Olympus Fluoview-1000 Benutzerhandbuch - Seite 21

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

Center on Human Development and Disability, Digital Microscopy Center

Use of Sequential, Simultaneous and Averaging.

3.1

Simultaneous capture collects a multi-channel image by scanning the sample with the laser lines for all

selected channels at the same time. The combined fluorescent emission spectra are separated and

directed to their respective detectors by a series of dichroic mirrors. The barrier filter at each channel

transmits narrow spectrum of wavelengths designated to represent the respective fluorophore with a

minimal amount of bleedthrough from other fluorescent labels in the specimen. However, emission

spectra from most fluorophores overlap to some degree, especially onto the longer wavelength side of

the emission peak. The result is that some emissions from any fluorophore will appear in the adjacent

channel(s) on the "red" side of the spectrum.

Sequential capture of multiple channels is accomplished by collecting only 1 channel at a time. The

specimen is scanned with a single laser line for each channel and the emission is captured with the

appropriate detector. Thus, the image from each label is collected by itself without the presence of

overlapping signals from other fluorophores present in the specimen. This eliminates most bleedthrough

but, acquisition time is longer and any sample movement will appear as mis-registration between

channels. If a laser line also significantly excites other fluorophores, their emissions may overlap

sufficiently to cause bleedthrough. As always, control specimens are important.

Simultaneous is faster than Sequential capture and is commonly used for positioning the sample. It may

be "good enough" for routine imaging of samples that exhibit tolerable levels of bleedthrough and is an

important tool for recording dynamic events. Sequential capture is slower, but produces images with

higher contrast and greater spectral accuracy.

3.2

Channels are recorded by sequentially scanning each line in the image for each channel in turn. If 3

channels are to be recorded, then each line is scanned 3 times, once for each channel, and the process

is repeated for each line in the image. The advantages of line sequential scanning are that all

channels update relatively quickly, as with a merged image, and motion in living cells is minimized

between the channels within each line. Sequential line capture is about 10% faster than sequential

frame capture.

The entire field is scanned for only the selected channel and only that channel will be updated in the

Live View window. Select the desired channel to image by clicking on its group in the sequential

panel.

May 11, 2011

Olympus Fluoview-1000 User's Guide