Sterling: Spinning Disk, TIRF, STORM microscope

Summary

Sterling is a multifunctional microscope and is equipped for 5 image modalities: spinning disk confocal imaging, Total Internal Reflection Fluorescence (TIRF) imaging, Stochastic Optical Reconstruction Microscopy (STORM), photo activation and stimulation, and bright field. Live cells that express fluorescent proteins at low levels are easily imaged using the spinning disk confocal mode due to the mild illumination method and a sensitive camera. Complete automation in combination with perfect focus system make this an ideal system to acquire live cell pro-longed time-lapses (< 72 h). Biological processes which occur at or near the membrane are best studied using the TIRF mode. In addition, single molecule studies (e.g. tracking or photo physics) are performed using the TIRF mode due to the high-power lasers. Furthermore, super-resolution images are obtained using the STORM modality. This system is fully equipped with stage top incubator for live cells, nonetheless, this system is also suited for fixed cells and non-biological samples. 

Image modalities

• Spinning disk confocal imaging
• TIRF (also HILO / ‘dirty’ TIRF)
• (3D) STORM
• Photo activation/stimulation
• Brightfield

Applications

Spinning disk confocal imaging

• Immunofluorescence. Images are acquired fast compared to a laser scanning confocal and the Piezo Z drive allows fast Z stacking.
• (Fast) live cell imaging. Spinning disk illumination is very mild for your sample, which is beneficial when imaging fluorescent proteins. The stage top incubation chamber regulates the temperature (30 – 40 ⁰C), CO₂ and humidity.
• Pro-longed time-lapse live cell imaging. The perfect focus system controls the focus very precisely and can be used over a long-time course (< 72 h).
• Filter FRET / ratiometric FRET. Filter FRET using blue, green, red and far red fluorescent dyes or fluorescent proteins can be performed.

TIRF modality

• Membrane processes in live cells. Processes that occur 200 µm from the cover slip can be very well studied with TIRF.
• Single molecule tracking in live cells. This can be done near the membrane in full TIRF or relatively higher using HILO TIRF.
• Single molecule photo physics. Non-biological samples are also compatible on this microscope.

STORM modality

• STORM and 3D STORM. These super resolution techniques can be performed on live and fixed biological samples. Our analysis workstations are also equipped with the full N-STORM analysis package.

Technical description

The Eclipse Ti2-E (Nikon) is equipped with automated stage (Nikon), perfect focus system (Nikon), Piezo Z stage (Mad City Lab Inc), stage top incubator (Tokai Hit) to control the temperature (30 – 40 ⁰C), CO₂ and humidity (including objective heater and temperature sensor). The following Nikon objectives are available for this system: Plan Apo lambda 10x NA 0.45 air, Plan Apo lambda 20x NA 0.75 air, S Fluor 40x NA 1.30 oil, Plan Apo lambda 60x NA 1.40 oil, Apo TIRF 60x 1.49 and SR HP APO TIRF 100x 1.49 NA. All imaging functions are integrated in the NIS-Elements software (High content analysis package). 

For spinning disk confocal imaging, the sample is excited by a 405 nm, 488 nm, 561 nm or 640 nm laser controlled by an Agilent laser box. The excitation light is focused through micro lenses of the excitation disk of the CSU-X1 (Yokogawa) with 50 µm pinholes. The excitation light is reflected to the sample with a quad dichroic mirror. The emission light is filtered using a single bandpass filter (455/50 nm, 525/36 nm, 600/50 nm or 705/72 nm) or a quad bandpass filter (open at 425 – 470 nm, 507 – 535 nm, 575 – 620 nm, and 665 – 742 nm). The emission is captured using a Prime 95B sCMOS camera (Photometrics). The spinning disk fluorescence mode can be combined with brightfield, in that case a diascopic LED (Nikon) is used and the light follows the same light path from the sample and the images are acquired on the same camera.

For TIRF imaging, the sample is excited by a 405 nm, 488 nm, 561 nm or 640 nm laser controlled by an Agilent laser box. The TIRF angle is controlled by the automated N-STORM illumination arm. An additional side camera observes the back focal plane and thereby aids in setting the correct angle and orientation of the laser beam. The excitation light is reflected by a quad dichroic and the emission is filtered by a quad emission filter (open at 422 – 478 nm, 502 – 549 nm, 581 – 625 nm, and 674 – 786 nm). The images are captured using an iXon Ultra 897 EMCCD camera (Andor). Two objectives are available for TIRF: Apo TIRF 60x 1.49 NA and SR HP APO TIRF 100x 1.49 NA.

For (3D) STORM imaging (N-STORM, Nikon), the setup is similar as for TIRF imaging, but only the 561 nm and 640 nm lasers are powerful enough for STORM imaging. The excitation light is concentrated with a 1x, 2x, 4x or 8x magnification lenses. For 3D STORM, an astigmatic lens is placed the light path in front of the camera. One objective is available for STORM: SR HP APO TIRF 100x 1.49 NA.

Photo activation and stimulation can be combined with the spinning disk confocal mode or the TIRF imaging mode. A pattern or region of interest is created by a digital mirror device (Polygon) which reflects light of the AuraII (lumencor) onto the sample plane. The AuraII contains 4 individual LEDs (395/25 nm, 470/24 nm, 555/28 nm, and 640/30). You can create almost any sequence and combination of illumination, activation/stimulation and acquisition with the illumination sequence function of the NIS-Elements software.

Location

Leichtag building, Room 470