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Description

Location: Room 1.12, MRC Building

Advantages: Speed, sensitivity, flexible photo-bleaching and photo-activation

Disadvantages: Lower resolution; poorer optical sectioning of thick fluorescent specimens

The Vox is a spinning disc confocal microscope designed for rapid acquisition of 3D and 4D data. The microscope consists of a Nikon TiE inverted stand attached to a Yokogawa CSU-X1 spinning disc scan head. The detector is a Hamamatsu C9100-13 EMCCD camera that multiplies the signal on the chip before reading out. This makes it more sensitive than a regular CCD but a large pixel size means that resolution is relatively low. The Ultraview Focus Drive (Prior NanoscanZ piezo focus) is generally used for acquiring stacks because it is fast and does not limit the rate of acquisition. It has a maximum range of 250 µm so any specimens thicker than this will have to be imaged using the microscope internal focus motor. The TiE stand includes the Nikon Perfect Focus System (PFS), which can maintain samples in precise focus indefinitely. This is most suitable for samples mounted at a glass/aqueous interface but there are some exceptions. Please check specimen and objective lens compatibility before using the PFS. The Vox also has a Photokinesis Unit (PKU) for spot or ROI illumination in techniques such as FRAP and photo-activation/photo-switching.

Suitable specimens: All culture vessels must have glass bottoms with the same thickness as a standard #1.5 cover-slip (170 µm). There are inserts for slides, Ibidi µSlides, Lab-Tek chambered cover slips (note - use Lab-Tek I not Lab-Tek II), 35 mm dishes, multi-well plates with square not chamfered corners. If you have any other requirements then you must speak to a member of the light microscopy facility before you start preparing your specimen. Note that CO2 supply is not available for all configurations so check what vessel you need before you start setting up your specimen.

In general spinning disc confocal systems perform optimally with adherent cells in culture and other relatively thin specimens. Thicker specimens with a large amount of out-of-focus fluorescence can be difficult to image because of a phenomenon called pinhole crosstalk.

• Using the Nikon TiE microscope
• Coming Soon - Familiarising yourself with the Vox user interface
• Further Information

Objective Lenses

There is also a 100x 1.3 NA Plan Fluor oil objective that can be used if the PFS range turns out to be too narrow when using the 100x Plan Apo.

There is a choice of three immersion oils:

Note that Cargille Type 37 is optimal for use at 37ºC but introduces spherical aberrations when imaging beyond about 10 μm in aqueous media because it is not refractive indexed matched to water. Zeiss Immersol W in combination with the 60x WI lens might be a better choice in this case but its RI is corrected for use at room temperature and won't be correct at 37 degrees.

Lasers and emission filters

The Vox has diode or DPSS lasers that emit the following wavelengths: 405 nm, 488 nm, 561 nm and 640 nm.

Emission Wheel Filters

Filter cubes in the microscope stand

The filter cubes below are for use when viewing your specimen down the microscope eyepieces. The filter turret must be in position 1 (EMPTY) when imaging with the spinning disc.