Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Summary

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm³, which can be particularly useful for integration with lab-on-a-chip platforms.

Abstract

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. ^1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.

Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. ⁷ LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of <1 μm x <1 μm x <3 μm in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm³, and can be particularly useful for lab-on-a-chip platforms.

Protocol

1. Imaging Setup

LOT can be assembled in a compact and lightweight field-portable architecture ⁸, and alternatively as an optofluidic microscope with sectional imaging ability. ⁹ In this report, however, we will describe the basic imaging setup for a bench-top implementation toward tomography of static samples.

1. Illumination Module: In LOT, partially coherent light sources such as light-emitting-diodes (LEDs) can be utilized. For experimental flexibility, we used a monochromator with a Xenon lamp (Cornerstone T260, Newport Corp.). The monochromator was adjusted to provide an output with ~1-1....

Discussion

It is important to emphasize that the unique geometry of lensfree on-chip holographic microscopy is the critical enabler to achieve pixel super-resolution and tomographic imaging. Since the recorded images are not assumed to be projection images as in incoherent contact imaging approaches ¹², but projection holograms, the diffraction of the transmitted light until it impinges on the detector can be digitally corrected by holographic reconstruction. Therefore, variations in the sample-to-sensor distanc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Linear X-Y stages	Newport Corp.	MFA-PP	Miniature Linear Stage
Motorized rotation stage	Thorlabs	PRM1Z8	Motorized Precision Rotation Mount
Multimode optical fiber	Thorlabs	AFS105/125Y	Multimode Fiber
Light source	Newport Corp.	6255	Ozone-free Xenon Lamp
Monochromator	Newport Corp.	74100	Cornerstone 260 1/4 m Monochromator
CMOS sensor array	Aptina Inc.	MT9P031STC	5 Megapixels CMOS Sensor
C. elegans sample	Carolina Biosupply	173500	Wild-type C. elegans
Levamisole	Sigma Aldrich	L9756-5G	Tetramisole hydrochloride