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There will be some sample-dependent situations where this might be the case, that confocal yields an image that is no better than widefield imaging. In fact, you should always ask yourself if confocal is even necessary to answer the question you're after in an imaging experiment. See for example:

Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.

Also, if the specimen is really thin, confocal might not be so beneficial... But having said that, a few basic questions about your setup are in order:

1. What are you imaging?

2. What pinhole size are you using and with what objective lens (magnification and NA)? If the pinhole size is too large relative to the lateral and axial resolution afforded by the objective, you'll end up with a widefield-like image. Also consider that confocal imaging generally breaks down at low magnification (see end sections of Amos, B., G. McConnell, and T. Wilson, Confocal microscopy, in Comprehensive biophysics, E.H. Egelman, Editor. 2012, Elsevier: Amsterdam. p. 3-23.)

3. Is the camera well focused to the pinholes of the disk? Check by stopping the disk.

4. Is the scan head well aligned to the microscope? Check by looking for eyepiece parfocality and centration, and also check that the excitation light enters the back of the objective centred (be laser safe when looking for this).

5. Can you try imaging a "standard" sample in your lab as a sanity check (ie: a positive control)? Do you obtain good optical sectioning behaviour with a sample like that? Pollen grains (Carolina, no commercial interest), or the mouse kidney section (Life Technologies, no commercial interest), are good for a confocal sanity check. Imaging the autofluorescence of sheet of white paper is also an option. 

Just some thoughts,

John Oreopoulos
Staff Scientist
Spectral Applied Research
A Division of Andor Technologies
www.spectral.ca



On 2016-05-19, at 11:45 PM, Rafael Jaimes wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
> 
> This has me stumped. I suspected it was a lack of light but I turned up the
> excitation light significantly and still haven't gotten good images with the
> spinning disk in place. The widefield mode is giving much better images
> which doesn't make any sense to me. I figured I'd ask for recommendations in
> case anyone has been in a similar boat, especially with the hardware I'm
> running:
> 
> Nikon Ti w/ Crest X-Light v1 spinning disk. Detector is a Andor Zyla 4.2
> PLUS sCMOS.