Imaging Modes of the Oxford Nanoimaging Nanoimager

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.

Nanoimager imaging modes
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Epifluorescence

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.

Total Internal Reflection Fluorescence (TIRF)

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.

Highly Inclined and Laminated Optical Sheet (HILO)

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.