Laser (light source)

Laser lines can be chosen via a selection device such as an AOTF (accusto-optical tunable filter) and should be matched with the fluorophores used in the specimen. Laser power can be adjusted via the an attenuation device (e.g. AOTF) and the tube current seeting in some gas lasers (e.g. Ar laser). The Argon laser should run at 30-50% of the maximum current, which will prolong the lifetime significantly Tip: start with low laser power to protect the sample by reducing photo bleaching and phototoxic effects

Beam splitter

This is a filter device that separates the excitation from the emitted light in the fluorescence beam path of the microscope. It can be either a dichroic mirror or an AOTF, and can be operated for different configurations via the software. In a filter-based system, the main dichroic mirror (HFT) separates the excitation and the emission light, and secondary dichroic mirrors (NFT) further separate the emitted light.

Scanner (scanhead)

The scanner is a unit based on two or more mirrors, which guide the focussed laser beam across the specimen, pixel by pixel and line by line. The user can set the following imaging parameters that greatly affect the image acquisition:

Scanning speed is defined by the pixel dwell time and is usually given for a line or an image frame.

The pixel dwell time is the time the focussed laser beam rests on a single pixel and illuminates it. Therefore, the longer the pixel time, the more photons can be collected per pixel, and the lower the scan speed will be. With longer dwell times photo bleaching and phototoxic effects will also increase.

The Pixel resolution is defined by the the resolution (i.e. pixels per line, e.g. 1024) and the pixel size, which is directly adjustable via the zoom. This also defines the sampling rate of the system. For optimal and loss-less image acquisition, the sampling rate should be twice the Nyquist sampling rate.

Frame size can be set via the software and affects the scanning speed. By reducing the number of lines to be scanned the scanning speed per frame can be reduced significantly. This can be achieved by either using an appropriate frame format (e.g. 1024x256) or by using the ROI tool.

 Objective lens

The optics are the heart piece of the microscope and mainly determine the optical image formation and the resolution of the system. The numerical aperture (NA) of an objective lens is a measure of its ability to collect light and resolve fine object details. It is defined by the refractive index (RI) of the immersion medium and the angular aperture of the lens. The higher the NA, the better the resolution of an objective. Objective lenses are designed for specific immersion media such as water, oil, air or glycerol and should only be used with the appropriate medium. The RI of the immersion medium and thus the lens used, should be matched with the RI of the medium the specimen is mounted in. For multi-fluorescence image acquistion only lenses that are apochromatically corrected should be used.

 Z-control

This allows to focus on any focal pane within the specimen and the motorised Z-stepper allows movement in the axial direction in small step sizes (>10 nm) at high precision. The Z-interval (step size) determines the distance between two adjacent optical planes and is defined by the Nyquist sampling rate (Z-dimension of the voxel). The pinhole size and not the Z-interval determines the thickness of the optical slice.

Pinhole

The pinhole is an adjustable iris in the intermediate image plane, which allows to exclude most of the out-of-focus light from the acquried image and thus provides optical sectioning capacity . It defines the thickness of the optical slice and is dependent on the wavelength and the properties of the obective lens. The pinhole size can be set via the software and the best trade-off between the efficiency of light collection and optical sectioning is if it is set to 1 Airy unit.

Photomultiplier tube (PMT)

These are highly sensitive detectors that collect pixelwise the photons emitted by the respective specimen detail and filtered by the system. Unlike a didgtal camera, a PMT has no spatial information of the image. PMTs are dynode-based amplifying devices operated by a high voltages. The higher the applied voltage (gain setting), the greater the amplification and thus sensitivity, but also the higher the noise in the acquired image. The amplifier offset defines black level setting of the image. Both, gain and off-set settings can be calibrated using the range indicator tool of the software.