Olympus Fluoview-1000 Podręcznik użytkownika - Strona 33

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

Center on Human Development and Disability, Digital Microscopy Center

disk. An iris set to this size passes approximately 80% of incident emissions and achieves a 20%

increase in lateral and axial resolution, relative to the widefield microscope. The 'Auto CA' button

(Figure 4.10) sets the aperture to 1 Airy disk diameter for the middle wavelength for multi-channel

imaging. A diameter less than the optimal will provide moderately increased resolution with greatly

reduced signal intensity. A larger than optimal diameter will decrease the resolution, while greatly

increasing the contributions of out of focus light to the signal. However, a relatively large pinhole may

be necessary to collect enough weak signals or to employ low laser intensities, conditions often

encountered when imaging live cells. Deconvolution can be used to regain the resolution "lost" in the

additional out of focus light.

6.5

Bleedthrough.

The emission spectrum of a fluorophore is a roughly bell-shaped curve describing the probability that

any emitted photon will possess a particular wavelength. Each fluorophore has a wavelength of

maximal probability with declining probability curves extending into both longer and shorter

wavelengths. Bleedthrough occurs when the emission curve from one fluorescent label overlaps an

adjacent channel with sufficient intensity to appear in that channel's image. Although, it is important to

select fluorophores with minimal spectral overlap, bleedthrough is affected by both the signal intensity

of the bleedthrough source and by the degree of amplification of the channel(s) in which the

bleedthrough is observed. High excitation intensity will cause the low probability tails of an emission

spectrum to reach detectable levels in adjacent channels for most combinations of labels. Additionally,

setting a channel's detector for high amplification levels will amplify signal from any overlapping

emission spectra to create a significant bleedthrough. Another source of bleedthrough arises from over-

labeling the specimen.

Bleedthrough also may occur when the excitation light intended for one channel also excites some other

fluorescent label possessing an overlapping emission spectrum. Although such overlaps of excitation or

emission spectra are usually minimal, they may still give artifactual images.

1. Reduce label concentration.

2. Select fluorescent labels with adequate separation of excitation and emission spectra

3. Minimize laser strength

a. high excitation power increases the output of photons across the entire emission spectrum

4. Use appropriate filters

a. longpass filters and wide bandpass filters allow signal overlap

b. modern filters with steep 'cut-on" and "cut-off" angles are recommended

1. Set the confocal for the multiple channels, same as to be used in the experiment;

a. ensure consistency with the filters and dichroics

2. Select to image with Sequential, Frame;

3. Select the positive control for each channel, in turn, and optimize parameters;

a. select the appropriate channel for the control

May 11, 2011

Olympus Fluoview-1000 User's Guide