What is a hologram and how are holograms made?

First, we narrow this definition to “optical hologram”, since holograms can record any type of wave, including acoustical and non-optical waves. An optical hologram is a recording of the information contained in light waves that have passed through or reflected from objects that were illuminated by a beam of light. Figure 2 shows such an illuminated object as it scatters light in the direction of the hologram. This scattered light, the “object wave”, is mixed with a “reference wave”, which interferes with it to form fringes (variations in intensity), known as a diffraction pattern. (Holography is conducted with laser illumination, which is coherent light, since coherent illumination makes interference possible. Mixing light from a standard light bulb does not result in interference fringes.) The recording of the diffraction pattern, produced by mixing an object and reference wave, is a hologram of the object.

Figure 2.

Recording a hologram of an object. ⁱⁱ

The diffraction pattern contains enough information about the object wave to enable reconstructing it in full detail, replicating the original object wave. If the hologram is then illuminated again by the reference wave, as in Figure 3, the diffraction pattern in the hologram will, through the process of diffraction, convert some of it into a wave that is identical to the original object wave that was used to produce it, so that what the viewer sees is that same as what was seen when the object was present.

The hologram that was just described is called an “off-axis” hologram, since the object wave and reference wave come from two different directions. If an object field is sufficiently transparent, such as a volume containing particles, holograms can be produced with a single optical wave passing through the volume with a small amount of it being scattered by the particles and the unscattered light serving as a reference wave, which then mixes with the scattered light to form the diffraction pattern that becomes the hologram. This process is called “in-line” holography, since the object and reference waves come from the same direction and overlap. In-line holography is the simplest kind of holography, since it can be done with a single beam of light.

Figure 3.

Reconstructing the original object wave. ⁱⁱ

Holoscope employs “in-line” holography. Figure 4 illustrates the complete recording and reconstruction process of the Holoscope in recording and reconstructing a three-dimensional image of a volume containing particles. In this figure, a laser beam is passed through a volume that contains three particles. The resulting diffraction pattern is recorded on a digital video camera and is displayed on the computer monitor as shown. Each circular diffraction pattern shown in this image is an in-line hologram of one of the three particles in the volume.

If this image were recorded on a transparent film, we could illuminate it with a reference wave and three images would appear in their original relative positions in space. In digital holography, the information is recorded in digital form and the reconstruction process is completed in computer memory, where the diffraction and propagation equations are used to simulate reconstructing the wavefront and viewing it plane-by-plane to see where the particles come into focus. In the reconstructed image, the top particle focuses first as we scan through the various planes from 9.5 cm to 12.2 cm. After the scan is completed, the particle positions in space are shown in the bottom-left 3D diagram.

Figure 4.

Recording and image reconstruction process in the Holoscope

Figure 5 shows the Holoscope with microscope slides and a cuvette, which are provided to contain the objects of interest that will be recorded in holograms.

Figure 5

-Holoscope with accessories

Holoscope set up

Figure 6 illustrates the Holoscope set up procedure, which begins by attaching to a computer, which provides power to the laser and the camera inside the Holoscope.

The sample under study is inserted into the Holoscope sample slot either on a microscope slide or in a solution in the cuvette and centered in the field of view. For cuvettes, adapter caps are provided to slide the cuvette into one of the slots. The sample position can be viewed on the monitor to assist with alignment.

Adjustment knobs on the front and side are provided to move the sample to the center of the monitor. When the sample is in position, the software provided with the Holoscope enable an operator to record the hologram and reconstruct and focus on images.

Figure 6.

Holoscope set up with a sample inserted: (a) Connecting Holoscope to a computer; (b) inserting sample into the Holoscope slots on a microscope slide or in a cuvette; (c) rotating adjustment knobs to move the sample.

The Holoscope features three slots for sample mounting shown in Figure 7. Slot 1 (top) is the closest to the image sensor, providing the smallest magnification, while Slot 3 (bottom), located furthest from the sensor yet closest to the light source, offers the greatest magnification in the resulting image.

Figure 7.

Three available slots for inserting a microscope slide or a cuvette.

In the following discussion, we illustrate Holoscope examining a 3D volume of particles. For this example, we created a 3D scene, a volume of water in the cuvette, dropped some chalk dust into the water, and recorded video holograms of the dust slowly settling to the bottom. The holograms can be used to view the dynamics of these particles and their threedimensional position over time by reconstructing their focused images at different propagation depths and times.

Holograms of the water and dust particle mixture were captured at three different times, shown in Figure 8, immediately after shaking the sample, 5 minutes later, and 10 minutes later, to illustrate how particle density affects the appearance and quality of the hologram and focused images.

The initial hologram displays densely packed overlapping diffraction patterns. As time passes and the particles settle, holograms from later intervals show clearer and more distinct diffraction patternsdue to a lower particle density.

Figure 8.

In-line holograms of dust particles suspended in water; (a) t = 0 mins; (b) t = 5 mins; (c) t = 10 mins.

Figure 9 shows the reconstructed and focused images produced, using the Holoscope’s software, from one of the holograms, at three different depths, and at one instant in time in the cuvette. A particle image that is in focus in one of the planes will be out of focus in other planes and can be observed as a diffraction pattern in the out-of-focus plane for that particle.

Figure 9.

Reconstructed images of the dust particles in water with different propagation depth; (a) 12.0 mm, (b) 16.5 mm, (c) 21.0 mm.

Holograms of Insects

We show here examples of holograms of insects to examine their intriguing shapes and movements in 3D. We placed a small caterpillar on a slide, inserted the slide in the middle sample slot of the Holoscope and recorded the hologram shown in Figure 10a. The hologram was then used to reconstruct and scan the 3D image recorded in the hologram. Figure 10b shows the image focused on the caterpillar legs. In this image plane, we can also see that particles that were on the slide coming into focus also, since they are in a plane that is close to that where the legs focus.

The adjustment knobs are used to center the caterpillar within the image frame, ensuring optimal viewing. The caterpillar’s movements can be observed in real-time on the preview monitor in the software interface.

Insects have many interesting microscopic features that require 3D scanning, which are more difficult to analyze with conventional microscopy. DHM enables recording and tracking of the relative movements with high resolution of such parts over time, which is difficult to achieve with conventional microscopy. Figure 11 shows reconstructed images of an aphid and a mosquito.

Figure 10.

Hologram and a reconstructed image of a caterpillar: (a) Hologram of caterpillar and (b) reconstructed image focused on the legs.

Figure 11.

Reconstructed images of an aphid and a mosquito: (a) Reconstructed image of an aphid and (b) reconstructed image of a mosquito’s head.

The legs of the aphid are clearly visible, and closer focusing reveals the small tube-like cornicles near the posterior end of the aphid’s body. When aphids are threatened by predators, they release a defensive fluid through these cornicles that contains chemicals that deter or repel attackers. Looking at mosquito’s head under the microscope, its eyes, palps, and a needle-like fiber called a proboscis are clearly displayed. Mosquitoes use their long antennae to sense things around them. The proboscis is like a needle they use for feeding. They also have palps near the needle that guide it to the blood vessels. These palps help the mosquito find a good spot to feed.