Imaging thick samples with a 3D microscope

Volumetric reconstructions with no moving parts

Most microscopes capture a flat, two dimensional image per snapshot. If your goal is to look closely within a thick sample, then typically your only choice is to focus the microscope objective up and down to try to get a clear view. Unfortunately, the resulting image will only offer sharp information from one particular plane at a time, and light from above and below this plane will mix into the image to reduce contrast. 

We have been developing a new way to capture and reconstruct thick samples in 3D without re-focusing or moving anything. Instead, we simply turn on different LEDs and capture multiple images, just like in Fourier ptychography. Then, we use a new algorithm to combine the captured data into a 3D "tomographic" reconstruction.

The movie below demonstrates the type of volumetric data that we can capture with a thick starfish embryo sample:

Comparing our approach (the first sequence, in color) to standard microscope refocusing (the second sequence, in grayscale), you'll first notice that our technique recovers more detail (e.g., we can resolve new features). Second, you'll see that our technique offers a more accurate reflection of the actual composition of the embryo.

We call our new measurement and reconstruction process "Fourier ptychographic tomography" (FPT, for short). Unlike the related method of diffraction tomography, FPT just measures normal intensity-only images, and does not need a reference beam to measure phase. This allows FPT to measure very small changes in the index of refraction of primarily transparent samples without using any interferometry (i.e., useing a phase-stable laser and a reference beam).