Imaging Instrumentation

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an advanced technique to make 3D images of biological cells in tissues with superior z-axis (vertical) resolution, generating easily interpretable data for reconstruction. FIB-SEM produces detailed images of cell features like protein complexes and membranes through an electron beam's sequential etching of a sample surface. Until the developments of Enhanced FIB-SEM by C. Shan Xu and Harald Hess, presented here, the image produced had been limited to a single cell or small volume of cells.

Although multiple electron microscopic techniques can achieve ~10 nm isotropic resolution, none are well-suited to image volumes at or above 1 mm³. Structural imaging at this scale opens new experimental achievements, like imaging large sections of vertebrate brains or an entire invertebrate nervous system. Unfortunately, current methods don’t meet the automatic resolution and throughput requirements needed to trace volumes spanning many cubic millimeters.

MIMMS (Modular In vivo Multiphoton Microscopy System) is a modular platform for performing two‐photon laser scanning microscopy (TPLSM) optimized for in vivo applications. The system generally uses commercially available core parts for movement of the objective in the X‐, Y­‐, and Z­‐axis linear translation and X‐axis rotation for in vivo experiments. The backbone of the design is a movable, raised optical breadboard, providing a large area for affixing optical equipment associated with the microscope.

This microscope images megapixel fields of view at over 1000 Hz within scattering tissue by scanning excitation lines at multiple angles and recovering images computationally. It can image large tissue volumes dramatically faster than other 2-photon microscopes.

Figure 1: Rendering of the SLAP Microscope system. A 3D pdf of the system above can be obtained here.

The Janelia PMT Controller is an updated design for use in high-performance microscopy systems. It was designed and built to meet the needs of the new large field of view 2 photon random access mesoscope, also available via license or purchase. You can find the complementary PMT Amplifier here.

Hardware Overview

Researchers at HHMI Janelia Research Campus have designed and built a 2-photon random access mesoscope (2P-RAM) that provides rapid imaging anywhere within a large tissue volume.

Cellular imaging can link activity in populations of neurons with behavior. But individual neurons are small, around 10μm in diameter. Most microscopes with subcellular resolution have small fields of view and, therefore, cannot image neurons in multiple brain areas simultaneously. Most large-field-of-view microscopes do not resolve single cells, especially in the axial dimension.

The standard reflection objective is designed to work in air with reasonably high resolution. It is a lightweight, simple structure (only one or two mirrors are needed) with little chromatic dispersion and a relatively long working distance. However, it cannot be used in an immersion medium other than air. Otherwise, the light passing through the objective will suffer from considerable aberrations, and those will reduce resolution at the sample.

To obtain the documentation, designs, and parts list, please contact innovation@janelia.hhmi.org.

See additional explanations from the scientists here: https://www.janelia.org/lab/keller-lab/microscopes.

MIMMS (Modular In vivo Multiphoton Microscopy System) is a modular platform for performing two‐photon laser scanning microscopy (TPLSM) optimized for in vivo applications. The system generally uses commercially available core parts for movement of the objective in the X‐, Y­‐, and Z­‐axis linear translation and X‐axis rotation for in vivo experiments. The backbone of the design is a movable, raised optical breadboard, providing a large area for affixing optical equipment associated with the microscope.

This technology advances imaging techniques of biomicroscopy using an innovative type of a wide-field Multi-Focus microscope to enable fast, high-resolution 3D imaging.