Scientists studying the brain’s neuroplasticity have needed to focus on tiny structures within neural tissue, but the resolution provided by conventional confocal and two-photon laser scanning microscopy has not been adequate for the task.The larger goal of Howard Hughes Medical Institute investigator Bernardo L. Sabatini and his group at Harvard Medical School is to observe how synapses – connections between neurons – work in healthy brains as well as how they look and function in neuropsychiatric diseases such as autism and Alzheimer’s. They need the ability to see the finer features of the synapses to form new hypotheses about how neurons signal to one another.A mouse hippocampal CA1 pyramidal neuron is filled with Alexa Fluor 594 dye. The red and yellow areas show stimulated emission depletion two-photon laser scanning microscope imaging, while the blue shows normal two-photon imaging. Photo courtesy of Jun Ding and Bernardo Sabatini.The investigators developed a hybrid technique called stimulated emission depletion two-photon laser scanning microscopy, which has enabled them to clearly image dendritic spines – tiny projections that extend from the edges of neurons. This adaptation of two-photon microscopy, which has allowed fluorescent imaging of neurons below the surface of the brain, has enabled them to improve spatial resolution threefold in the radial direction. The technique allows them to examine slices of brain tissue that are thick enough to show how the connections between nerve cells are formed and regulated. It reveals greater detail in the structure of dendritic spines located approximately 100 μm below the surface of brain slices, according to Sabatini.The group was able to image beyond the diffraction limit, a fundamental rule in physics resulting in blurred microscopic images of specimens – such as a protein within a cell – that are less than half the wavelength of light. The researchers overcame the limit by using near-IR lasers for both pulsed two-photon excitation and continuous-wave stimulation emission depletion, a super-resolution technique. The latter technique uses one laser light to excite a small spot within the sample and another laser to lower the excitation level in a ring surrounding the spot. This shrinks the portion of the sample in which fluorescence occurs to such a small region that spatial resolution is greatly improved.The depletion ring is created with a vortex phase plate – a small piece of glass with a spiral-shaped coating – that is used to make the light cancel out in the center of the ring. Stimulated emission depletion microscopy typically can operate only near the surface of tissue and cells. However, by combining it with two-photon microscopy, Sabatini and his colleagues are able to use it at much greater depths.After adjusting and restructuring the microscope parts, the investigators were able to view inside cells deep within brain tissue to clearly see dendritic spines and other smaller structures. They found the fluorophore Alexa Fluor 594 suitable for use with the technique.Sabatini said that they expect to see even higher resolution – by another factor of three – by using power more efficiently to pulse the depletion light in time with the excitation. The current setup uses brief pulses of light to illuminate the spots, while the depletion beam stays on constantly, he added.To make the setup more accessible for beginners, they are writing computer programs that automate the process of aligning the laser beam.