These techniques are time-consuming processes to obtain the 3-D information of objects
We have presented a scanless 3-D fluorescence imaging scheme based on the transport of intensity equation (TIE) technique that uses the Fresnel backpropagation of a complex wave function calculated from the phase retrieved by TIE and its corresponding intensity image
In the proposed imaging technique, the phase distribution corresponding to defocus fluorescence images is retrieved by a phase retrieval algorithm based on TIE

Summary

Introduction

Fluorescence imaging is an important technique to get the functional information of a biological sample for cellular and microbiological investigations, as has been proved by several studies.[1,2,3,4,5,6,7,8,9,10,11,12,13] Most of the reported fluorescence imaging techniques are either two-dimensional in nature or involve sectioning to get three-dimensional (3-D) information, such as laser scanning confocal microscopy and other related techniques.[1,2,3,4,5,6,7,8,9,10,11] These techniques are time-consuming processes to obtain the 3-D information of objects. Further techniques have been developed by using digital holography in fluorescence microscopy to record and retrieve the 3-D information of a fluorescent object.[12,13] by adopting digital holography or other interferometric systems for 3-D fluorescence imaging, the imaging systems become more complicated due to the involvement of an additional reference beam. It is desirable to further investigate 3-D fluorescence imaging techniques to develop simple, compact, and cost-effective solutions

Methods

Results

Conclusion