Olympus Fluoview-1000 Benutzerhandbuch - Seite 36

Blättern Sie online oder laden Sie pdf Benutzerhandbuch für Mikroskop Olympus Fluoview-1000 herunter. Olympus Fluoview-1000 50 Seiten.

V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

Increased laser power – stronger excitation of fluorophores, with disadvantages of photobleaching,

bleedthrough and autofluorescence.

Longer dwell time - increases the signal by exciting each sample point in the specimen for a longer

period of time, but provides similar disadvantages as higher laser power;

Higher HV – amplify the signal at the risk of detector noise.

Gain – multiplies the signal, however, background and noise will be amplified. Not recommended.

Photon Counting - accumulate very weak signals to detect individual photon events with very high S/N.

Change lenses – higher NA objectives focus more light into the image plane for greater excitation levels,

and collect more light from the emission due to their greater acceptance angle; Fluor and Achromat

objectives have greater light transmissivity than Plan, Planapochromat and lenses with phase contrast

rings. On the other hand, lower magnification lenses acquire light from a larger area.

6.10

Using Zoom

Zoom is essential for getting the maximum resolution from a confocal microscope. However, use a few

standard zoom settings, such as 2X, 4X, 8X, for your work. Resist the urge to use random zoom settings

that make more work for yourself by constantly needing to recalculate scale bars and re-scale images to

allow direct comparisons between images.

The zoom setting on a confocal is not a digital zoom. It physically alters the size of the area scanned by

the laser spot and in doing so, changes the sampling rate for the image. Zoom provides additional

resolution without the need to increase magnification, as with a widefield microscope and camera.

A zoom setting of 2.0X will improve the image quality by collecting images from the central

portion of the objective's field of view. Zoom allows avoiding chromatic and spherical

aberrations that are most serious at the edges of the lens. Objective lenses without flatfield

corrections, such as Achromats, benefit greatly from using zoom.

The Nyquist Theorem requires that an object must be sampled at a frequency at least 2.3 times its

size in order to be completely resolved. In other words, a 0.5 µm diameter mitochondrion, or

two fluorescently labeled membranes separated by 0.5 µm, cannot be completely resolved unless

viewed with pixels that are at least 0.217 µm, or smaller (0.5 µm/2.3).

If the object to be resolved is 6 µm, then the sampling interval only needs a pixel size of 2.6 µm.

Bear in mind however, that software recognition may require higher resolution than the human

eye.

Similarly, if an objective lens has a lateral resolution of 0.5 µm, then the confocal zoom and box

size must be set to a pixel size of 0.217 µm in order to record the finest details that can be

May 11, 2011

Olympus Fluoview-1000 User's Guide