Biological cells and tissues feature extremely complex three-dimensional (3D) structures spanning a large range of length scale. Living organisms are also very dynamic; molecular processes take place in a timescale covering over 15 orders of magnitudes while the organisms move and metabolize. For example, molecular processes such as protein, DNA, and RNA conformation changes take place in a timescale ranging from 100 fs to 100 s. (For discussion, see, e.g., Whitesides, G. M., Nat. Biotechnol. 21, 1161-1165 (2003); Williams, S. et al., Biochemistry 35, 691-697 (1996); Gilmanshin, R., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 3709-3713 (1997); Callender, R. H., et al., Annual Review of Physical Chemistry 49, 173-202 (1998); Trifonov, A. et al., Journal of Physical Chemistry B 109, 19490-19495 (2005); Cheatham, T. E., Curr. Opin. Struct. Biol. 14, 360-367 (2004); Millar, D. P., Curr. Opin. Struct. Biol. 6, 322-326 (1996); and Brauns, E. B., et al., Physical Review Letters 88 (2002), the disclosures of which are incorporated herein by reference.) The understanding of these processes not only has fundamental biological significance, but also will enable the treatment of a host of human diseases. Essentially, probing these processes requires a “whole-field” technique, which means taking 3D microscopic pictures in a rapid succession with high sensitivity and specificity without scanning, i.e. a four-dimensional (4D) microscope is needed.
Since holography was invented 60 years ago, an aberration-free, three-dimensional microscope has long been sought. (See, e.g., D. Gabor, “A new microscopic principle”, Nature 161, 777-778 (1948), the disclosure of which is incorporated herein by reference.) In fact, soon after Leith and Upatniek demonstrated the first 3D image by holography, holographic microscopy was proposed. With the recent advent of digital holography, holographic 3D microscopy received renewed and rapidly increasing attention. (E. N. Leith and Upatniek J., J. Opt. Soc. Am. 54, 1295-& (1964); E. N. Leith, et al., J. Opt. Soc. Am. 55, 981-& (1965); U. Schnars and W. Juptner, Appl. Opt. 33, 179-181 (1994); I. Yamaguchi and T. Zhang, Opt. Lett. 22, 1268-1270 (1997); E. Cuche, et al., Appl. Opt. 38, 6994-7001 (1999); F. Dubois, et al., Appl. Opt. 38, 7085-7094 (1999); P. Marquet, et al., Opt. Lett. 30, 468-470 (2005); L. Miccio, et al., Appl. Phys. Lett. 90, 3 (2007); L. Toth and S. A. Collins, Appl. Phys. Lett. 13, 7-& (1968); W. Xu, et al., Opt. Lett. 28, 164-166 (2003); and T. Zhang and I. Yamaguchi, Opt. Lett. 23, 1221-1223 (1998), the disclosure of which are incorporated herein by reference.) Holography is a “whole-field” technique, which means recording all 3D pixels simultaneously in one laser shot without scanning, an extremely valuable asset for biomedical imaging. When combined with modern high-repetition rate pulsed laser and fast imaging devices, a time sequence of consecutive 3D images can be captured, forming a 4D microscope. Indeed, successful 4D holographic imaging has been achieved recently in the context of fluid velocity measurement. (See, e.g., Y. Pu and H. Meng, Appl. Opt. 44, 7697-7708 (2005), the disclosure of which is incorporated herein by reference.)
Despite the technical advancements, holographic microscopes are not widely deployed in biomedical research because of the lack of specificity. In a microscopic setting with biological samples, holography alone suffers severe background scatterings from irrelevant cell structures. A holographic microscope would capture all scattering entities in the viewing field faithfully, but indiscriminately. On the other hand, the signals of interest (often from small nanostructures like protein molecules) are usually very weak and buried in the strong ambient scatterings from much larger organelles.
A key to achieve the specificity required for molecular biomedical imaging is to create a contrast between the useful signal and the ambient scatterings. Contrast imaging is routinely achieved through tagging the molecule or nanostructure of interest with fluorescent agents, such as fluorescent dyes, green fluorescent proteins (GFPs), and quantum dots (QDs). By converting the light signal into a different frequency, the unwanted ambient scattering can be easily removed with proper optical filters.
Recent advances in fluorescence microscopy have profoundly changed how cell and molecular biology is studied in almost every aspect. (See, e.g., Lichtman, J. W. & Conchelo, J. A., Nat. Methods 2, 910-919 (2005); Michalet, X. et al., Annu. Rev. Biophys. Biomolec. Struct. 32, 161-182 (2003); Jares-Erijman, E. A. & Jovin, T. M., Nat. Biotechnol. 21, 1387-1395 (2003); Bastiaens, P. I. H. & Squire, A., Trends Cell Biol. 9, 48-52 (1999); Suhling, K., et al., Photochem. Photobiol. Sci. 4, 13-22 (2005); Chalfie, M., et al., Science 263, 802-805 (1994); Pollok, B. A. & Heim, R., Trends Cell Biol. 9, 57-60 (1999); Jaiswal, J. K., et al., Nat. Methods 1, 73-78 (2004); Michalet, X. et al., Science 307, 538-544 (2005); Zipfel., W. R., et al., Nature Biotechnol. 21, 1368-1376 (2003); Shi, S. H. et al., Science 284, 1811-1816 (1999); Maletic-Savatic, M., et al., Science 283, 1923-1927 (1999); Miyawaki, A., et al., Proc. Natl. Acad. Sci. U.S.A. 96, 2135-2140 (1999); Akerman, M. E., et al., Proc. Natl. Acad. Sci. U.S.A. 99, 12617-12621 (2002); and Alivisatos, P., Nat. Biotechnol. 22, 47-52 (2004), the disclosures of which are incorporated herein by reference.) However, as an incoherent optical process, fluorescence lacks the capability of 3D representation. Should 3D information be required, point-scanning technique has to be performed over the entire volume of interest. The elapsed time for the scanning (i.e. the 3D framing time) is usually in the order of seconds to minutes, which severely constrains the use of this technique. Attempts to extend fluorescence microscopy into three spatial dimensions over time (4D microscopy) are thus incapable of capturing dynamic events. (See, e.g., D. Gerlich and J. Ellenberg, Nat. Cell Biol. 5, S14-S19 (2003), the disclosure of which is incorporated herein by reference.)
Accordingly, a new holographic imaging methodology and system is needed that allows for dynamic 4D imaging with high contrast and ample spatial and temporal resolution.