Inverted Microscope 2: A Comprehensive Guide for Physics Students

Inverted microscopes, such as the Olympus IX83 and Nikon ECLIPSE Ti2, are advanced imaging systems designed for a variety of research applications. These microscopes offer several observation methods, including fluorescence (with blue/green and ultraviolet excitation), differential interference contrast (DIC), phase contrast, and brightfield, making them versatile tools for physics students and researchers.

Olympus IX83: Motorized Precision and Automated Workflows

The Olympus IX83 is a fully-motorized inverted microscope that can be expanded with additional modules to meet growing research needs. It offers a two-deck system for high-speed, fully automated device selection during live cell research and advanced image acquisition, as well as a one-deck system with a large field number and TruFocus compatibility for live cell imaging. The IX83 features motorized focus, a Z drift compensator, and a variety of observation tubes and condensers to suit different imaging requirements.

Motorized Focus and Z Drift Compensation

The IX83’s motorized focus system allows for precise and repeatable focusing, with a minimum step size of 0.01 μm. This high-precision focusing is crucial for applications such as 3D imaging, where accurate z-positioning is essential. Additionally, the Z drift compensator helps maintain focus during long-term experiments, ensuring that the sample remains in focus even as environmental conditions change.

Observation Tubes and Condensers

The IX83 offers a range of observation tubes and condensers to accommodate various imaging techniques. The observation tubes include options for binocular, trinocular, and multi-port configurations, allowing for flexible camera and eyepiece placement. The condensers, such as the UIS2 condenser, provide optimized illumination for brightfield, phase contrast, and DIC imaging, ensuring high-quality images across different observation methods.

Nikon ECLIPSE Ti2: Unparalleled Field of View and Intelligent Imaging

inverted microscope 2

The Nikon ECLIPSE Ti2, on the other hand, boasts an unparalleled 25mm field of view (FOV) that maximizes the sensor area of large-format CMOS cameras and significantly improves data throughput. Its exceptionally stable, drift-free platform is designed for super-resolution imaging, and its hardware-triggering capabilities enhance high-speed imaging applications. The Ti2 also offers intelligent functions that guide users through imaging workflows and automatically record the status of each sensor during acquisition, providing quality control for imaging experiments and enhancing data reproducibility.

Large Field of View and Sensor Optimization

The ECLIPSE Ti2’s 25mm field of view is significantly larger than the typical 18-22mm field of view found in many inverted microscopes. This expanded FOV allows for the use of large-format CMOS cameras, which can capture more of the sample in a single image. This, in turn, improves data throughput and reduces the need for tile-scanning, which can be time-consuming and introduce stitching artifacts.

Stable, Drift-Free Platform for Super-Resolution Imaging

The ECLIPSE Ti2’s exceptional stability and drift-free platform make it an ideal choice for super-resolution imaging techniques, such as Structured Illumination Microscopy (SIM) and Stochastic Optical Reconstruction Microscopy (STORM). The stable platform ensures that the sample remains in focus and properly aligned throughout the imaging process, which is crucial for achieving high-resolution, artifact-free images.

Hardware Triggering and Intelligent Workflow Guidance

The ECLIPSE Ti2’s hardware-triggering capabilities enhance high-speed imaging applications by allowing for precise synchronization between the microscope and external devices, such as cameras and light sources. Additionally, the microscope’s intelligent functions guide users through imaging workflows and automatically record the status of each sensor during acquisition, providing quality control for imaging experiments and enhancing data reproducibility.

Quantifiable Metrics for Microscope Performance and Image Quality

Inverted microscopes like the Olympus IX83 and Nikon ECLIPSE Ti2 can provide a range of quantifiable metrics to ensure the quality and reliability of microscope performance and image data. These metrics include:

Resolution

The lateral and axial resolution of a microscope can be characterized using a point spread function (PSF), which measures the response of the microscope to a point source of light in the x, y, and z dimensions. The PSF can be used to calculate the Rayleigh criterion for the microscope’s resolution, which is given by the formula:

$d = 0.61 \lambda / NA$

where $d$ is the minimum resolvable distance, $\lambda$ is the wavelength of the light, and $NA$ is the numerical aperture of the objective lens.

Field Illumination

Field illumination determines the intensity quantification over the whole field of view of the system, which is crucial for tile-scan reconstructions and preventing inhomogeneous sample bleaching. Uniform field illumination can be achieved through careful alignment of the illumination system and the use of appropriate condenser lenses.

Coregistration

Coregistration characterizes the chromatic mismatch between different color channel images of the same fluorescent emitter in the x, y, and z directions. This is important for accurate multi-color imaging and quantitative analysis of co-localized signals.

Illumination Power Stability

Illumination power stability at different time scales determines the reproducibility and quantification of the experiment. Fluctuations in illumination power can lead to artifacts and inconsistencies in the acquired data, so it is essential to monitor and maintain stable illumination throughout the imaging process.

Stage Drift and Positioning Repeatability

The imaging quality depends on the stability of the sample in x, y, and z, which can be characterized by drift monitoring and stage positioning repeatability. Excessive drift or poor stage positioning can compromise the quality of the acquired images and the reliability of the experimental results.

Camera Sensor Noise and Offset

The noise and offset characterization of the camera sensor influences quantification, especially for weak signals. Proper characterization of the camera’s performance is crucial for accurate intensity measurements and signal-to-noise ratio calculations.

By understanding and monitoring these quantifiable metrics, physics students and researchers can ensure the quality and reliability of their microscope-based experiments, leading to more robust and reproducible results.

Conclusion

Inverted microscopes like the Olympus IX83 and Nikon ECLIPSE Ti2 offer advanced imaging capabilities and a range of quantifiable metrics for ensuring the quality and reliability of microscope performance and image data. These microscopes are suitable for various research applications, including live cell imaging, high-content applications, confocal and super-resolution imaging, and laser applications. By mastering the technical details and quantifiable metrics of these inverted microscopes, physics students can unlock the full potential of these powerful imaging tools and conduct cutting-edge research in their field.

References:

  1. Huisman, M., Hammer M., Rigano A., Farzam F., Gopinathan R., Smith C., Grunwald D., Strambio-De-Castillia C. (2022). Quality assessment in light microscopy for routine use: Tools, acquisition protocols, and automated analysis methods to assess quality control metrics for light fluorescence microscopy. NCBI.
  2. Olympus Life Science. (2013). IX83 Inverted Microscope.
  3. Nikon Instruments. (n.d.). ECLIPSE Ti2 Series | Inverted Microscopes.
  4. Zeiss Microscopy. (n.d.). Axio Observer 7 – Inverted microscopes.