Re: Using a mirror for axial resolution testing

[Nuno Moreno]

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Hi,

we just refurbished the air conditioning in our imaging facility. You should consider:

. Room pressurized to avoid dust to get in
. Air return as close as possible to the floor (to take the dust out)
. Hepa filtering (H13 minimum)
. Have the sensor close to the microscope and not in the pipeline
. Air outlet that spreads as much as possible
. Humidity control
...and very important do not have units inside, just air pipping


I do think that people don't pay attention to it and put millions in a room with poor filtering and other bad environment conditions. This that can degrade optical components faster and make you cleaning filters (and others) more often. Cleaning is always a risk.

Best,
Nuno Moreno, PhD
Instituto Gulbenkian de Ciência
http://lnkd.in/6VXcrM





On Oct 8, 2013, at 8:51 AM, Csúcs Gábor wrote:

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> *****
>
> Dear All,
>
> Not a very scientific (though who knows) but still an important (in terms
> of comfort) question about the climatization of microscopy rooms. We are
> just in the middle of discussions about the arrangement of our new
> microscopy rooms. Most of the things are clear, however the planning team
> suggested a solution for the climatization that is new to me and I wanted
> to enquire whether anyone of you had experience with similar systems. In
> the "conventional" arrangement the cold air comes in somewhere at the
> ceiling and I think there is a general consensus that it is better if it
> is well distributed and not simply blown in into one direction. Our
> planning team however suggests a new solution, where the cold air would be
> blown in (through some canvas tubes) close to the bottom/floor. The warm
> air (that goes anyway upward) is sucked at the ceiling. According to them,
> although this systems creates a height-dependent temperature gradient
> (cold bottom, stable 22 C at the microscope level and warmer at the
> ceiling) but with this one can avoid the continuous mixing/turbulence
> where both the blowing in and the sucking away happens on the ceiling
> (conventional solution). Now, in theory this sounds good but we are
> somewhat skeptical how well this system works in practice and what the
> users say if their feet has colder (approx. 16-18 C) temperatures then
> their body. In a few weeks we will have the opportunity to check a similar
> installation, but I'd really appreciate if you could share your
> experiences with us. Obviously this is a important decision for us so any
> feedback is welcome.
>
> Thanks     Gabor

[Watkins, Simon C]

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Recently (last year) we expanded our Center to include 5 new scope rooms.
These were all around 100-200 sq ft each and were to be built within a
converted classroom space. Previously we had thought of this conversion
but balked because of the cost of the changes in air handling needed.  In
this case I used Watt Misers (power calculator widgets) to measure the
power usage by systems and was truly stunned by how things have changed.
Essentially if you are using current Diode based lasers launches, flat
screen panels and diode illuminators your power use and hence heat
production is quite low, lots and lots of power cordsŠ most of which lead
to 12 volt transformersŠ Which essentially mean little power is consumed.
For example a scope with a 4 line launch, 7 color diode illuminator,
inverted scope, 3 cameras, a small stage type warm/co2 stage, computer and
2x 30 inch screens used less than 400 watts when at full workload. By
comparison just one argon laser used @ 2.5kW (I think).  The only heat
generator in the new space are the Ti Sapphire lasers which we vent into
the corridor and the larger heated chambers we use in the MPE animal
warming boxes.  This is a particularly sensitive issue because the
super-res systems, specifically our SIM system (also in this space)
demands very stable temperatures.  Nowadays the biggest heat producers are
the operators not the systems. As far as heat/cooling air delivery we use
solutions where air is pumped in through the ceiling diffused with baffles
and leaves either through another air vent in the ceiling or a vented
panel in the door, because of decreased heat load the airflow is never
that great (except in rooms with gas or MPE lasers). However, and because
of variable cooling need between spaces all rooms still have independent
thermal control and it does not vary over a 24 hour cycle.
Hope this helps
S

Simon Watkins Ph.D

Professor and Vice Chair Cell Biology
Professor Immunology
Director Center for Biologic Imaging
University of Pittsburgh
Bsts 225 3550 terrace st
Pittsburgh PA 15261
Www.cbi.pitt.edu <http://Www.cbi.pitt.edu/>
412-352-2277






On 10/8/13 3:51 AM, "Csúcs  Gábor" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All,
>
>Not a very scientific (though who knows) but still an important (in terms
>of comfort) question about the climatization of microscopy rooms. We are
>just in the middle of discussions about the arrangement of our new
>microscopy rooms. Most of the things are clear, however the planning team
>suggested a solution for the climatization that is new to me and I wanted
>to enquire whether anyone of you had experience with similar systems. In
>the "conventional" arrangement the cold air comes in somewhere at the
>ceiling and I think there is a general consensus that it is better if it
>is well distributed and not simply blown in into one direction. Our
>planning team however suggests a new solution, where the cold air would be
>blown in (through some canvas tubes) close to the bottom/floor. The warm
>air (that goes anyway upward) is sucked at the ceiling. According to them,
>although this systems creates a height-dependent temperature gradient
>(cold bottom, stable 22 C at the microscope level and warmer at the
>ceiling) but with this one can avoid the continuous mixing/turbulence
>where both the blowing in and the sucking away happens on the ceiling
>(conventional solution). Now, in theory this sounds good but we are
>somewhat skeptical how well this system works in practice and what the
>users say if their feet has colder (approx. 16-18 C) temperatures then
>their body. In a few weeks we will have the opportunity to check a similar
>installation, but I'd really appreciate if you could share your
>experiences with us. Obviously this is a important decision for us so any
>feedback is welcome.
>
>Thanks     Gabor

[mmodel]

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The dye method can be used with any objective that uses a coverslip; without a coverslip it would difficult even to do it with beads (the mirror would probably be the only practical option). But I don't think it's often necessary to check every single objective on the confocal - if the point is to check a new objective for aberrations it's much easier to get a stack of a bead in widefield.

In my opinion, most  usefulness of the concentrated dye method comes from the fact that it creates a sample with uniform and reproducible quantum yield, so that fluorescent samples can be calibrated and measured ("Measurement of wheat germ agglutinin binding with a fluorescence microscope". Cytometry 75A, 874-881, 2009).

Best,

Mike
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of James Pawley [[hidden email]]
Sent: Tuesday, October 08, 2013 12:24 PM
To: [hidden email]
Subject: Re: Using a mirror for axial resolution testing

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Hi all,

May I add two points to this important discussion?

1. My usual point: Maybe it is relevant that when
looking at the refection from a mirror, one has
an essentially infinite level of signal (the
reflection of a mW laser beam) that is millions
if not billions of times larger than that
available from viewing a fluorescent bead that is
only 100 nm in diameter. Because of this, it is
common to cylindrically average the signal in the
case of the bead, a process that can mask the
effect of asymmetries in the OTF such as the
astigmatism caused by imperfect beam splitters or
improper alignment.

Apart from this, Poisson noise will have a much
greater effect in terms of introducing
uncertainty..

2. My other usual point: Is spherical aberration
involved? Beads are made of plastic and the RI of
plastic may or may not be the same as that of the
surrounding medium or that for which the
objective was designed. Of course, one assumes
that this has little effect when the diameter of
the bead is 50nm, but as such beads usually give
only about 1/8th the signal of 100nm beads made
of the same material and only about 1/64th the
signal of 200nm beads, point 1 (above) can drive
the user to larger beads (and larger pinhole
settings). I have no calculation to describe the
blurring effect of using any particular design of
bead in any specific mounting medium, however, I
would expect such blurring to be more pronounced
in the z than in the x-y direction because some
of the rays from focus planes on the far side
have to pass through the bead. This would be even
more important is the entrance pupil of the
objective is not fully and evenly filled by the
laser beam.

Perhaps one should also point out the
peculiarities of the light signal reflected from
a flat RI interface, such as that between
coverslip and water, compared to that produced by
reflection from a metallic mirror. The former
will contain proportionally more signal from
"high-NA" rays than from those approaching the
interface at closer to normal incidence and there
is also the fact that the amount reflected at an
RI interface varies strongly with the angle
between the polarization of a high-NA ray and the
orientation of the surface. The asymmetric
doughnut-shaped apodization resulting from these
effects can make the "z-resolution" look better
(or worse) than it would be otherwise.

I can see the
"thin-layer-of-fluorescent-immersion-oil"
specimen being useful for measuring the
performance of oil-immersion lenses, but how does
one use it to measure the performance of water or
glycerine objectives?

The
"thin-layer-of-fluorescent-molecules-deposited-on-glass"
specimens do not have this problem, but I would
guess that the signal levels might be lower and
there could be orientation effects related to the
alignment between the (possibly non-random)
dipole axes of the dye and the electric field
orientation of a convergent and polarized light
beam.

More to the point, who has compared the PSF of a
bead near the coverslip surface with one embedded
even a few µm inside a watery (living?)
biological specimen? My few attempts to do so
have revealed pronounced asymmetries that are
readily visible by eye (as long as the eye is
observing and the stored image collected from the
CCD). In other words, apart from its use as a
(very important) check on instrument performance,
do we really need to know the PSF or FWHM etc to
such precision?

Regards,

Jim Pawley

>*****
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>*****
>
>Reto,
>you pointed out correctly that the integrated
>intensity in any 2d plane of a widefield PSF is
>identical, in theory. Although, to the trained
>eye it gives away the singularity in reciprocal
>space, but this by itself may not fully explain
>the sectioning capabilities. The widefield
>microscope indeed has no sectioning ability, but
>strictly only at the zero frequency component!
>In other words, you can not focus on a plane
>object or fluorescent sheet without detail.
>However you can focus on small dust particles on
>it that exhibit higher frequency components.
>This all becomes clear when looking at the OTF.
>There you can see that the "sectioning strength"
>actually depends on the frequency and it has a
>maximum in about the middle of the radius of
>this torus, while as said before, there is a
>singularity (Dirac) in its origin.
>
>Now having said that, when the bead used in the
>measurements approximates a Dirac, its spectrum
>is a constant, leaving the product with the OTF
>unchanged and what you see in the inverse FT
>simply is the PSF. It is straight forward that
>the axial extend of that PSF corresponds to the
>sectioning ability at around the reciprocal of
>the pass-band frequencies of the OTF torus (...
>and YES of a widefield microscope).
>
>I am not sure how helpful this information is to
>some, as for specimens with varying frequency
>content, your sectioning will vary too. Lower
>frequencies usually dominate giving the well
>known response and the notion of no sectioning.
>Using deconvolution one can increase the
>sectioning capabilities, but really only in the
>nonlinear (iterative with positivity constraint)
>case where the lower pass band frequencies, that
>got lost lost due to the inner part of the torus
>become restored, if indeed they were present in
>the specimen to begin with.
>
>Hope that helped
>Regards
>Lutz
>
>__________________________________
>L u t z   S c h a e f e r
>Sen. Scientist
>Mathematical modeling / Computational microscopy
>Advanced Imaging Methodology Consultation
>16-715 Doon Village Rd.
>Kitchener, ON, N2P 2A2, Canada
>Phone/Fax: [phone]
>Email: [hidden email]
>Website: http://home.golden.net/~lschafer/
>___________________________________
>
>-----Original Message----- From: Reto Fiolka
>Sent: Monday, October 07, 2013 10:47 AM
>To: [hidden email]
>Subject: Re: Using a mirror for axial resolution testing
>
>*****
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>*****
>
>Hi John
>
>This is my humble opinion:
>
>Measuring resolution with beads in a shift invariant imaging system (image is
>convolution with PSF) in any dimension is considered legitimate provided you
>are using sufficiently small beads.
>
>
>Sufficiently small: if your bead is 10 times smaller than the PSF you are
>measuring, then your error is below 1% (rough estimation assuming gaussian
>profile for PSF and bead fluorophore distribution: sqrt(1^2+0.1^2)=1.005 ). So
>a 50nm bead for axial resolution in confocal microscopy is safe.
>
>Any peer reviewed journal that I am aware of accepts such a measurment
>when you introduce a new technique.
>
>Brad Amos probably means that having a finite FWHM in the axial PSF does not
>mean that you have a good optical sectioning capability. That the widefield
>microscope has no sectioning capability is
>included in its PSF: the out of focus
>rings conserve the same energy as is found in the focal plane, hence there is
>no sectioning. However just measuring the
>profile along the central axis will not
>reveal this, it might actually look as the intensity would decrease as you go
>away from the focal plane.
>
>Best,
>Reto

--
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Haller, Edward]

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Something I haven't seen addressed yet is harmonic noise produced by the air handler system. Make sure that whatever system that you use is silent, and that the air flow is not directed toward your vibration isolation tables. Both conditions can set up harmonic noise in your imaging systems. I run an E. M. core, but this also translates to high magnification optical microscopy. Noisy air handlers and pulses of air hitting your imaging equipment can affect your images.

Ed

Edward Haller, Lab Manager
University of South Florida
Integrative Biology Department
Electron Microscopy Core
SCA 110
4202 East Fowler Avenue
Tampa, FL 33620
813-974-2676
[hidden email]
Office: ISA 1046
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Csúcs  Gábor [[hidden email]]
Sent: Tuesday, October 08, 2013 3:51 AM
To: [hidden email]
Subject: Room climatization

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Dear All,

Not a very scientific (though who knows) but still an important (in terms
of comfort) question about the climatization of microscopy rooms. We are
just in the middle of discussions about the arrangement of our new
microscopy rooms. Most of the things are clear, however the planning team
suggested a solution for the climatization that is new to me and I wanted
to enquire whether anyone of you had experience with similar systems. In
the "conventional" arrangement the cold air comes in somewhere at the
ceiling and I think there is a general consensus that it is better if it
is well distributed and not simply blown in into one direction. Our
planning team however suggests a new solution, where the cold air would be
blown in (through some canvas tubes) close to the bottom/floor. The warm
air (that goes anyway upward) is sucked at the ceiling. According to them,
although this systems creates a height-dependent temperature gradient
(cold bottom, stable 22 C at the microscope level and warmer at the
ceiling) but with this one can avoid the continuous mixing/turbulence
where both the blowing in and the sucking away happens on the ceiling
(conventional solution). Now, in theory this sounds good but we are
somewhat skeptical how well this system works in practice and what the
users say if their feet has colder (approx. 16-18 C) temperatures then
their body. In a few weeks we will have the opportunity to check a similar
installation, but I'd really appreciate if you could share your
experiences with us. Obviously this is a important decision for us so any
feedback is welcome.

Thanks     Gabor

[Chris Guerin]

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Hi Gabor/everyone:

        We recently built a new facility and our architect came up with a very effective, relatively low cost and really efficient method which controls our rooms to a stable temp of less than 1 degree. We move lots of air through these rooms with no detectable currents nor hot/cold spots. The rooms use a system where the air enters through a false wall which has large fabric panels acting as diffusers (see https://dl.dropboxusercontent.com/u/27902941/DSCN9949.JPG), and then exits through passive ducting in the ceiling on the opposite wall. This not only supplies perfect laminar airflow but also positive pressure.

        I've seen facilities with similar systems to what you describe, they seem to work but unless the air is moving through the canvas fabric and not out a hole somewhere I would still expect some disturbances. Anyone who wishes to see our solution firsthand is very welcome in Ghent.

best wishes,

Chris

Christopher Guérin, Ph.D.
Senior Expert Scientist
Leader, IRC Microscopy Core
Manager, VIB Bio-imaging Core, Gent
V.I.B., I.R.C., Univ. Gent
Fiers-Schell-Van Montagu' building
Technologiepark 927, B-9052 Ghent (Zwijnaarde) - Belgium
tel : [phone]
[hidden email]
web: www.bioimagingcore.be
http://www.vib.be/en/research/scientists/Pages/Chris-Guerin-Lab.aspx

[Jules Girard]

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Dear all,
I've been following that interesting discussion with a bit of delay. Although
most has been already said, I feel that a clearer answer can be given to the
initial question (I was about to say more concise, but I now figure out that my
contribution is not so short...). Let me try to do so.

Definitions are important here :
-Resolution (axial and lateral), is the capability of an imaging system to resolve small details of an object, which can be quantified in different ways. In Fourier
space, it is related to both the extent (support) and the magnitude of the OTF.
-(Axial) Optical Sectioning is the capability of an imaging system to get rid of
out of focus signal.

In my opinion, the most complete way to quantify resolution is to think in the
frequency space and use OTF, especially when deconvolution is considered
and/or with samples that are not pointillistic (i.e. that have an inhomogeneous
spatial spectrum). But a more common and often convenient way to quantify
resolution is to stay in direct space and use PSF + Rayleigh criterion/FWHM.
This is however an arbitrary and restrictive way of defining/measuring
resolution.
Brad Amos previous answer seems to be confusing axial and lateral resolution.
Unlike what is often thought, lateral resolution is scarcely improved by confocal
microscopy compared to WF (I generally consider that improvement to be
inexistent), as it has been shown by Guy Cox and Colin Sheppard [1]. The
sqrt(2) factor is valid only for very small pinhole and infinite signal : in practice
that improvement is exactly compensated by the signal (over noise ratio)
decrease. The improvement is however clear for axial resolution, as it can be
seen with comparison of 3D OTF in both WF and confocal case (cf. that great
99 paper by Gustafsson [2]). The improvement is much better than a sqrt(2)
factor here, even with a reasonable pinhole size.

You need Optical Sectioning when you have a 'thick' or autofluorescing sample,
to avoid getting your in focus signal drown in blurry background signal. It has
been pointed out already, but let me remind here that it is sometimes possible
to axially resolve two objects even without any sectioning power: in the WF
case, the out of focus signal is quickly spread laterally, and that one can
generally distinguish two beads axially aligned in a z-stack. In Fourier space,
this can be seen as a contribution of "diagonal" frequencies (i.e. with both
lateral and axial direction components) that are out of the dark cone (or
"singularity" as Lutz was calling it) of the WF OTF. Ideally, with a well known
PSF, a confined object and no shot noise increase, one could deconvolve such
blurry 3D image and not care about optical sectioning anymore (except for the
object features that are mainly described with spatial frequencies situated in
that dark cone, i.e. flat layers of fluorophores more or less orthogonal to the z
axis).

To sum up :
-If you care more about resolution, I would say that using small beads is more
appropriate. If your bead is 100nm or smaller, you get a good approximation of
your PSF from your direct measurement and you can use it for whatever
arbitrary criterion you want to use (Rayleigh, FWHM, Sparrow or OTF...).
-If you care more about optical sectioning, then you should use a thin
fluorescent layer : the spreading of the out of focus light will be compensated
by the quasi infinite lateral extent of your sample (within the limit pointed out
by James Pawley) and you should get a I(z) function that is suitable to define
optical sectioning. Alternatively, you could also use a PSF measurement from a
small bead and integrate the signal you get at each z position (i.e. project your
I(x,y,z) measured function onto a I(z) one). The result should theoretically be
the same than with the thin layer, but you will have a much lower signal and
and a lot of pixels to sum when going out of focus, leading to some poor SNR.

Best,

Jules

[1] G. Cox, C.J.R. Sheppard, Practical limits of resolution in confocal and non-
linear microscopy., Microsc. Res. Tech. 63 (2004) 18-22.
[2] M.G.L.
Gustafsson, Extended resolution fluorescence microscopy, Curr. Opin. Struct.
Biol. 9 (1999) 627-628.

--
Jules Girard, PhD

Postdoctoral Researcher
in
Physics of Living Systems group
Department of Physics and Astronomy
VU University Amsterdam

Room U-059
De Boelelaan 1081
1081 HV Amsterdam, The Netherlands

Tel : [phone]
Email: [hidden email]