(a) Schematic of the confocal rainbow volume holographic imaging system showing the multiplexed hologram that is used for illumination and imaging. Each hologram collects light from a different depth in the tissue sample. Wavelengths are dispersed across the lateral object field. (b) Image energy as a function of depth (z) in the sample. FWHM with z is 15 µm.
High-resolution three-dimensional optical imaging instruments such as confocal microscopes and optical coherence tomography systems are important tools in biomedical research.1 Volume holographic spatial-spectral imaging represents a different approach to subsurface tissue imaging.2 In this method, a multiplexed hologram is recorded with each hologram, corresponding to a different depth within a tissue sample. When the tissue is illuminated, one can simultaneously project images from different tissue depths onto a camera surface. Lateral spatial resolution is determined by the imaging properties of a high-NA microscope objective.
The system has been used with laser illumination and one-axis scanning as well as with a broadband illumination source such as an LED. In the former case, scanning is required to set the field of view (FOV), making it difficult to use in clinical settings. In the latter case, one determines the FOV by the spectral bandwidth; however, the wavefront selectivity capability of the system is compromised due to a position-wavelength degeneracy that develops in object space. Multiple images can still be obtained through contrast enhancement but the background level remains high.
Sun and Barbastathis suggested using a second illumination grating to break the position-wavelength degeneracy by spreading out the wavelengths from a broadband source across the object space.2 However, this approach had very coarse depth sectioning properties (~200 µm) that would not be useful for cellular imaging. A new and elegant approach is to use the same highly selective multiplexed hologram for imaging and to control the illumination spectrum.3
A multiplexed hologram is formed with each hologram collecting light from a different depth within the tissue sample. This hologram has high angular (~0.02°) resolution that results in high wavefront selectivity when used with narrow band illumination. When the multiplexed hologram is illuminated with a broadband source, the wavelengths are dispersed. The objective lens focuses different wavelengths to varying positions in the object field, thereby breaking the position-wavelength degeneracy. In addition, this is accomplished for each multiplexed grating at different imaging depths within the tissue sample. The high selectivity of the hologram acts similarly to a confocal imaging effect; however, it is effective at multiple depths within the tissue sample.
The system used a 0.55 NA imaging lens in combination with a 650 nm LED with a 30-nm bandwidth. The resulting z-psf FWHM was approximately 15 µm —which will allow imaging of typical biological cells (~20 µm). The lateral FOV was 300 x 100 µm and the group 7, element 6 feature of the AF Resolution target was resolved (2.5 µm).
The confocal rainbow volume holographic imaging system achieves a major step in subsurface tissue imaging by realizing high resolution depth selectivity in a compact, non-scanning instrument. The simplicity and high image resolution of this technique will make a tremendous advance in many clinical imaging applications.
This work was supported by the National Institute of Health Grant RO1CA134424.
Jose M. Castro, Paul J. Gelsinger-Austin, Jennifer K. Barton and Raymond K. Kostuk are with the department of computer and electrical engineering at the University of Arizona, U.S.A.
References and Resources
1. J. Fujimoto and D. Farkas. Biomedical Optical Imaging (Oxford, 2009).
2. W. Sun and G. Barbastathis. Opt. Lett. 30, 976 (2005).
3. J.M. Castro et al. Appl. Opt. 50, 1382 (2011).