Imaging has always played a fundamental role in the biosciences. In recent years, significant advances have been made in the performance, availability and cost of imaging equipment. For instance, sensitive high frame rate CCD cameras are now available from many vendors and laser technology continues to improve. Despite this progress, the microscope and its associated optics remain at the core of an imaging system. Nikon has been manufacturing inverted microscopes for biomedical research since the 1960s when they released the model MD, the latest incarnation of this microscope is the TE2000. Two major objectives were achieved during the development of the TE2000: to provide multimode imaging capability on one platform and to improve the image quality generated by the different optical modes.
To facilitate different imaging configurations, Nikon employs infinity optics, the current industry standard for both biomedical and industrial applications. Objectives on infinity optical systems generate parallel light waves whereas normal objectives produce light focused to a set point. An additional lens subsequently refocuses the parallel light produced by an infinity-corrected objective, indeed, the objective and the refocusing lens can be considered as two halves of a conventional compound lens. This arrangement creates a light path that contains optically ‘accessible’ parallel light waves. As such, additional optical components, including epi-fluorescence illuminators, prisms and polarizers, can be included in the parallel portion of the light path without changing the final focal point. Older, fixed focal point microscopes were very sensitive to auxiliary optical elements and often required corrective optics to re-establish their effective focal point which could degrade image quality.
To exploit the infinity optics on the TE2000, a series of modules are available from Nikon to configure the imaging modes of the microscope. These modules can be ‘layered’ - in a so-called ‘stratum structure’ – to generate a microscope that is capable of multiple imaging modalities. For instance, a TE2000 platform can be configured with two turrets each containing six filter blocks: the bottom turret can be used for total internal reflection (TIRF) illumination whereas the top turret can be used with a conventional lamp for simultaneous epi-fluorescence illumination. Similarly, a microscope can be configured for confocal and TIRF imaging or laser tweezers and fluorescence illumination. Layering optical components sometimes requires that the stage be raised to create space for optical components, however, as outlined above, the infinity optics accommodate these changes. This overall design concept consequently provides ‘off-the-shelf’ imaging modules for many optical methods in a simple, ready-to-go format.
To improve image quality from the TE2000 microscope, Nikon have developed new features as well as relying on continued advances in objective and filter technology. The Noise Terminator, for example, is engineered into the filter cube and turret to suppress background signal thus increasing the signal-to-noise of images. This extremely simple feature - explained pictorially at Nikon’s MicroscopyU website – works by diverting stray excitation light that passes through the dichroic mirror away from the detection light path. No additional optical components are required and thus, there is no effect on the throughput of emitted light. In terms of objectives, Nikon recently developed a 100x with 1.45 numerical aperture (NA) lens. This objective is extremely popular in the lab, in addition to its designed purpose of total internal reflection fluorescence, this objective has outstanding light-throughput and resolution for normal fluorescence work. Advances in objectives come quickly though - Nikon have already released a 1.49 NA TIRF objective that supercedes our 1.45 NA. Nikon also offer an extensive range of objectives optimized for alternative applications.
The TE2000 family of microscopes has three models: the basic TE2000-S, the TE2000-U and the range topping, motorized TE2000-E. The TE2000-S has two ports (eyepieces and a detector) whereas the TE2000-U and –E have four and five ports respectively to permit more detection methods. The TE2000-S and -E also feature a built-in intermediary 1.5x lens to increase resolution on CCD chips. Our lab currently has three TE2000 microscopes, a TE2000-U for dedicated fluorescence work (widefield and TIRF), a TE2000-S for phase contrast, differential interference contrast and fluorescence work and a second TE2000-S set up for fluorescence and phase contrast. All three setups are very popular in the lab. The ergonomics are excellent and the overall design is extremely user-friendly. Extending the capabilities of these microscopes has been very easy and, as projects dictate, these scopes can be upgraded. All models also accept motorized accessories that can be controlled from a PC and a wide range of dedicated accessories such as micromanipulators and incubators are available from Nikon for many applications. Size and cost compare favorably with other manufacturer’s microscopes.
Overall, the TE2000 is an excellent microscope. The image quality of our TE2000 systems is significantly improved over older microscope designs and, without too much effort, fluorescence imaging of single molecules can be achieved. This ‘work station’ can be readily configured and is capable of broad and multiple imaging modalities. The convenience of being able to purchase optimized modules for different imaging methods is also an exceptional feature in comparison to older generation microscopes.