http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585212.html
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>Post images on
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>*****
>
>dear Zdenek,
>I guess this comparison, we've got from enduser, would be interesting for
>others.
>using same protocol.
>
>"..
>- Throughput confocal performance ratio (CF/WF %), taking into account both
>excitation and collection efficiency:
>
>o Visitech 1% efficiency
>o Yoko CSU X1 2.8% efficiency
>o Crest X-Light 5.5%-6.3% efficiency
>
>Note: we have been using 200nm beads, 100X 1,49 obj and SpectraX LED system
>(not even lasers), Evolve CCD
>.."
>
>regards.
>
>Andrea Latini
>President
>CrestOptics Srl
>
>
>
>On Mon, 23 May 2016 21:21:14 +0200, Zdenek Svindrych <
[hidden email]> 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.
>>*****
>>
>>Dear Andrea,
>quite off the original topic, but, whatever...
>
>As already mentioned, it's very important to separate the overall efficiency
>into excitation efficiency and collection efficiency.
>
>5% excitation efficiency would be tolerable, e.g. with a spinning disk
>without microlenses...
>
>But in detection path "every photon counts", 80% efficiency should be
>possible with > 1AU pinholes.
>
>What's missing in the protocol is "measure the illumination intensity in the
>BFP (or image plane) of the objective in both cases and do the math"...
>
>And what would be the useful piece of information for the customer? Perhaps
>something like "increase tour illumination power 27.35 times (don't quote me
>here :-) to get the same average illumination intensity if you want to
>compare spinning disk to widefield..."
>
>Best, zdenek
>
>
>
>
>
>
>---------- Původnà zpráva ----------
>Od: Andrea Latini <
[hidden email]>
>Komu:
[hidden email]
>Datum: 23. 5. 2016 14:40:42
>Předmět: Re: [QUAR] Re: widefield getting better images than spinning disk
>
>"*****
>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.
>*****
>
>dear Nico,
>the spinning disk itself acts as an optical filter on thick samples.
>Spinning Disk fill factor means 'Open Area Ratio' - holes area / blocked
>area.
>by inserting the disk within the optical path the theoretical throughput
>collection value would be 8.2% with respect to widefield (both excitation
>and emission pass through spinning disk of course), on 4 um beads, GFP
>excitation channel.
>
>the value 5-6% on 4um beads (and other values I'm reporting), has been
>measured by using "Measuring and interpreting point spread functions to
>determine confocal microscope resolution and ensure quality control Richard
>W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume 6, Pages:
>1929–1941(2011)" method.
>
>briefly (Macro is running in NIS Element SW):
>"
>
>1.) Sample: subresolution beads prepared according to „Measuring and
>interpreting point spread functions to determine confocal microscope
>resolution and ensure quality control
>Richard W Cole,Tushare Jinadasa,& Claire M Brown; Nature Protocols, Volume
>6, Pages: 1929–1941(2011)
>2.) Technical setup of Microscope:
>a. The microscope is set up for a typical point spread function
>acquisition to characterize the resolution/imaging quality using a high-end
>100x oil objective.
>b. A regular fluorescence light source is used for all acquisitions in
>combination with a filter cube. This is essential and the bottom principle
>of the test. The same illumination with the same sample is used to compare
>wide field intensity with the confocal intensity of bead images. This test
>assesses only the emission light transmission of the spinning disc device.
>i. Option one preferred: left and right port of the microscope is used so
>that one port holds the spinning disc device with camera and the other port
>an identical second camera in simple wide field mode.
>ii. Alternatively one can run the acquisition of test data first with the
>spinning disc +camera and then remove the scan head and use the same camera
>alone on the same port where the spinning disc scan head was mounted.
>iii. Toggle between wide field mode and spinning disc mode if the to be
>tested scan head provides such an option and (important!) if the wide field
>mode does not have more than a single mirror in the light path (otherwise
>the transmission loss of the additional optical elements in the wide field
>mode of the scan head results in a lower intensity measurements . This in
>turn would artificially “improve� the result so that one would think the
>ratio wide field intensity / confocal intensity is higher and therefore
>pretending to be the better instrument)
>
>3.) Procedure
>Time required: about 1 -2 hours
>Overview of work flow: acquisition of image z-stacks of beads at 3 different
>exposure times in each mode (confocal and wide field) while illumination
>remains constant.
>Prepare collapsed stack images (max projection)
>rolling ball background reduction
>find beads and set ROIs
>calculate mean or median intensity (in our hands it does not make a
>difference)
>plot intenstity vs. exposure time for both modes
>determine slope
>ratio of slopes = result
>
>In detail:
>a.) Set up beads in focus with the scanning device. The signal should be
>chosen at an intensity (illumination light intensity ) , so that with the
>same illumination wide field images are possible (sufficiently short shutter
>time should be available)
>b.) Stacks of images are taken at 3 different exposure times of different
>view fields to avoid the issue of bleaching,
>c.) Repeat the steps a) and b) in wide field mode
>d.) Run macro in ImageJ (enclosed) to analyze bead intensity
>
>Find different ROIs in each maximum projection :
>
>run("Z Project...", "projection=[Max Intensity]");
>run("Subtract Background...", "rolling=5");
>run("Threshold...");
>title = "WaitForUserDemo";
>msg = "If necessary, use the \"Threshold\" tool to\nadjust the threshold,
>then click \"OK\".";
>waitForUser(title, msg);
>getThreshold(lower, upper)
>run("Analyze Particles...", "size=1-4 exclude summarize");
>
>
>or: you run the macro above on the stack with the lowest exposure time
>and then – to keep the ROIs the same for all stacks- yu run the macro below
>on the remaining stacks
>
>run("Z Project...", "projection=[Max Intensity]");
>run("Subtract Background...", "rolling=5");
>roiManager("measure")
>
>
>e.) Prepare plots of the so calculated intensities vs the exposure time
>f.) Calculate slope
>g.) Ratio the slope.
>
>"
>
>
>regards.
>
>Andrea
>
>Andrea Latini
>President
>CrestOptics Srl
>
>
>On Mon, 23 May 2016 08:42:07 -0700, Nico Stuurman <
[hidden email]>
>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.
>>*****
>>
>>Dear Andrea,
>>
>>> collection efficiency CF/WF is 5% to 6% in our systems with 70um pinholes
>(fill factor 8.2%).
>>
>>Could you please explain what that number means, or better, how it is
>>measured? The way it is written, I have the impression that when a very
>>thin film of fluorescent material is excited by the same amount of light
>>you get 5-6% intensity in confocal mode and 100% in wide-field mode.
>>Obviously, that would be pretty bad (and according to the discussion on
>>this list, you should not lose more than a few percent of the
>>in-focus-signal through the pinholes), so I assume that you mean
>>something else.
>>
>>Best,
>>
>>Nico"