Focus On Neurobiological Imaging

Nikon supports a variety of microscope-based neurobiological imaging applications.

The neurobiological imaging landscape is rapidly shifting due to technical advances in established fields such as tissue clearing and multiphoton in vivo imaging, but also the emergence of exciting new technologies such as optogenetics and super-resolution microscopy.  Nikon is committed to the development, refinement, and support of technologies enabling cutting-edge neurobiological research.

Deep in vivo and Intravital Imaging

Multiphoton imaging provides superior optical sectioning in large specimens.

Multiphoton microscopies exploit high-power femtosecond pulsed infrared lasers to excite fluorescence deep inside challenging samples. The power density required for absorption of multiple photons is only realized at the focus of the laser, confining excitation to a small focal volume and thus minimizing out-of-focus fluorescence. Combined with ability of IR light to penetrate strongly scattering media, multiphoton imaging provides both superior depth penetration and optical sectioning, important for imaging thick and scattering tissues.

  • The A1R MP+ multiphoton system provides the deep optical sectioning performance of a multiphoton system along with the high-speed imaging ability of our new high definition (HD) resonant scanner. Resonant scanning enables video rate imaging (30 frames per second and greater), important for capturing fast dynamics such as calcium signaling. Other advanced features include dual beam scanning for simultaneous multichannel imaging, auto laser alignment, non-descanned detection, 1300 nm compatibility, and more.
  • Our CFI75 Water Dipping Series of microscope objectives provide exceptional IR transmission, high NA, long working distances, and large fields of view. The 25xW MP features an industry-leading NA = 1.10 and 2.0 mm working distance. Our LWD 16xW lens can be used for imaging at 5.6x, 32x, and 64x magnifications together with a dedicated magnification module for our FN1 upright microscope.

In vivo imaging of an anesthetized YFP-H mouse (4-week-old) via open skull method. Visualization of the entire layer V pyramidal neurons and the deeper hippocampal neurons. Deep imaging achieved for 3-dimensional imaging of hippocampal dendrites up to 1.4 mm into the brain.

Captured with episcopic GaAsP NDD for 1300 nm and CFI75 Apochromat 25XC W 1300 objective (NA 1.10, WD 2.0 mm)

Excitation wavelength: 1040 nm

Photos courtesy of: Drs. Ryosuke Kawakami, Terumasa Hibi and Tomomi Nemoto, Research Institute for Electronic Science, Hokkaido University

① Pyramidal cells in layer V

② White matter

③ Alveus

④ Hippocampal pyramidal cells

⑤ Hippocampus 3D zoom image

Electrophysiology

Researchers require a stable and accessible microscope platform for performing sensitive electrophysiology experiments.

Electrophysiology is the study of the electrical properties of biological systems, such as neurons, using microelectrodes to read and manipulate electrical signals. Sample accessibility is a top priority for electrophysiologists as placement of electrodes requires micron-level precision. The microscope must be exceptionally stable and not interfere with other measuring apparatus. The Nikon FN1 upright microscope features a slim and accessible I-shaped profile for maximum sample accessibility and system customization, perfect for patch clamp experiments. Our sliding nosepiece for the FN1 makes it easy to retract and switch objectives while avoiding collisions with sensitive instrumentation. Our CFI60 Water Dipping Series of objectives lenses provide steep manipulator approach angles, high NA, long working distances, and inert ceramic tips.

Optogenetics

Patterned illumination enables powerful optogenetics based applications.

Optogenetics is a family of experimental techniques providing light-gated control of biological processes, famously including the stimulation of single neurons expressing exogenous channelrhodopsins. Furthermore, optical reporters such as fluorescent calcium and voltage sensors may be used to read out activity in an all-optical experimental design. However, control of the spatiotemporal distribution of stimulation must be precise, and be independent from the readout illumination. Patterned optogenetic stimulation is most robustly achieved using a Digital Micromirror Device (DMD), an array of hundreds of thousands of independently controllable micro-scale mirrors. Nikon offers multiple DMD solutions as well as point-scanning stimulation devices for optogenetics research*.

Read our application note on optogenetics to learn about using a DMD to control signaling in developing embryos.

* Available products may vary depending on world area.

Cleared Tissue Imaging

Objectives designed for imaging cleared and refractive index-matched samples.

Clearing and refractive index (RI) matching techniques such as CLARITY have fundamentally changed how we approach tissue imaging. Previously, researchers would mechanically section large tissues and sequentially image individual slices. Unfortunately, this time-consuming approach disrupts the 3D context of the sample, obscuring critical relationships. Tissue clearing makes it possible to characterize the continuous three-dimensional structure of whole specimens quickly, providing greater insight than previous. However, optimal imaging of cleared tissues requires specialized optics featuring low magnification, high numerical aperture, and the ability to tune optical corrections in the objective for different refractive indices corresponding to different clearing methods. Nikon has recently introduced two new microscope objectives to address these needs:

  • Our CFI60 10xC Glyc glycerin immersion objective can correct for refractive indices between 1.33 – 1.51, able to match the RI of most common clearing reagents, and features NA = 0.5.
  • Nikon’s CFI90 20xC Glyc glycerin immersion objective features a unique 90 mm parfocal distance, allowing for both an incredibly high NA of 1.0 and a large field of view, all while maintaining an ultra-long working distance of 8.2 mm. This combination of capabilities results in an objective ideal for imaging large tissues in combination with the large image scanning/stitching tools available in the NIS-Elements software.

PFC

Striatum

Hippocampus and Amygdala

Ultrastructural Imaging with Super-Resolution

Super-resolution STORM illuminates previously unseen neuronal details with nanoscale resolution and high specificity.

Unlike standard diffraction-limited microscopies, super-resolution techniques provide sub-organelle level resolution. Ultrastructural cellular details previously only observed by electron microscopy (EM) have been resolved by super-resolution, and with the superior molecular specificity and multiplexing capability of fluorescence imaging. STochastic Optical Reconstruction Microscopy (STORM) uses the concept of single molecule localization to pinpoint single fluorescent emission events with single nanometer precision. This technique was used to discover both the periodic structure of the axon cytoskeleton (unobservable by EM) and the architecture of the axon initial segment scaffold. Several research groups have applied STORM towards quantifying synaptic protein distribution. Nikon makes the STORM technique accessible to neuroscientists via our N-STORM system. Furthermore, fast super-resolution imaging at up to 15 frames per second is now possible with our N-SIM S structured illumination system.

Christophe Leterrier Ph.D., NeuroCyto, Marseille, France

Large-Image Stitching

Powerful tools advanced image acquisition and analysis

NIS-Elements software provides comprehensive control of acquisition, analysis, and visualization of your data. Neurobiology applications frequently require working with large samples. NIS-Elements makes it easy to perform both large image scanning/stitching as well as z-stacking, simplifying the acquisition of large 3D (and 4D) datasets. Powerful visualization tools provide stunning images for data sharing and presentation. Graphical programming tools such as JOBS simplifies custom acquisition and analysis workflows including advanced conditional workflows (e.g. automated acquisitions that depend on analysis results). Time-consuming analysis steps such as image segmentation can be simply integrated into intelligent and automated image analysis routines.