12 Nonlinear Label-Free Super-Resolution Microscopy Using Structured Illumination
307
12.8 Conclusion
We conclude this chapter by stating that label-free super-resolution multiphoton
microscopy is a vibrant and timely research topic. Label-free techniques are minimally invasive and require very little sample preparation. Therefore, compared to conventional fluorescent microscopy modalities, nonlinear label-free techniques could
be especially suitable for applications in clinical environments. Several label-free
super-resolution techniques based on SIM approach have already been proposed and
demonstrated. Although none of the existing works have yet experimentally demonstrated resolution better than 100 nm, we are confident that this landmark will be
soon reached. Once that landmark has been achieved, label-free SIM microscopy
becomes more competitive in its capabilities with conventional SIM, after which
novel imaging applications benefiting of label-free modalities will surely follow.
The recent technological development of suitable laser sources and detectors has
been impressive, and has driven the prices of commercial products down facilitating
the progress in the field. Especially for SIM-based super-resolution approaches, the
recent progress in sensitive CMOS camera technology is noteworthy and is motivating the users to move away from traditional electron-multiplying CCD cameras.
In overall, we can envision a bright future for nonlinear label-free super-resolution
microscopy, which will provide new possibilities both for basic research in biology
and medicine as well as for biomedical applications.
Acknowledgements This work was supported by the Academy of Finland (Grant No. 308596).
References
1. R. Erni, M.D. Rossell, C. Kisielowski, U. Dahmen, Atomic-resolution imaging with a sub50-pm electron probe. Phys. Rev. Lett. 102(9), 96101 (2009)
2. A.H. Zewail, 4D ultrafast electron diffraction, crystallography, and microscopy. Annu. Rev.
Phys. Chem. 57(1), 65–103 (2006)
3. B.R. Masters, P. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008)
4. E. Abbe, Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch.
für Mikroskopische Anat. 9(1), 413–418 (1873)
5. H.L.F. Helmholtz, H. Fripp, On the limits of the optical capacity of the microscope. J. Microsc.
16(1), 15–39 (1876)
6. S.W. Hell, J. Wichmann, Stimulated-emission-depletion fluorescence microscopy. Opt. Lett.
19(11), 780–782 (1994)
7. R. Heintzmann, C.G. Cremer, Laterally modulated excitation microscopy: improvement of
resolution by using a diffraction grating. Proc. SPIE 3568, 185–197 (1999)
8. M.G.L. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence
imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. 102(37), 13081–13086
(2005)
9. M.J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3(10), 793–795 (2006)
10. S.W. Hell, Far-field optical nanoscopy. Science 316(101), 1153–1158 (2007)
307
12.8 Conclusion
We conclude this chapter by stating that label-free super-resolution multiphoton
microscopy is a vibrant and timely research topic. Label-free techniques are minimally invasive and require very little sample preparation. Therefore, compared to conventional fluorescent microscopy modalities, nonlinear label-free techniques could
be especially suitable for applications in clinical environments. Several label-free
super-resolution techniques based on SIM approach have already been proposed and
demonstrated. Although none of the existing works have yet experimentally demonstrated resolution better than 100 nm, we are confident that this landmark will be
soon reached. Once that landmark has been achieved, label-free SIM microscopy
becomes more competitive in its capabilities with conventional SIM, after which
novel imaging applications benefiting of label-free modalities will surely follow.
The recent technological development of suitable laser sources and detectors has
been impressive, and has driven the prices of commercial products down facilitating
the progress in the field. Especially for SIM-based super-resolution approaches, the
recent progress in sensitive CMOS camera technology is noteworthy and is motivating the users to move away from traditional electron-multiplying CCD cameras.
In overall, we can envision a bright future for nonlinear label-free super-resolution
microscopy, which will provide new possibilities both for basic research in biology
and medicine as well as for biomedical applications.
Acknowledgements This work was supported by the Academy of Finland (Grant No. 308596).
References
1. R. Erni, M.D. Rossell, C. Kisielowski, U. Dahmen, Atomic-resolution imaging with a sub50-pm electron probe. Phys. Rev. Lett. 102(9), 96101 (2009)
2. A.H. Zewail, 4D ultrafast electron diffraction, crystallography, and microscopy. Annu. Rev.
Phys. Chem. 57(1), 65–103 (2006)
3. B.R. Masters, P. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University Press, 2008)
4. E. Abbe, Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch.
für Mikroskopische Anat. 9(1), 413–418 (1873)
5. H.L.F. Helmholtz, H. Fripp, On the limits of the optical capacity of the microscope. J. Microsc.
16(1), 15–39 (1876)
6. S.W. Hell, J. Wichmann, Stimulated-emission-depletion fluorescence microscopy. Opt. Lett.
19(11), 780–782 (1994)
7. R. Heintzmann, C.G. Cremer, Laterally modulated excitation microscopy: improvement of
resolution by using a diffraction grating. Proc. SPIE 3568, 185–197 (1999)
8. M.G.L. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence
imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. 102(37), 13081–13086
(2005)
9. M.J. Rust, M. Bates, X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3(10), 793–795 (2006)
10. S.W. Hell, Far-field optical nanoscopy. Science 316(101), 1153–1158 (2007)