Posted by
Andrea Latini on
URL: http://confocal-microscopy-list.275.s1.nabble.com/widefield-getting-better-images-than-spinning-disk-tp7585177p7585211.html
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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:
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>
>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
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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:
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>Post images on
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>
>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"