Super‐resolution Microscope from Oxford Nanoimaging
Oxford Nanoimaging has redesigned super‐resolution instrumentation. We have removed the superfluous elements of traditional fluorescent microscopes and by doing so have created an instrument which is optimally designed to generate high quality single‐molecule data. This bottom up design removes instrument complexity and delivers a cost‐effective solution, eliminating all requirements for optical tables and dedicated laser laboratories. The instrument operates on a standard laboratory bench, delivering super‐resolution capabilities to a broader range of scientific researchers. The instrument supports various modes of operation: single‐molecule localization‐based super‐resolution for quantitative cellular imaging, TIRF and epifluorescence for diffraction‐limited fluorescence imaging, single‐molecule FRET for measuring molecular interactions in the 2–10 nm range and single particle tracking PALM in cells.
Super-resolution techniques break the 200 nm limit on image resolution imposed by the diffraction of light. The Nanoimager achieves super-resolution by single-molecule localization (dSTORM and PALM). These techniques involve localizing only subsets of fluorophores in consecutive frames with high precision (typically better than 20 nm laterally and 50 nm axially), and reconstructing an image from the positions of the localizations.
The Nanoimager offers four laser lines with powers up to 1 W and the highest available power densities of any commercial instrument, calibrated at the sample plane and reported in real time. The two emission channels enable simultaneous dual-color imaging (four color imaging is possible through interlaced lasers). It supports colocalization studies and the capture of dynamic information for different molecular species.
3D information can be captured using the method of astigmatism for super-resolution detail, and by imaging sequential sections for greater depth.
The Nanoimager microscope unit has a footprint of just 21 cm by 21 cm. The compact design is space-saving, but also reduces aberrations and loss of light in the optical path.
The Nanoimager geometry inherently compensates for drift. It houses specialist materials within the solid microscope body which significantly reduce thermal drift. Multiple software components further eliminate the effect of thermal drift. In addition to the anti-drift features, it has full vibration dampening which is supported by its compact design.
Together, this produces the most stable instrument on the market with no need for an optical table.
The Nanoimager boasts the largest commercially available field of view, which is evenly illuminated throughout. The large field of view facilitates high-throughput imaging of single molecules and rapid accumulation of sample statistics.
As a Class 1 laser product, the Nanoimager can be safely operated in any room or laboratory
The advanced sample stage has exceptional positional accuracy and reproducibility.
Autofocusing allows automated data acquisition over multiple fields of view and the rapid overview feature negates the need for oculars.
Whole-body heating to 37°C supports live-cell imaging and avoids the disruptive effect of temperature gradients.
Intelligent data analysis using our custom software suite is provided for all the Nanoimager modes of operation, including super-resolution imaging, smFRET and sptPALM.
The ease of acquiring high‐content data with the Nanoimager is supported by a range of useful analysis features. A common problem across single‐molecule biology is the difficulty of interpreting complex results. To support researchers, the Nanoimager software suite contains a range of features that analyse and present data in an accessible way, as well as providing quantitative conclusions about the sample.
smFRET traces are analysed in real time, and can be viewed as individual traces or as a population average across a single acquisition or a whole series of acquisitions. Traces can be grouped into different behaviours and 2‐D histograms of stoichiometry versus FRET efficiency are presented for use in alternating laser excitation (ALEX) mode.
Single‐particle tracking PALM data can be acquired and analysed with the Nanoimager, providing instant diffusion analysis and visualization of tracks. In super‐resolution mode, the super‐resolved pointillistic image is rendered in real time, with various options for filtering the localizations, enlarging particular features and selecting one or two colors at a time. Moreover, for quantitative analysis, the user can analyse the distribution of localizations along a line; can measure the time‐dependency of positions, standard deviation of fitted functions and photon count for all localizations in a given area; can quantify the degree of clustering of molecules, and can measure colocalization of molecules detected in the two separate emission channels.
The Nanoimager is designed to make fluorescence imaging easier. Given a sample with a fluorescently labeled component such as a cellular feature, or a population of single molecules attached to a surface, the Nanoimager can capture the labeled species in three different modes of operation. These different modes are used in the capture of super-resolution images as well as smFRET traces.
Epifluorescence or wide‐field imaging is perhaps the most common type of fluorescence imaging, where a parallel beam of light passes directly upwards through the sample. The high magnification of the Nanoimager (1 pixel = 117 nm) and the large field of view are advantages for epifluorescence experiments in comparison to other fluorescence microscopes. Epifluorescence is preferred for imaging samples over 10 µm deep. However, this method does result in higher background signals due to excited molecules outside of the focal plane.
The highest possible signal‐to‐noise ratio (SNR) is achieved by the Nanoimager using total internal reflection fluorescence (TIRF) microscopy. Only a thin, 200 nm layer of the sample is excited near the coverslip, but virtually all of the excited molecules are in focus and the background signal is significantly reduced. This type of imaging is thus ideal for studying molecules attached to a surface or on a membrane.
The final mode of imaging is highly inclined and laminated optical sheet (HILO) imaging, where the laser is directed at a sharp angle through the sample. This affords an imaging depth of up to 10 µm, at a SNR only slightly lower than that of TIRF. One click of a button in the Nanoimager user interface changes the illumination from epifluorescence to HILO or TIRF mode.
Each of these modes is capable of imaging at high temporal resolution, with full frames taking only milliseconds to record. For even higher temporal resolution, a reduced area can be imaged at up to 5 kHz frame rate. To support these imaging capabilities, the Nanoimager uses a latest‐generation sCMOS camera, which combined with tailored software compares favorably to alternative options such as EMCCDs in most common applications, including super‐resolution imaging. The objective lens is a high‐numerical aperture, high‐magnification oil immersion lens.
|Imaging modalities||Single-molecule imaging based 3D localization microscopy|
|Förster resonance energy transfer (FRET) spectroscopy|
|Achievable resolution||Lateral: exceeding 20 nm|
|Axial: exceeding 50 nm|
|Simultaneous imaging channels||2 (< 10 nm channel mapping accuracy)|
|Total number of imaging colors||Up to 4 lasers|
|Field of View||50×80 μm per channel|
|Software features||Real-time 3D localization analysis and rendering (sCMOS optimized)|
|Real-time FRET trace analysis|
|Clustering and co-localization analysis|
|Residual drift correction|
|Scripting interface and OMERO compatibility|
|Acquisition Speed||100 fps full frame|
|5 kHz with frame height cropped to 2%|
|3‐D Imaging Technique||Astigmatism|
|Time for super-resolution full frame||Seconds to minutes (depends on number of localizations and on laser power)|
|Focus system||One-shot autofocus|
|Mechanical stability||<1 μm/K drift|
|<1 nm vibration amplitude (1 Hz to 500 Hz)|
|Illumination modes||Closed-loop, continuous illumination angle adjustment between epi-illumination and total internal reflection|
|Closed-loop adjustments of laser power density at sample plane|
|Temperature control||Resistive heating, whole instrument (for live cell imaging)|
|Environmental conditions||Sensor array (temperature, humidity, acceleration)|
|Dimensions (width×depth×height)||Microscope: 21×21×15 cm|
|Light engine: 21×42×45 cm|
|Camera||Latest generation sCMOS|
|82 % peak QE|
|1.6 electrons RMS read noise at standard scan|
|Objective||Oil immersion, NA = 1.4 to NA = 1.49|
|Laser Options||Violet||405 nm (150 mW)|
|Blue||473 nm (300 or 1000 mW)|
|488 nm (200 mW)|
|Green||532 nm (300 or 1000 mW)|
|561 nm (200 or 300 mW)|
|Red||640 nm (300 or 1000 mW)|
|Near Infrared||730 nm (300 or 1000 mW)|
|Laser Types||DPSS and Diode|
|Other Light Sources||LED for bright-field imaging|
|NIR auto-focus laser|
|Sample Stage||20/20/10 mm X/Y/Z travel range, closed-loop piezo stage with 1 nm encoder resolution|
|PC Requirements||PC or laptop included (32GB RAM, nVidia GeForce GTX 1080m)|
|Nanoimager software included, along with all future updates|