Skip to main content Accessibility help

Spatially-controlled illumination microscopy: For prolonged live-cell and live-tissue imaging with extended dynamic range

  • Venkataraman Krishnaswami (a1) (a2), Cornelis J. F. Van Noorden (a1), Erik M. M. Manders (a2) and Ron A. Hoebe (a1)


Live-cell and live-tissue imaging using fluorescence optical microscopes presents an inherent trade-off between image quality and photodamage. Spatially-controlled illumination microscopy (SCIM) aims to strike the right balance between obtaining good image quality and minimizing the risk of photodamage. In traditional imaging, illumination is performed with a spatially-uniform light dose resulting in spatially-variable detected signals. SCIM adopts an alternative imaging approach where illumination is performed with a spatially-variable light dose resulting in spatially-uniform detected signals. The actual image information of the biological specimen in SCIM is predominantly encoded in the illumination profile. SCIM uses real-time spatial control of illumination in the imaging of fluorescent biological specimens. This alternative imaging paradigm reduces the overall illumination light dose during imaging, which facilitates prolonged imaging of live biological specimens by minimizing photodamage without compromising image quality. Additionally, the dynamic range of a SCIM image is no longer limited by the dynamic range of the detector (or camera), since it employs a uniform detection strategy. The large dynamic range of SCIM is predominantly determined by the illumination profile, and is advantageous for imaging both live and fixed biological specimens. In the present review, the concept and working mechanisms of SCIM are discussed, together with its application in various types of optical microscopes.


Corresponding author

*Author for correspondence: Ron A. Hoebe, Core Facility Cellular Imaging, van Leeuwenhoek Centre for Advanced Microscopy (LCAM), Academic Medical Centre (AMC), University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: 3156664743; Email:


Hide All
Bell, A., Brauers, J., Kaftan, J. N., Meyer-Ebrecht, D., Böcking, A. & Aach, T. (2009). High dynamic range microscopy for cytopathological cancer diagnosis. IEEE Journal of Selected Topics in Signal Processing 3, 170184.
Bernas, T., Zarebski, M., Cook, R. R. & Dobrucki, J. W. (2004). Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux. Journal of Microscopy 215, 281296.
Brakenhoff, G. J., Blom, P. & Barends, P. (1979). Confocal scanning light microscopy with high aperture immersion lenses. Journal of Microscopy 117, 219232.
Caarls, W., Rieger, B., De Vries, A. H. B., Ardnt-Jovin, D. J. & Jovin, T. M. (2011). Minimizing light exposure with the programmable array microscope. Journal of Microscopy 241, 101110.
Carlton, P. M., Boulanger, J., Kervrann, C., Sibarita, J. B., Salamero, J., Gordon-Messer, S., Bressan, D., Haber, J. E., Haase, S., Shao, L. & Winoto, L. (2010). Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proceedings of the National Academy of Sciences of the United States of America 107, 1601616022.
Chakrova, N., Canton, A. S., Danelon, C., Stallinga, S., & Rieger, B. (2016a). Adaptive illumination reduces photobleaching in structured illumination microscopy. Biomedical Optics Express 7, 42634274.
Chakrova, N., Heintzmann, R., Rieger, B., & Stallinga, S. (2015). Studying different illumination patterns for resolution improvement in fluorescence microscopy. Optics Express 23, 3136731383.
Chakrova, N., Rieger, B., & Stallinga, S. (2016b). Deconvolution methods for structured illumination microscopy. JOSA A 33, B12B20.
Chen, B. C., Legant, W. R., Wang, K., Shao, L., Milkie, D. E., Davidson, M. W., Janetopoulos, C., Wu, X. S., Hammer, J. A., Liu, Z., English, B. P., Mimori-Kiyosue, Y., Romero, D. P., Ritter, A. T., Lippincott-Schwartz, J., Fritz-Laylin, L., Mullins, D. R., Mitchell, D. M., Bebenek, J. N., Reymann, A., Böhme, R., Grill, S. W., Wang, J. T., Seydous, G., Tulu, U. S., Kiehart, D. P. & Betzig, E. (2014). Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998.
Chu, K. K., Lim, D. & Mertz, J. (2007). Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination. Optics Letters 32, 28462848.
Chu, K. K., Lim, D. & Mertz, J. (2010). Practical implementation of log-scale active illumination microscopy. Biomedical Optics Express 1, 236245.
Chuang, C. H., Carpenter, A. E., Fuchsova, B., Johnson, T., De Lanerolle, P. & Belmont, A. S. (2006). Long-range directional movement of an interphase chromosome site. Current Biology 16, 825831.
Croix, C., Shand, S. & Watkins, S. (2005). Confocal microscopy; comparisons, applications and problems. Biotechniques 39 (Supplement, S2–S5).
Daetwyler, S., & Huisken, J. (2016). Fast fluorescence microscopy with light sheets. Biological Bulletin 231, 1425.
De Vos, W. H., Hoebe, R. A., Joss, G. H., Haffmans, W., Baatout, S., Van Oostveldt, P. & Manders, E. M. (2009). Controlled light exposure microscopy reveals dynamic telomere microterritories throughout the cell cycle. Cytometry Part A 75, 428439.
De Vos, W. H., Houben, F., Hoebe, R. A., Hennekam, R., Van Engelen, B., Manders, E. M. M., Ramaekers, F. C. S., Broers, J. L. V. & Van Oostveldt, P. (2010). Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins. Biochimica Biophysica Acta 1800, 448458.
Dixit, R. & Cyr, R. (2003). Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant Journal 36, 280290.
Editorial (2013). Artifacts of light. Nature Methods 10, 1135.
Eggeling, C., Willig, K. I., Sahl, S. J. & Hell, S. W. (2015). Lens-based fluorescence nanoscopy. Quarterly Reviews of Biophysics 48, 178243.
Ettinger, A. & Wittmann, T. (2014). Fluorescence live cell imaging. Methods Cell Biology 123, 7794.
Fahrbach, F. O., Simon, P., & Rohrbach, A. (2010). Microscopy with self-reconstructing beams. Nature Photonics 4, 780785.
Fahrbach, F. O. & Rohrbach, A. (2012). Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media. Nature Communications 3, 632.
Fu, Q., Martin, B. L., Matus, D. Q., & Gao, L. (2016). Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy. Nature Communications 7, 11088.
Gao, L. (2015). Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet. Optics Express 23, 61026111.
Grzelak, A., Rychlik, B. & Bartosz, G. (2001). Light-dependent generation of reactive oxygen species in cell culture media. Free Radical Biology Medicine 30, 14181425.
Gustafsson, M. G. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy 198, 8287.
Gustafsson, M. G., Shao, L., Carlton, P. M., Wang, C. R., Golubovskaya, I. N., Cande, W. Z., Agard, D. A. & Sedat, J. W. (2008). Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophysical Journal 94, 49574970.
Hanley, Q. S., Verveer, P. J., Gemkow, M. J., Arndt-Jovin, D. & Jovin, T. M. (1999). An optical sectioning programmable array microscope implemented with a digital micromirror device. Journal of Microscopy 196, 317331.
Harnett, M. T., Makara, J. K., Spruston, N., Kath, W. L. & Magee, J. C. (2012). Synaptic amplification by dendritic spines enhances input cooperativity. Nature 491, 599602.
Heintzmann, R. (2003). Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron 34, 283291.
Heintzmann, R., Hanley, Q. S., Arndt-Jovin, D. & Jovin, T. M. (2001). A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images. Journal of Microscopy 204, 119135.
Hell, S. W. & Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Optics Letters 19, 780782.
Helmchen, F. & Denk, W. (2005). Deep tissue two-photon microscopy. Nature Methods 2, 932940.
Hoebe, R. A. (2010). Controlled light exposure microscopy. PhD thesis, University of Amsterdam, Amsterdam, The Netherlands.
Hoebe, R. A., Van Noorden, C. J. F. & Manders, E. M. M. (2010). Noise effects and filtering in controlled light exposure microscopy. Journal of Microscopy 240, 197206.
Hoebe, R. A., Van Oven, C. H., Gadella, T. W., Dhonukshe, P. B., Van Noorden, C. J. F. & Manders, E. M. M. (2007). Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nature Biotechnology 25, 249253.
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. (2004). Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 10071009.
Ji, N., Shroff, H., Zhong, H. & Betzig, E. (2008). Advances in the speed and resolution of light microscopy. Current Opinion in Neurobiology 18, 605616.
Keller, P. J., Schmidt, A. D., Wittbrodt, J., & Stelzer, E. H. (2008). Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 10651069.
Khodjakov, A. & Rieder, C. L. (2006). Imaging the division process in living tissue culture cells. Methods 38, 216.
Krishnaswami, V., Van Noorden, C. J., Manders, E. M. & Hoebe, R. A. (2014). Towards digital photon counting cameras for single-molecule optical nanoscopy. Optical Nanoscopy 3, 1.
Magidson, V. & Khodjakov, A. (2013). Circumventing photodamage in live-cell microscopy. Methods in Cell Biology 114, 545560.
Marx, V. (2015). Probes: paths to photostability. Nature Methods 12, 187190.
Mikhailov, A., Shinohara, M. & Rieder, C. L. (2005). The p38-mediated stress-activated checkpoint: a rapid response system for delaying progression through antephase and entry into mitosis. Cell Cycle 4, 5762.
Planchon, T. A., Gao, L., Milkie, D. E., Davidson, M. W., Galbraith, J. A., Galbraith, C. G. & Betzig, E. (2011). Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nature Methods 8, 417423.
Schilling, Z., Frank, E., Magidson, V., Wason, J., Lončarek, J., Boyer, K., Wen, J. & Khodjakov, A. (2012). Predictive-focus illumination for reducing photodamage in live-cell microscopy. Journal of Microscopy 246, 160167.
Staudt, T., Engler, A., Rittweger, E., Harke, B., Engelhardt, J. & Hell, S. W. (2011). Far-field optical nanoscopy with reduced number of state transition cycles. Optics Express 19, 56445657.
Stelzer, E. H. (2015). Light-sheet fluorescence microscopy for quantitative biology. Nature Methods 12, 2326.
Stephens, D. J. & Allan, V. J. (2003). Light microscopy techniques for live cell imaging. Science 300, 8286.
Uetake, Y., Lončarek, J., Nordberg, J. J., English, C. N., La Terra, S., Khodjakov, A., & Sluder, G. (2007). Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells. Journal of Cell Biology 176, 173182.
Vettenburg, T., Dalgarno, H. I., Nylk, J., Coll-Lladó, C., Ferrier, D. E., Čižmár, T., Gunn-Moore, F. J. & Dholakia, K. (2014). Light-sheet microscopy using an Airy beam. Nature Methods 11, 541544.
Verveer, P. J., Hanley, Q.S., Verbeek, P.W., Van Vliet, L.J. & Jovin, W.M. (1998). Theory of confocal fluorescence imaging in the programmable array microscope (PAM). Journal of Microscopy 189, 192198.
Waterman-Storer, C. M., Desai, A., Bulinski, J. C. & Salmon, E. D. (1998). Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Current Biology 8, 1227.
Williams, E. S., Stap, J., Essers, J., Ponnaiya, B., Luijsterburg, M. S., Krawczyk, P. M., Ullrich, R. L., Aten, J. A. & Bailey, S. M. (2007). DNA double-strand breaks are not sufficient to initiate recruitment of TRF2. Nature Genetics 39, 696698.

Spatially-controlled illumination microscopy: For prolonged live-cell and live-tissue imaging with extended dynamic range

  • Venkataraman Krishnaswami (a1) (a2), Cornelis J. F. Van Noorden (a1), Erik M. M. Manders (a2) and Ron A. Hoebe (a1)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed