Compared to 2-D techniques, tomographic technology provides 3-D images with improved resolution in the z-axis. Further, image acquisition speed is often fast enough to provide useful temporal resolution or 4-D.
Two new imaging systems were exhibited at the ASCB Annual Meeting, held December 3–7, in San Francisco. Both instruments provide optical resolution better than the diffraction limit, which places them in the super-resolution realm.
Tomocube (Daejeon, South Korea) utilizes optical diffraction tomography (ODT) to form 3-D images made from many 2-D images obtained from the refractive index distribution of live cells. At its heart, the HT-1 is a laser interferometer that compares the variation in light in the sample beam path to the steady light in the reference light path.
Tomography involves measuring many images, each focusing on slightly different parts of the sample and then reconstructing a sample image from the multiple composites. While conventional tomography uses moving light sources and detectors, the HT-1 uses a digital micro-mirror device (DMD) (Figure 1) as a tunable diffraction grating. The DMD can be programmed to change the focus of the sample beam. Thus, the sample is illuminated and scanned without moving the light source, sample or detector. Light from any particular setting (or image) passes through the sample to the interferometer. A stack of 2-D images is reconstructed to 3-D using the Helmholtz equation, which is useful for oscillating phenomena.
Figure 1 – Sample light path of the HT-1. Laser light passes through a dispersive lens to the DMD, which selectively focuses the light on successive slices of the sample, producing a pseudo rotation indicated by the arrow above the sample. This tomographic optical system is rugged and low cost. (Figures 1, 2 and 3 reproduced with permission from Tomocube.)
Supported applications of the HT-1 include time course, label-free images of single cells such as necrosis and apoptosis, and 4-D imaging of organelles and vesicles during cell growth and death (see Figures 2 and 3).
Figure 2 – 4-D images of necrosis of a hepatocyte recorded with the HT-1 tomograph. Figure 3 – Recording of changes in a Hela cell following intimation of apoptosis.
Models include the HT-1S, which offers lateral resolution of 166 nm and 1 μm vertical; vertical resolution can be reduced to 332 nm with computer-aided reconstruction. The HT-1H offers 110-nm resolution in x-, y- and 368-nm vertical, which can be reduced to 220 nm for z-axis with reconstruction. Temporal resolution is 250 frames/sec for 2-D imaging and 2.5 frames/sec for 3-D.
3-D Cell Explorer-f
Nanolive (Ecublens, Switzerland) introduced a holographic tomograph platform for the rapid and label-free and fluorescent imaging of individual living cells in real time. For label-free work, the 3D Cell Explorer-f maps out the refractive index of the cells using a rotating tomographic head. This can be don in open air with little concern about ambient lighting or thermal effects. Rapid response facilitates continuous monitoring of live cells for days; temporal resolution is 0.6 frames/sec. x, y resolution is 200 nm with a 90 × 90 μm field of view. Fast temporal resolution also makes 4-D experiments practical.
Staining extends normal 2-D fluorescence experiment to 3-D cell tomography. Sample types include organelles, proteins and drugs using accepted labeling protocols. Up to 12 markers can be detected in one run, which opens up possibilities in multiplexing and co-localization studies.
The holographic tomograph of the 3D Cell Explorer-f makes 96 slices over a depth of field (Δz) of 500 nm, permitting localization of organelles at different cell depths.
Three-dimensional tomography is a potentially disruptive technology that should replace most 2-D microscopes, as demonstrated by the innovative systems shown at this conference.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].