On Wed, 29 May 1996, Dennis McKearin wrote
>         I am interested in learning the pro & cons of confocal vs
> deconvolution methods of microscopy. Specifically, I wish to know how the
> two methods compare for double and triple labeling when stained samples are
> 200-300 microns thick. What limitations exist between the two techniques -
> do both provide images of equal resolution and sensitivity or is one
> superior, especially as applied to multiple labeling experiments? Are
> sample bleaching rates equal?
>         Thank you for your time and help.
>
> Sincerely,   Dennis McKearin
 
Well, just to get things rolling on this thread, I'll put my $.02 in.
Recognize that both technologies are in the process of evolution, so what
I say is probably outdated by about 6 months. Also, I will confine my
comments to my own experiences - work done with the BioRad, Zeiss and
Meridian confocal microscopes and my experiences reading deconvolution
related articles and implementing some of the techniqes described. I
hear, for example that there are now video-rate scanning confocal
microscopes, but I have no idea what the trade-of involved in
implementing this is. Thus, with everything following preceeded with an
understood "in my experience and opinion, often limited or misguided"...
 
Confocal microscopes rely on the geometry of emmited light paths to
filter out out-of-focus illumination from a specimen. Usually this has
been implemented by scanning a laser beam across the specimen with a
series of scanning mirrors, with emitted light descanned through much of
this pathway. The detectors at the end of all this are usually
photomultipliers. Generally, there is a significant trade-off between
image quality, scanning speed and sensitivity, as expected. The beauty of
the technique is that the formed image is "real" in the sense that it is
not computationally derived from estimates (although very good ones) of
what light "should" be doing in your optical system. A side-by-side
comparison of images of the same specimen using the two techniques
suggests that confocal microscopes can attain slightly higher resolution,
partially due to a phenomenon achieved by using coherent laser light
called "super resolution" - see A Handbook of Confocal Microscopy,
incidentally), and partially due to the difficulties in estimating
(mathematically) the spread of light very close to an in focus point. As
a result, microtubules in confocal microscopes (properly adjusted and
used) seem to be thinner and more clearly resolved than can be achieved
by (commercial) deconvolution software. Limitations, particularly for
issues related to multispectral imaging, are related to the use of lasers
to illuminate multiple fluorophores - you can do this but it won't come
cheap and may in fact involve buying (and maintaing) more than one laser,
and the relatively skewed spectral efficieny of photomultiplier tubes
used for imaging - some of the confocal microscopes I've used see
fluorescein-like dyes MUCH better than rhodamine-like dyes. Multispectral
imaging seems to be particularly difficult so implement fast and
efficiently, in some implementations on currently available instruments,
multiple fluorophores are illuminated with the same excitation spectrum,
chosen so as to excite more than one fluorophore - although neither are
excited at anything like peak efficiency, thus requiring higher
illumination levels and longer/slower scanning times to obtain an image
of the same quality that could be achieved by scanning the specimen with
filters optimized for individual fluorophores. Scanning is also slow,
although higher scan rates are accomplished by either scanning at lower
resolution or by scanning smaller areas.
 
Deblurring (can) use standard epifluorescent illuminators and captures
all of the emitted light coming from the specimen, forming images with
standard imaging devices such as SIT camaras or CCD arrays. The
techniques relies on the ability to mathematically estimate the
contribution of adjacent areas in the specimen to a given image and
subtracting this information.A consideration is that the technique is
only as good as the starting images - noise images from low-light level
imaging devices do not work as well as images collected over long
integration times with cooled CCD camaras. Also images from specimen
areas burried deeply in a specimen that's not very transparent are also
going to be a problem, while confocal microscopes (won't get much light
from this area either) but may be more likely to build up an acceptable
image over time. For a discussion of these methods you are of course
refered to articles by Agard and Sedat, UCSD? or Fay at UMass Worchester,
(my sincere apologies to others I am not mentioning at the momment
because -hey I AM on vacation and my brain doesn't have to work that
well). In principle, deconvolution could be used on images which are
captured VERY quickly - much faster than could be achieved by scanning
techniques, and with the judicious use of filters and optics could obtain
multispectral images simultaneously or nearly so. In practice, of course
whis would be quite difficult to do with a living specimen (which is the
only thing you really need to do this with) because of the need to obtain
relatively noise-free images. Although deconvolution is computationally
demanding and can require hours of computation to implement some
algorithms, this can all be done after the fact, allowing rapid
collection of data. In my experience, imaging devices can also be
substantially more sensitive than confocal microscopes allowing much
lower illumination levels to achieve a reasonable image. Deconvolution
can also be implemented somewhat less expenxively than buying a confocal
microscope - although after adding the cost of the microscope, software
and camaras up I'd hesitate to call the technology cheap.
 
Both technologies have limitations in dealing with out-of-focus
information in thick specimens, ie if your area of interest is located
behind a brick, neither technique works well. Deconvolution is
particularly susceptable to neighboring bright objects punching
subtractive holes in one's image.
 
So, ideally I'd like to have both around :-). If you need to get images
of lots of different fluorophores from the same specimen and budgets are
tight I'd have to give the deconvolution systems a close look. My
personal feeling is that confocal microscopy is easier to use for the
uninitiated and perhaps more powerful for penetrating thick and
semi-opaque specimens, but slower, more expensive and less adaptable in
the hands of an experienced digital microscopist. (You might for example
see what Dr. S. Inoue has been up to).
 
Since I've stuck my neck out really far here, I'll be very interested in
other peoples opinions.
 
Regards,
 
 
Eric.