Inverted Research Microscope Eclipse Ti2

 Expand Your Vision (0:30)

As research trends evolve towards large-scale, systems-level approaches, there is an increasing demand for faster data acquisition and higher throughput capabilities. Development of large-format camera sensors and improvements in the data processing capabilities of PCs have facilitated such research trends. The Ti2, with its unprecedented 25mm field of view, provides the next level of scalability, enabling researchers to truly maximize the utility of large-format detectors and future-proof their core imaging platform as camera technologies continue to develop at a rapid pace.

Single FOV of cultured neurons, captured with a CFI Plan Apo lambda 60x objective and DS-Qi2 camera.

Photo courtesy of J. Rappoport, Nikon Imaging Center, Northwestern Univ.; Sample courtesy of S. Kemal, B. Wang, and R. Vassar, Northwestern Univ.

Bright Illumination Over a Wide Area

High-power LEDs deliver bright illumination across the Ti2’s large field of view, ensuring clear, consistent results from demanding applications such as high-magnification DIC. Incorporation of a fly-eye lens design provides uniform illumination from edge to edge for quantitative high-speed imaging and seamless tiling of images in stitching applications.

High-power LED illuminator

Built-in fly-eye lens

A compact epi-fluorescence illuminator designed for large FOV imaging is equipped with a quartz fly-eye lens and provides high transmittance across a broad spectrum, including UV. Large diameter fluorescence filters with hard coatings deliver large FOV images with a high signal-to-noise ratio.

Large FOV epi-fl illuminator

Large diameter fluorescence filter cubes

Large Diameter Observation Optics

The diameter of the observation light path has been enlarged in order to achieve a field number of 25 at the imaging port. The resulting large FOV is capable of capturing approximately double the area of conventional optics, enabling users to gain maximum performance from large-format sensors such as CMOS detectors.

Enlarged tube lens

Imaging port with large 25 field number

Objectives for Large FOV Imaging

Objectives with superior image flatness ensure high quality images from edge to edge. Utilizing the maximum potential of the OFN25 objective significantly accelerates data collection.

Cameras for Large-Volume Data Acquisition

The DS-Qi2 high-sensitivity monochrome camera and DS-Ri2 high-speed color camera are equipped with large 36.0 x 23.9 mm, 16.25 megapixel CMOS image sensors, enabling maximum performance with the Ti2’s large 25mm FOV.

D-SLR camera technology optimized for microscopy

DS-Qi2

DS-Ri2

Nikon’s high-precision CFI60 infinity optics, designed for use with a variety of sophisticated observation methods, are highly regarded by researchers for their superb optical performance and solid reliability.

Apodized Phase Contrast

Nikon’s unique apodized phase contrast objectives with selective amplitude filters dramatically increase contrast and reduce halo artifacts to provide detailed high-definition images.

BSC-1 cells captured with CFI S Plan Fluor ELWD ADM 40xC objective

Apodized phase plate is incorporated in APC objectives

External Phase Contrast (Ti2-E)

The motorized external phase-contrast system enables users to combine phase contrast with epi-fluorescence imaging without compromising fluorescent light transmission by bypassing the need to use phase-contrast objectives. For example, very high NA, liquid immersion objectives can be used for phase-contrast imaging. Using this external phase contrast system, users can easily combine phase contrast with other imaging modalities, including weak-fluorescence imaging such as TIRF and laser tweezer applications.

Epi-fluorescence and external phase contrast images: PTK-1 cells labeled with GFP-alpha-tubulin captured with CFI Apo TIRF 100x Oil objective. Photo courtesy of Alexey Khodjakov, Ph.D Research Scientist VI / Professor, Wadsworth Center

DIC (Differential Interference Contrast)

Nikon’s highly-regarded DIC optics provide uniformly clear and detailed images with high resolution and contrast throughout the magnification range. DIC prisms are individually tailored for each objective lens to provide the highest-quality DIC images for every sample.

Epi-fluorescence and DIC images:
25mm FOV image of neurons (DAPI, Alexa Fluor® 488, Rhodamine-Phalloidin), captured with CFI Plan Apo lambda 60x objective and DS-Qi2 camera
Photo courtesy of Josh Rappoport, Nikon Imaging Center, Northwestern Univ.;
Sample courtesy of S. Kemal, B. Wang, and R. Vassar, Northwestern Univ.

DIC prisms matched to individual objectives are mounted in the nosepiece

NAMC (Nikon Advanced Modulation Contrast)

This is a plastic-compatible, high-contrast imaging technique for unstained, transparent samples such as oocytes. NAMC provides pseudo-three-dimensional images with a shadow-cast appearance. The direction of contrast can be easily adjusted for each sample.

NAMC image: Mouse embryos, captured with CFI S Plan Fluor ELWD NAMC 20x objective

NAMC objective lenses contain rotatable modulators

Auto Correction Collar (Ti2-E)

Changes in sample thickness, cover glass thickness, refractive index distribution in the sample, and temperature can lead to spherical aberration and image deterioration. The highest quality objectives are often equipped with correction collars to compensate for these changes, and precise positioning of the collar is critical in achieving high resolution, high contrast images. This new automated correction collar utilizes a harmonic drive and automatic correction algorithm that enable users to easily achieve precise collar adjustment to achieve maximal objective performance every time.

Super-resolution image (DNA PAINT): CV-1 cells expressing α -tubulin (green) and TOMM-20 (magenta) captured with CFI Apo TIRF 100x Oil objective.

Harmonic drive mechanism for high-precision control of correction collar movement

Epi-Fluorescence

The λ series objectives, utilizing Nikon’s proprietary Nano Crystal Coat technology, are perfect for demanding, low-signal, multi-channel fluorescence imaging that requires high transmission and aberration correction over a wide wavelength range. Combined with new fluorescence filter cubes that offer improved fluorescence detection and stray light countermeasures such as the Noise Terminator, the λ series objectives demonstrate their power in weak signal observations such as single-molecule imaging and even luminescence-based applications.

Luminescence image: HeLa cells expressing BRET-based calcium indicator protein, Nano-lantern (Ca2+). Sample courtesy of Prof. Takeharu Nagai, The Institute of Scientific and Industrial Research, Osaka University

Conventional coating

Nano Crystal Coat

The Nikon Perfect Focus System (0:33)

Even the slightest change in temperature and vibrations in the imaging environment can greatly impact focus stability. The Ti2 eliminates focus drift using both static and dynamic measures to enable faithful visualization of the nanoscopic and microscopic world during long time-lapse experiments.

Mechanically Redesigned for Ultra-High Stability (Ti2-E)

High stability Z-focusing mechanism remains adjacent to the nosepiece even in expanded configurations

In order to improve the focusing stability, both Z-drive and PFS autofocusing mechanisms have been completely re-designed.

The new Z-focusing mechanism is smaller and positioned adjacent to the nosepiece to minimize vibrations. It remains adjacent to the nosepiece even in an expanded (staged-up) configuration, ensuring stability for all applications.

The detector portion of the Perfect Focus System (PFS) has been detached from the nosepiece in order to reduce mechanical load on the objective nosepiece. This new design also minimizes heat transfer, which contributes to a more stable imaging environment. Towards this end, the power consumption of the Z-drive motor has also been reduced. Combined, these mechanical redesigns result in an ultra-stable imaging platform, perfectly suited for single-molecule imaging and super-resolution applications.

Fourth Generation Nikon Perfect Focus System (Ti2-E)

The newest generation of the Perfect Focus System (PFS) automatically corrects focus drift caused by temperature changes and mechanical vibrations that can be caused by a variety of factors, including the addition of reagents to the sample and multi-position imaging.

The PFS detects and tracks the position of a reference plane (e.g. coverslip surface in the case of immersion objectives) in real time to maintain focus. Unique optical-offset technology allows users to maintain focus at an arbitrary position offset from the reference plane. The user can simply focus on the desired plane and then engage the PFS. The PFS then automatically and continuously maintains focus by means of a built-in linear encoder and high-speed feedback mechanism, providing highly-reliable images even during long-term, complex imaging tasks.

PFS is compatible with a wide range of applications, from routine experiments involving plastic culture dishes to single-molecule imaging and multi-photon imaging. It is also compatible with a wide range of wavelengths, from ultraviolet to infrared, providing a means for maintaining perfect focus even for multi photon and optical tweezer applications.

Spectra of PFS dichroic

Built-in sensors detect the status of microscope components

It is no longer necessary to memorize complex microscope alignment and operation procedures. The Ti2 integrates data from sensors to guide you through these steps, eliminating user error and enabling researchers to concentrate on their data.

Continuous display of microscope status (Ti2-E/A)

Shift Your Perspective (0:19)

A collection of built-in sensors detects and relays status information for a variety of components in the microscope. All of the status information is recorded in the metadata when you acquire images with a computer, so you can easily recall acquisition conditions and/or check for configuration errors.

In addition, a built-in internal camera allows users to view the back aperture, facilitating confirmation of phase ring alignment and extinction cross in DIC. It also provides a laser-safe method for aligning lasers for applications such as TIRF.

Status lights

Microscope status can be viewed on a tablet and also determined based on status lights on the front of the microscope, enabling status determination in a dark room.

Guidance for Operational Procedures (Ti2-E/A)

The Ti2’s Assist Guide function provides interactive step-by-step guidance for microscope operation. The Assist Guide can be viewed on a tablet or PC, and integrates real time data from built-in sensors and an internal camera. The Assist Guide is designed to help users through alignment procedures for both experiment setup and troubleshooting.

Automatically Detect Errors (Ti2-E/A)

The Check Mode allows users to easily confirm, on either a tablet or PC that all the correct microscope components are in place for their chosen observation method. This capability eliminates time and effort normally required for troubleshooting when the desired observation method is not achieved. This functionality is particularly advantageous when multiple users are involved, each with the potential to make unexpected changes to the microscope settings. Custom check procedures can also be pre-programmed.

Hardware Triggering (Ti2-E)

Modern live cell imaging experiments demand optimal system performance, lost seconds can be the difference between failure and discovery.  Most microscope software allows for control of imaging devices, including fast piezo z drives, filter wheels, and more.  However, performance at the advertised device specifications is seldom realized in a real-world setting.  So how does one maximize the overall speed performance of their system?

Microscope devices are typically controlled using serial communication with the software, which introduces latencies due to checks/callbacks, lack of parallelization, and other factors.  Nikon Instruments offers a comprehensive suite of solutions for maximizing imaging speed via hardware triggering:  a control method using direct I/O voltage-mediated connections to drive system devices.  The Eclipse Ti2-E is the world’s first completely hardware triggerable microscope stand. Native filter turret(s), filter wheel(s), and Z drive may be triggered using I/O connections on the microscope control box.

Triggered experiments using the Ti2-E are designed in NIS-Elements and signals are managed using a National Instruments data acquisition device (NIDAQ), which has a far more precise timing clock than a PC.  Software and hardware setup of devices for triggering is further simplified with our new NI-BB kit, a central hub providing predefined NIDAQ connections for all your triggered devices, including the Ti2-E.

Hardware triggering easily doubles (or more) the image acquisition rate compared to software control for a number of common experimental configurations (Table 1).

Table 1:  Comparison of experiment durations with typical hardware and experiment parameters.  Hardware triggering displays a significant improvement in overall experiment time.  In these examples, the camera exposure time is longer than the camera readout time for most examples.  *The last row illustrates a comparison when the camera exposure time is the same as its readout time, and the camera can run in overlapped (100% duty cycle) mode.

Intuitive Operation

The placement of all of the buttons and switches are based on the type of illumination they control. Buttons that control diascopic observation are positioned on the left side of the microscope and those that control epi-fluorescence observation are on the right side. Buttons that control common operations are on the front panel. This use of zoning provides an easy-to-remember layout, a desirable feature when operating the microscope in a dark room.

❶ Shuttle Switch (Ti2-E)

Shuttle switches have been incorporated into the design to control devices such as the fluorescence filter turret and objective nosepiece. These types of switches emulate the feel of manually rotating these devices, for intuitive control. Additional functionality can be incorporated into these shuttle switches so that a single switch can operate multiple related devices. For example, the shuttle switch for the fluorescence filter turret not only rotates the turret but also opens and closes the fluorescence shutter when the user presses the switch. It is also possible to program these switches to operate a barrier filter wheel and the external phase contrast unit.

❷ Programmable Function Button (Ti2-E/A)

Conveniently located Function buttons allow customization of the user interface. Users can select from more than 100 functions, including control of motorized devices such as shutters and even signal output to external devices via the I/O port for triggered acquisition. Mode functions, which enable instant changing of observation methods by storing the settings of each motorized device, can also be assigned to these buttons.

❸ Focusing Knob (Ti2-E)

A focus acceleration button and a PFS engagement button are provided adjacent to the focusing knobs. The two buttons are easily identified by touch because of their different shapes. Focusing speed is automatically adjusted for the objective in use, enabling stress-free operation by maintaining an ideal focusing speed.

Intuitive Control with Joystick and Tablet (Ti2-E)

The Ti2 joystick not only controls stage movement, but the majority of motorized functions on the microscope, including PFS activity. It can display XYZ coordinates and the status of microscope components, providing an effective means for the user to remotely control the microscope. Motorized functions of the Ti2 can also be controlled from a tablet, connected by wireless LAN to the microscope, providing a versatile graphical interface for microscope control.