Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes

Abstract

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D ‘object plane’. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Optimization method to suppress background for imaging multiple planes

“…For the detection part, in order to be simultaneously detected, several optical strategies have been performed that enable simultaneous imaging multiple planes throughout a 3D volume using refractive 25 or di®ractive optics. 26,27 Nevertheless, in each case, the°u orescence signal imaged in each plane is reduced by the degree of parallelization.…”

“…10 This method enables simultaneous imaging through a 3D volume using di®raction 25 or refraction optics. 26 Nevertheless, the°uorescence signal imaged in each plane is reduced by the number of multiple light sheets. A major disadvantage of the method is the high background from cross-talk of neighboring light sheets because the distance between light sheets is too short.…”

Three-dimensional fluorescence imaging using the transport of intensity equation

“…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.…”

Imaging subcellular dynamics with fast and light-efficient volumetrically parallelized microscopy

“…To overcome the limitations of serial image acquisition, several optical strategies have been developed that enable simultaneous imaging of multiple planes throughout a 3D volume using refractive [16] or diffractive optics [17-20]. Nevertheless, in each case, the fluorescence signal imaged in each plane is reduced by the degree of parallelization, and most fluorescence is detected as out-of-focus blur.…”

“…For shallow sample volumes, pLSFM achieves 3-fold spatial parallelization without crosstalk between image planes, and is thus 3 to 4.5-fold less lossy than refractive or diffractive methods, respectively [16-20]. Unlike techniques that introduce aberrations [8, 12], sacrifice numerical aperture [11], or rely on short dwell times [13, 14], resolution and sensitivity are not compromised.…”