Zeiss Axioplan 2 imaging and Axiophot 2 Guía de usuario descargar pdf (Pagina 5)

1.7 Sample preparation

1.7.1 Selection of fluorochromes

If you want to achieve the best possible resolution, use a fluorochrome whose emission spectrum is

covering as short wavelengths as possible. The resolution of the microscope is determined by the

numerical aperture (NA) of the objective used and the wavelength of the light detected. The larger

the NA, and the shorter the wavelength, the better the resolution.

For thick tissues, image acquisition deeper in the tissue is often a problem because of light

scattering in the tissue. The longer the wavelength of the light, the less it scatters, and the better its

penetration into tissues. Thus, use fluorochromes whose emission spectra are in the far-red region if

you need to image deep inside tissue.

For multilabel imaging, select fluorochromes that are spectrally wide apart. E.g. Alexa488 and

Alexa594 usually have cross-talk between channels if imaged simultaneously, so they should be

imaged sequentially.

1.7.2 Mounting samples

You should either use a mounting medium that will harden or, in the case of non-hardening one,

seal the edges of the cover glass with nail polish or some other sealant. Let the sealant dry well

before you come to the microscope - nail polish is one of the most harmful substances for optics.

Also, unsettled mounting medium, or one containing too much water (slides not dry enough when

mounted), or too much mounting medium, can affect imaging by causing shadows and/or stripes in

the image.

If you are using oil immersion objectives, the refractive index of the mounting medium used should

be close to the refractive index of the immersion oil (1.518). For more information on mounting

media and antifade reagents, visit: http://www.uhnresearch.ca/facilities/wcif/PDF/Mountants.pdf.

Most of the objectives are designed for coverslip thickness 0.17 mm, which corresponds to number

1.5 coverslips.

1.8 Image processing and analysis

MIU has two Imaging Workstations that have software for further image processing and analysis,

such as deconvolution, cell counting, and more. Visit the MIU web site or contact MIU for more

information. Use of Imaging Workstations is free of charge, and MIU staff gives training and help

with the software. MIU also provides customized image analysis tools in case the commercial and

free software do not satisfy your image analysis needs.

2 Turning on the equipment

2.1 Zeiss Axioplan 2 imaging microscope

The main power switch of the microscope (Figure 1) is the green switch located on the right side of

the microscope.

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Zeiss Axioplan 2 imaging and Axiophot 2 Guía de usuario Pagina 5