Neuronal signals can vary on millisecond timescales, with communicating neurons often separated by
hundreds of microns. Imaging such fast dynamics over extended volumes presents a challenge for
standard fluorescence microscopes. For example, a new generation of genetically encoded voltage
indicators are becoming available whose response times are on the order of milliseconds.
To address this challenge, we propose to develop a new type of microscope that can perform near-
1kHz-rate high resolution volumetric imaging over 1mm x 1mm x 0.2mm scales. Our proposed solution,
called Multi-Z confocal microscopy, is based on two key ideas. First, it combines high-NA detection with
low-NA illumination. The former leads to high signal collection efficiency; the latter leads to axially
extended illumination over an extended range of Z depths. Second, it detects multiple signals from this
extended depth range using multiple confocal pinholes that are axially distributed. The pinholes are
reflecting, so that signal rejected by one pinhole is sent to the next pinhole, and so forth. In this manner,
no signal is lost, and signal collection efficiency remains high.
Two versions of our microscope will be developed, based on line-scan and sheet-scan illumination.
The former provides better optical sectioning and will be designed for calcium imaging. The latter
provides much higher speed (near kHz-rate) and will be designed for voltage imaging. In contrast to
conventional line-scan or light-sheet microscopes, our lines and sheets are oriented parallel to the optical
axis rather than perpendicular to the axis. The versatility of both versions of our microscope will be
augmented with the addition of optogenetic stimulation and combined confocal reflectance contrast.
We have enlisted the help of Drs. Alberto Cruz-Martin (BU, Biology) and Xue Han (BU, BME), who
both specialize in in-vivo mouse imaging and have expertise in the genetic or viral delivery of novel
probes, animal preparation, head fixation, behavior protocols, etc.. For voltage imaging, we will test a
state-of-the-art indicator called SomArchon1 (provided by the Dr. Ed Boyden lab). The ability to image
large sample volumes at 1kHz rates is of general applicability and can be broadly impactful. Our goal will
be to demonstrate the effectiveness of our Multi-Z microscopy technique by performing calcium and
voltage imaging over entire populations of neurons in behaving mice.