ABSTRACT
Using Ca(2+) imaging in freely behaving mice that repeatedly explored a familiar environment, we tracked thousands of CA1 pyramidal cells' place fields over weeks. Place coding was dynamic, as each day the ensemble representation of this environment involved a unique subset of cells. However, cells in the â¼15-25% overlap between any two of these subsets retained the same place fields, which sufficed to preserve an accurate spatial representation across weeks.
Subject(s)
Action Potentials/physiology , CA1 Region, Hippocampal/physiology , Calcium/metabolism , Pyramidal Cells/physiology , Animals , Environment , Memory/physiology , MiceABSTRACT
The light microscope is traditionally an instrument of substantial size and expense. Its miniaturized integration would enable many new applications based on mass-producible, tiny microscopes. Key prospective usages include brain imaging in behaving animals for relating cellular dynamics to animal behavior. Here we introduce a miniature (1.9 g) integrated fluorescence microscope made from mass-producible parts, including a semiconductor light source and sensor. This device enables high-speed cellular imaging across â¼0.5 mm2 areas in active mice. This capability allowed concurrent tracking of Ca2+ spiking in >200 Purkinje neurons across nine cerebellar microzones. During mouse locomotion, individual microzones exhibited large-scale, synchronized Ca2+ spiking. This is a mesoscopic neural dynamic missed by prior techniques for studying the brain at other length scales. Overall, the integrated microscope is a potentially transformative technology that permits distribution to many animals and enables diverse usages, such as portable diagnostics or microscope arrays for large-scale screens.
Subject(s)
Microscopy, Fluorescence/instrumentation , Miniaturization , Neurons/metabolism , Animals , Calcium/metabolism , Calcium Signaling , Male , Mice , Molecular Imaging , SemiconductorsABSTRACT
We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.
Subject(s)
Brain/blood supply , Brain/cytology , Capillaries/cytology , Lenses , Micro-Electrical-Mechanical Systems/instrumentation , Microscopy, Fluorescence, Multiphoton/instrumentation , Animals , Computer-Aided Design , Equipment Design , Equipment Failure Analysis , Mice , Miniaturization , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A central goal in biomedicine is to explain organismic behavior in terms of causal cellular processes. However, concurrent observation of mammalian behavior and underlying cellular dynamics has been a longstanding challenge. We describe a miniaturized (1.1 g mass) epifluorescence microscope for cellular-level brain imaging in freely moving mice, and its application to imaging microcirculation and neuronal Ca(2+) dynamics.
Subject(s)
Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Miniaturization/methods , Movement/physiology , Animals , Brain/physiology , Fiber Optic Technology/instrumentation , Fiber Optic Technology/methods , Mice , Miniaturization/instrumentation , Time FactorsABSTRACT
Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.
Subject(s)
Lenses , Microscopy, Fluorescence, Multiphoton/instrumentation , Computer Systems , Equipment Design , Equipment Failure Analysis , Mechanics , Microscopy, Fluorescence, Multiphoton/methods , Miniaturization , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.
Subject(s)
Fiber Optic Technology/methods , Microscopy, Fluorescence/methods , Animals , Humans , Optical FibersABSTRACT
We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9 g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.
