Light Field Microscope

Vincent Lee & Leanna Morinishi

Introduction

Background and Motivation

Traditional light microscopy has been used to illuminate miniature biological specimens since the mid-1600s. Since, researchers have come to realize and (attempt to) circumvent the inherent limitations of the technique. One such limitation is that of superimposed features on the captured image. With no depth information from the subject, it is difficult to extract data specific to the focal plane from the rest of the image.

Confocal microscopy excludes light from above or below the focal plane of the image using a pinhole placed where the light from the focal plane comes to a waist. It also provides a method of visualizing 3-dimensional images and has better spatial resolution than light field microscopy, but images require scanning the entire sample at every depth of interest. Light field has the advantage of capturing relative depth information in a single photo. This additional visual information comes at a cost, however. Diffraction places an upper limit on the lateral and axial resolution of the images. We thus sacrifice spatial resolution to obtain angular resolution.

The light field microscope allows angular resolution information to be recorded in a single image. The light field is a function that represents the amount of light traveling in every direction through every point in space. By recording a sample’s light field, one can produce perspective and parallax views of the specimen. This is accomplished by inserting a microlens array in a conventional bright field microscope, and analyzing the images in silico. A microlens array is an optical component containing a spread of thousands of lenses with diameters in the micron scale. Applying 3D deconvolution to the focal stacks from the array produces a series of cross sections which allow for a 3D reconstruction of the sample.

The objective of this project is to create a light field microscope from a Lytro™ camera and the Thorlabs materials available in the 20.345 classroom, record associated documentation and code, and describe recommended experiments for use in a teaching undergraduate laboratory.

Prior Work

Design

Optical Setup

The microscope design went through a few iterations and continues to evolve. We originally tried to use a 2" lens telescope to magnify the image. At first the telescope was before the tube lens, but since that contributes to aberration it was placed after the tube lens. While doing this, we realized that the image should be in focus just before the Lytro™ optics.

We tried to put a different tube lens in the system to magnify the image with a single lens, however the led to a considerable amount of spherical aberration.

Image Processing

Reverse engineering the Lytro™ images

Creating .jpg image stacks from -stk.lfp

Creating .raw from .lfp

Creating .tif from .raw

Dealing with the hexagonal microarray

List of parts

Methodology

Disassembling the Lytro

Aligning the beam path

Making sample slides of fluorescent beads

Acknowledgments

1. Nirav Patel, for reverse engineering the Lytro image and lfpsplitter.
2. Frank Warmerdam, Andrey Kiselev, Bob Friesenhahn, Joris Van Damme and Lee Howard for raw2tiff tools.