4f Koehler Transmitted Illumination Condenser for Teaching and Low-Cost Microscopic Imaging

ABSTRACT Transmitted light imaging is an important tool in biophysics for applications that include sample analysis, recording samples whose viability is compromised by high levels of illumination (e.g., live cell tracking), and finding regions of interest in a sample. Koehler transillumination is a powerful illumination method used in commercial microscopes; yet commercial Koehler condensers are expensive, are difficult to integrate into tabletop systems, and make learning the concepts of Koehler illumination difficult because of their closed-box nature. Here, we show a protocol to build a simple 4f Koehler illumination system that offers advantages with respect to commercial condensers in terms of simplicity, cost, and compatibility with tabletop systems, such as open-source light sheet fluorescence microscopes. We include step-by-step instructions that can be followed by advanced undergraduate or graduate students without experience in optics on how to align and assemble the illuminator, along with a list of the necessary parts for assembly. We also include supplemental material that describes 4 supporting educational activities students can conduct with the apparatus and helps in the understanding of key concepts relevant to Koehler illumination and optics. The performance of the system is comparable to that of commercial condensers and significantly better, in terms of illumination homogeneity and depth of field (optical sections are possible), than that of LED flashlights, such as those found in low-cost diagnostic devices and tabletop systems.

In-Column Energy Filtering Transmission Electron Microscope (EFTEM) - Integrated Analysis of Energy Loss Signals

In an EFTEM the full range of signals generated by interaction of the primary electron beam with the specimen can be detected. Thus operating such a system and generating combined digital information usually is a rather complex issue. The demands of the users on the other hand are to achieve results fast, easily and reproducibly. Moreover it should be possible to tailor the integral system according to dedicated needs. In general it should be no problem to use modern digital equipment and just let an integral computer control everything. However, in order to make such digital settings really useful, there should be no D/A conversion in between the data paths of the microscope because any analogue system tends to drift and the changes of lens parameters between different modes of operation should be minimised to overcome hysteresis. Fully digitised in-column EFTEMs like the LEO EFTEMs with OMEGA filter and Koehler illumination including also multiple parallel and serial remote capabilities provide optimum preconditions to fulfil the demands.

Laser Koehler Epi-Illumination for Raman and Fluorescence Microscopic Imaging

A comparison of microscopic Raman images acquired with an optical-fiber critical (Nelson) illumination system, an optical-fiber Koehler laser illumination system, and Koehler laser illumination without an optical fiber demonstrates performance differences between the three illumination methods. Best images are obtained with optical-fiber Koehler illumination.

Cryo imaging using an energy-filtering TEM (EFTEM): Optimum use of phase-contrast transfer function (PCTF)

Lack of contrast is one of the numerous problems arising from imaging of vitrified biological macromolecules in a TEM. This is due to the similar density of biological material and of vitreous ice and to the high background of inelastic scattering in the ice which is about four times that of carbon.Consequently in a CTEM images have to be recorded 1-3 μm underfocus to maximise phase contrast which in the same sense decreases the reliability of density information.Elastic filtering using an (EFTEM) allows closer to focus imaging still achieving considerable contrast. For 3D reconstruction of molecular densities the largest source of error is likely to arise from contributions of the PCTF. Thus, such images have to be corrected for the PCTF, which is much morereliably done for elastically filtered images close to focus.Thin vitreous ice films containing the virus particles were prepared on holey carbon grids and examined with cryo EM procedures. Images of frozen-hydrated cucumber mosaic virus (CCMV) particles ( Ø of roughly 25 nm) were recorded in Elastic Brightfield mode using the Zeiss EM 912 OMEGA with integrated imaging spectrometer and Koehler Illumination. Magnification was 50.000x, HT 120 kV, energy width 7 eV, total dose 800 electrons /nm2.

Electron spectroscopic imaging and diffraction

A new Zeiss TEM with an imaging Omega filter is a fully digitized, side-entry, 120 kV TEM/STEM instrument for materials science. The machine possesses an Omega magnetic imaging energy filter (see Fig. 1) placed between the third and fourth projector lens. Lanio designed the filter and a prototype was built at the Fritz-Haber-Institut in Berlin, Germany. The imaging magnetic filter allows energy-filtered images or diffraction patterns to be recorded without scanning using efficient area detection. The energy dispersion at the exit slit (Fig. 1) results in ∼ 1.5 μm/eV which allows imaging with energy windows of ≤ 10 eV. The smallest probe size of the microscope is 1.6 nm and the Koehler illumination system is used for the first time in a TEM. Serial recording of EELS spectra with a resolution < 1 eV is possible. The digital control allows X,Y,Z coordinates and tilt settings to be stored and later recalled.

Design of an analytical TEM with integrated imaging ω spectrometer

Conventional transmission electron microscopy (CTEM) can be used for high resolution imaging of specimens and for the analysis of minute specimen areas. The capabilities of such an instrument are strongly improved by the integration of an imaging electron energy loss spectrometer. All imaging and diffraction techmques are provided in such an energy filtered transmission electron microscope (EFTEM).In addition to the well-known objective lens for Koehler illumination, the new Zeiss EFTEM features a projective lens system which integrates a new imaging ω-spectrometer comprising four individual magnets and one hexapole corrector Fig.l and Fig. 3 show the design of this microscope.