SPECIAL SECTION: MICROSCOPY IN BIOLOGY CURRENT SCIENCE, VOL. 105, NO. 11, 10 DECEMBER 2013 1537 *For correspondence. (e-mail: balaji@cns.iisc.ernet.in) Optical microscopy methods for understanding learning and memory Aditya Singh, Suraj Kumar, Vikram Pal Singh and J. Balaji* Centre for Neuroscience, Indian Institute of Science, Bangalore 560 012, India Structural dynamics of dendritic spines is one of the key correlative measures of synaptic plasticity for encoding short-term and long-term memory. Optical studies of structural changes in brain tissue using con- focal microscopy face difficulties of scattering. This results in low signal-to-noise ratio and thus limiting the imaging depth to few tens of microns. Multiphoton microscopy (MpM) overcomes this limitation by using low-energy photons to cause localized excitation and achieve high resolution in all three dimensions. Multi- ple low-energy photons with longer wavelengths mini- mize scattering and allow access to deeper brain regions at several hundred microns. In this article, we provide a basic understanding of the physical pheno- mena that give MpM an edge over conventional microscopy. Further, we highlight a few of the key studies in the field of learning and memory which would not have been possible without the advent of MpM. Keywords: Learning, memory, optical microscopy, two-photon excitation. SANTIAGO Ramón y Cajal through his neuroanatomical studies using Golgi stain and light microscopy put forth the neuronal hypothesis 1 . Observing the Golgi stained slices of various brain regions (Figure 1), Cajal suggested that the membranous protrusions found on the dendrites of the neuron may serve as the putative sites for synapse formation. He further thought that these protrusions, known as synaptic spines, could be plastic and can possibly serve as information storage sites. However, for a long time it was difficult to demonstrate whether the dendritic spines are plastic. Developments in the field of depth- resolved live cell fluorescence imaging and confocal microscopy made it possible to demonstrate the dynamic nature of the spines 2 . Despite these findings it was not clear if the spines continue to remain dynamic after development and if plasticity-inducing events trigger new spine formation. In order to answer these questions it is necessary to follow the spine dynamics, while such plas- ticity-inducing changes are occurring in a neuron. Imag- ing spines present inside a scattering live brain tissue required a technique that is benign and minimally affected by scattering. One of the major limiting factors in addressing this was lack of such microscopic methods to image live brain slices with near-micron resolution. Advent of two-photon microscopy 3 helped overcome this limitation and made it possible to show that the new spines are formed after long-term potentiation (LTP) induction in the CA1 region 4 . In general, the hypothesis of correlation between new spine formation and memory storage has gained favourable evidence 5–8 . Despite these developments, the significance of new spine formation and its contribution to information storage have been debated. Several recent studies utilizing two-photon microscopy of mice cortical regions have established that the spine density is altered following a variety of learning tasks 6,9 . Much of these developments in our understand- ing of plasticity at the synapse would have been difficult to come by if not for the pertinent developments in opti- cal microscopy. One such development is the use of two- photon microscope to extend the depth of imaging to reach the upper layers of the cortex 10 . Scattering of light by brain tissue limits the imaging depth in light microscopy 11 . Maximal imaging depth, de- fined as the depth at which the signal generated from the focal volume becomes indistinguishable from the back- ground noise is affected by light scattering 12 . Multiphoton microscopy (MpM) uses multiple photons of lower Figure 1. Santiago Ramón y Cajal observing brain slices to sketch neuronal structures under bright-field compound microscope.