I agree with previous responders that testing is definitely worth doing.
It is a good idea to use both the mirror slide and sub-resolution multicolor
beads.
The mirror slide will indicate the chromartic aberration and also the
curvature of the field. When I was testing lenses on our confocal (Olympus),
there was generally a reasonably small chromatic aberration (within their
specifications, less than 250 nm) with the exception of the 405 nm laser,
which was ~500 nm in some of the 'identical' 100x/1.4 oil immersion
objectives, but more than that with some of the objectives. Also, the Plan
correction may not be as good for the 405 nm wavelength. - A saw a fairly
obvious curvature of the field in the 405 nm channel.
The sub-resolution beads (I used Invitrogen Tetraspeck beads 100nm; no
commercial interest) should indicate if the lateral registration of channels
is uniform across the whole field of view, and reveal alignment problems. I
was surprised in my tests to see that in a two-channel image the
colocalization is perfect in one corner of the image, but in the opposite
corner there was a lateral shift of several pixels.
If aligning the microscope does not solve the problem completely, one could
map these differences measured from the bead data stacks and apply affine
transforms to warp one channel of the sample datasets. There was a post
about this few years ago (quoted below); I never got around to implement it
on our confocal.
Stan Vitha
Microscopy and Imaging Center
Texas A&M University
>Subject: Re: 3D registration of two channels
>From: Kevin Braeckmans <[log in to unmask]>
>Reply-To: Confocal Microscopy List <[log in to unmask]>
>Date: Sun, 24 Sep 2006 07:10:22 +0200
>Content-Type: text/plain
>You are right, it is also our experience that you can increase the overlay
>accuracy by a more complex transformation than a simple translation. In our
>case it is in a non-confocal application, but the principle stays the same.
>By using a general affine transformation instead of a simple translation we
>can increase the average overlay accuracy from approx. 0.5 micron to approx.
>10 nm (this is in 2D). You could even use more complex transformations
>(projective or even non-linear ones), but 10 nm is around the accuracy with
>which we can determine the (Gaussian) centres of fluorescent beads.
>(An affine transformation is a general combination of translation, scaling
>along x, scaling along y, rotation, shear (i.e. transformation of angles),
>and reflection along x and/or y.)
>Before the actual experiments we take a dual channel image of Tetra Speck
>beads. An image processing program we wrote in Matlab then calculates the
>affine transformation parameters based on the (Gaussian) centres of the
>beads. Finally, the dual channel movies are automatically overlayed using
>those transformation parameters.
>If you like you can send me a dual channel image of the Tetra Speck beads
>(at least 4 beads in the image as widely spread as possible) and I could
>check by how much you can improve the accuracy by using the more general
>afiine transformation (maybe your confocal is already good as it is and a
>translation is all you need).
>Also, I think about 2 weeks ago someone on the list asked about the
>registration accuracy of other confocal users. Maybe this person meanwhile
>can share the information obtained from other confocalists what is the
>overlay accuracy that is generally obtained on confocal microscopes?
>Best regards,
>Kevin
>Kevin Braeckmans, Ph.D.
>Lab. General Biochemistry & Physical Pharmacy
>Ghent University
>Harelbekestraat 72
>9000 Ghent
>Belgium
>Tel: +32 (0)9 264.80.78
>Fax: +32 (0)9 264.81.89
>E-mail: [log in to unmask]
=============
On Thu, 20 May 2010 17:42:18 -0600, Craig Brideau <[log in to unmask]>
wrote:
>Violet lasers are relatively recent. Microscope optics are still
>playing catch-up to the shorter wavelengths so they don't perform as
>well for violet lines. If one of your sources is close to 400nm this
>can be a serious problem. On the other hand, if you are using fairly
>standard wavelengths that the objectives are supposed to be
>chromatically corrected for, then you shouldn't have any focal shift
>issues. The exception being, as mentioned by others, if you are using
>mismatched media.
>
>Craig
>
>On Thu, May 20, 2010 at 2:43 PM, Glen MacDonald
><[log in to unmask]> wrote:
>> Hi Carl,
>> This sounds like a classic issue with chromatic aberration. Which
channel(s) exhibit the axial shift? Even in some planapo lenses, 405
excitation is shifted relative to the 488 line. Is this shift consistent
across the field? If less in center then you can zoom in to avoid it.
>>
>> Glen
>> On May 20, 2010, at 1:28 PM, Martin Wessendorf wrote:
>>
>>> Dear Carl--
>>>
>>> Always good to check the characteristics of the lens. However, I expect
that if you're using a good quality lens that's clean, the lens not the
problem. (If the lens is dirty--oil/mounting medium/etc on a dry lens or
glycerol, etc, on an oil lens--it's a different story: that can introduce
significant chromatic aberration.)
>>>
>>> Misalignment of the pinholes in the z-axis would produce z-shift.
However, my recollection is that on the 510, you can only align one of the
pinholes (--the meta pinhole) in z. Not sure why. When I've done 3-color
mirror scans on a 510 with good quality oil-immersion lenses, they generally
show some degree of misregistration in the z-axis--up to about a micron.
>>>
>>> I haven't noticed any place in the Handbook that deals with this
issue--Jim, if you're out there, am I missing something?
>>>
>>> Good luck!
>>>
>>> Martin Wessendorf
>>>
>>> Craig Brideau wrote:
>>>> I recommend coated beads for this task. They have an inner core that
>>>> tests one wavelength, and an outer coating that tests the other. Find
>>>> the difference in 'center of mass' between the solid core and the
>>>> hollow sphere around it and you should have your wavelength
>>>> displacement difference.
>>>> If your optics are good this should be a non-issue anyway. It's
>>>> always good to check for truth in advertising though, at least where
>>>> 'achromatic' lenses are concerned...
>>>> Craig
>>>> On Thu, May 20, 2010 at 1:11 PM, Carl Boswell
>>>> <[log in to unmask]> wrote:
>>>>> Hi listers,
>>>>> When one tries to assess colocalization, the question of where
something is
>>>>> in "z" is confounded by the differences in focal point based on emission
>>>>> wavelength. I see no difficulty in assessing how much z shift there is,
>>>>> using subresolution beads labelled with different dyes. My problem comes
>>>>> with trying to apply any correction factor derived from the beads back to
>>>>> the original image data and thus to the colocalization determination. Any
>>>>> ideas?
>>>>>
>>>>> We'll be using a Zeiss 510 Meta, and I haven't seen anything in the
software
>>>>> that allows an adjustment in "z" position for a particular
>>>>> channel/wavelength.
>>>>>
>>>>> Thanks,
>>>>> Carl
>>>>>
>>>>> Carl A. Boswell, Ph.D.
>>>>> Molecular and Cellular Biology
>>>>> University of Arizona
>>>>> 520-954-7053
>>>>> FAX 520-621-3709
>>>
>>> --
>>> Martin Wessendorf, Ph.D. office: (612) 626-0145
>>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>>> University of Minnesota Preferred FAX: (612) 624-8118
>>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>>> Minneapolis, MN 55455 e-mail: [log in to unmask]
>>
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