Samuel,
 
This is an excellant paper discussing when to use wide-field or confocal . . .

	
Swedlow JR, Hu K, Andrews, Roos DS, Murray JM

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy

	
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2014-9. 

Michael

	 
	 
	-----Original Message----- 
	From: Samuel Connell [mailto:[log in to unmask]] 
	Sent: Fri 12/3/2004 9:37 AM 
	To: [log in to unmask] 
	Cc: 
	Subject: Re: confocal pinhole size and deconvolution
	
	

	Search the CONFOCAL archive at
	http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
	
	On 12/3/04 7:51 AM, "Karl Garsha" <[log in to unmask]> wrote:
	
	> Search the CONFOCAL archive at
	> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
	>
	> Hi Jeremy,
	>   There are situations in which widefield-based deconvolution systems
	> will provide large advantages over traditional confocal imaging; there
	> are also situations in which the lscm would stand out as the instrument
	> of choice.  When photons are in short supply, well engineered,
	> integrated widefield deconvolution systems should produce better raw
	> data for deconvolution algorithms to work with.  This translates into
	> drastically better results where QE is a problem.
	> -Karl
	>
	> Jeremy Adler wrote:
	>
	>> Search the CONFOCAL archive at
	>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
	>>
	>> (1) Since confocal images are generally short of photons, the option of
	>> acquiring more photons by opening the pinhole and then deconvolving the image
	>> set to restore the quality, seems attractive.
	>> Given that image quality requires both resolution and contrast is this
	>> strategy viable ?.
	>>
	>> (2) Since deconvolution ideally requires a Z series that covers the likely
	>> size of a PSF and, if only single image is really required, is it better to
	>> acquire the Z series and deconvolve or to use the same acquisition time to
	>> improve the quality (acquiring more photons) from a single image plane ?.
	>> This choice arises when the fluorophore of interest bleaches rapidly in
	>> single photon confocal imaging.
	>>
	>> --
	>> Dr Jeremy Adler
	>> Dev. Biol.
	>> Wenner-Gren Inst.
	>> Stockholm University
	>> S-106 91 Stockholm
	>> Sweden
	>>
	>> +46 (0)8 16 1568
	>>
	>> --
	>>
	>>
	>
	> --
	> Karl Garsha
	> Light Microscopy Specialist
	> Imaging Technology Group
	> Beckman Institute for Advanced Science and Technology
	> University of Illinois at Urbana-Champaign
	> 405 North Mathews Avenue
	> Urbana, IL 61801
	> Office: B650J
	> Phone: 217.244.6292
	> Fax: 217.244.6219
	> Mobile: 217.390.1874
	> www.itg.uiuc.edu
	     I have read about this topic, and I know this has probably been
	discussed before on this list ad nauseum, however, I'd love to see a concise
	discussion of specifically what Karl just mentioned.  In what situations
	would one recommend either a well engineered widefield deconvolution system
	or a well engineered lscm?  Karl mentioned the situation in which photons
	are in short supply.  What other categories of sample or experimental
	question belong more on one system or another?  If people would like to
	chime in on where they feel multiphoton systems fit into this, feel free.
	Thanks,
	--
	Samuel Connell
	Imaging Facility and Technology Manager
	La Jolla Institute for Allergy and Immunology
	10355 Science Center Dr.
	San Diego, CA 92121
	858-558-3508
	[log in to unmask]