[OpenSPIM] OpenSPIM Digest, Vol 46, Issue 2

Sat Jun 10 00:10:33 CDT 2017

Stephanie,

You are correct. The tube lens (the 25mm) of the 2nd telescope needs to be
focused at the back focal plane (BFP) of the illumination objective. Move
the focus from the BFP and the light sheet exiting the objective will
either converge or diverge i.e. the sheet won't be collimated in the XY
plane and it'll look fan shaped. Conversely, even if you do move the
objective/chamber along the axis of the objective, the XZ plane will remain
in focus at 3.5 mm from the nose of the objective as the laser is
collimated in that plane at the BFP. Of course this behavior assumes all of
the upstream optics are in proper relationship to each other. You could
have the upstream alignments wrong and still get a visibly collimated sheet
with the 25 mm tube lens not focused in the BFP but your light sheet
quality will be somewhere between slightly off to just plain crap as well
as potentially being tilted out of and/or within the detection plane. The
final four optical elements are indeed functionally tied together. The
cylindrical lens shares a focal plane with the following 50mm which shares
a focal plane with the 25mm which shares a focal plane with the objective
all of which only work if the laser coming into the cylindrical is
collimated and enters into the center and is on axis.

Your next question might well be: where is the BFP of an objective? As most
objective houses won't tell you, you have to find it experimentally. This
can be done pretty easily. Assuming your system is assembled, mirror align
the laser, all lenses removed from their mounts, to the center and along
the axis of the illumination objective. Fill the sample chamber with water
containing a fluorophore appropriate to your laser. With the laser and
camera on and the objectives correctly placed with respect to each other,
you should see the laser focal point more or less in the center of the
camera image. Please note that your alignment could be pure genius and
still look stuffed up if your camera and tube lens aren't aligned with and
centered on the detection objective axis. Move the illumination objective
to center the laser focal point in the FOV and move the detection objective
to bring the beam into best focus. Try using profile plot with live update
in Fiji while dragging the ROI around to find the highest intensity point
i.e the center. If the beam is tilted within or out the FOV plane, the
laser is entering the objective at an angle to the axis (or the objectives
aren't square to each other). Too high or too low, and the beam is parallel
to the objective axis but not _on_ the axis. The laser back reflection off
a properly aligned objective should line up with the laser spot on the
mirror face. It's also fair to note that the sample chamber may be poorly
mounted and the objective ports not be perfectly in plane and at 90 degrees
to each other. This is important to consider because if you start with a
well aligned beam i.e. in plane, 50 mm from the optics board and centered
through the lens mounts, and then tilt the beam to match a poorly placed or
built chamber, which is certainly an optical element in this build, you'll
never get a really good alignment after putting the optics back in the
system as they are all mounted on fixed height posts and now might not
co-axially align with the new beam path.

Once you're satisfied with the location of the beam and chamber, place a
point reference either on the monitor (a Post-It corner works) or an ROI on
the live imaging window to mark the center point of focus for later use.
Place the 25 mm lens in question on the rail. Turn the laser back on and
live image while you slide the lens back and forth; you are in the BFP when
the light sheet exiting the objective is collimated. It really helps if you
have a thin object in the beam path as the shadows cast are very good
indicators of collimation and the expanded beam will be much fainter (and
possibly impossible to resolve clearly) than the focused beam. Look for
zero convergence/divergence of the shadows. Measure the distance from the
lens mount to the objective for your future reference. Note: if your lens
mount is rotated about the pillar mount axis or tipped at some small angle,
you won't be able to discern the issue until you mount the 50 mm lens.
Which then leaves you with a two variable question but...

When you place the 50 mm telescope lens onto the rail you can co-localize
its focal point with the 25 mm lens focal point by sliding it along until
the laser is in focus and located at your previously marked center point.
The center will be harder to find as the NA of the beam will be smaller
than the raw laser and thus the Rayleigh length longer. Again Fiji profile
can help. If the beam is tilted in the plane and/or out of plane, the laser
and the lens or lens pair don't share an axis. Adjust the lens, actually
more likely both lenses, as best as you can and move the beam via the
mirrors only as a last resort. This is the fiddly frustrating bit of
putting together a base level OpenSPIM as finding the right set of
sub-optimal alignments of mostly fixed optics that will work in the end is
a deeply iterative process.You can continue the alignment from this point
by working backwards through the remaining optics though I would leave the
cylindrical for last and you'll need to place the beam expander lens pair
as a group. Using a 1" 50mm achromat lens in place of the cylindrical for
initial alignment can be useful to get mounting angles close because
cylindrical lenses can be very difficult to center properly.

Finish up your alignment with a fine tune of light sheet planar and focal
parameters via imaging the center of an agarose cylinder sample containing
fluoro beads or simply dumping enough beads into chamber water (without
fluorophore of course). It'll be very obvious if your sheet is tipped out
of the detection focal plane. This is really the only way to get
cylindrical rotation correct. While you're at it, fine focus the detection
objective. A large number of out of focus beads in front of and/or behind
the focal plane indicates a loss of optical confinement and can mean your
sheet is badly formed. Take the opportunity to adjust the corner mirror to
understand what changes are reflected in light sheet propagation. You can
also use the airy discs of out of focus beads to tell you if your detection
objective, tube lens and camera are well aligned. I don't recommend using
an agarose cylinder for the initial build process as the agarose cylinder
will steer the beam potentially leaving your baseline alignment crooked. Of
course you're correct if you think the same issue is at work during sample
imaging. Nature of the beast.

Well it isn't a short answer but hopefully it's a useful one. Have a good
weekend.

Cheers,

- David

David Potter
Lattice Light Sheet Microscopy Manager
Monash Micro Imaging - Advanced Optical Microscopy
Monash University Clayton, VIC 3800, Australia

On 10 June 2017 at 03:00, <openspim-request at openspim.org> wrote:

> Send OpenSPIM mailing list submissions to
>         openspim at openspim.org
>
> To subscribe or unsubscribe via the World Wide Web, visit
>         http://openspim.org/mailman/listinfo/openspim
> or, via email, send a message with subject or body 'help' to
>         openspim-request at openspim.org
>
> You can reach the person managing the list at
>         openspim-owner at openspim.org
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of OpenSPIM digest..."
>
>
> Today's Topics:
>
>    1. distance telescope illumination objective (Dr S. Hoehn)
>    2. Re: distance telescope illumination objective (Weber, Michael)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 09 Jun 2017 14:42:58 +0100
> From: "Dr S. Hoehn" <S.Hoehn at damtp.cam.ac.uk>
> To: OpenSPIM at openspim.org
> Subject: [OpenSPIM] distance telescope illumination objective
> Message-ID: <a76138ed3fecf6377274c4ef4de4b2db at cam.ac.uk>
> Content-Type: text/plain; charset=US-ASCII; format=flowed
>
> Dear all,
>
> do I understand correctly that there has to be a specific distance
> between the last telescope lens and the illumination objective?
>
> So I could NOT move the sample chamber further away from the telescope?
> I was thinking about doing this since we have a wider bread board and a
> longer path would make it easier to align the laser.
>
> Thank you,
>
> Stephanie
>
>
> Dr. Stephanie Hoehn
> DAMTP, Biological Physics
> Centre for Mathematical Sciences
> Wilberforce Road
> Cambridge
> CB3 0WA
> United Kingdom
> Office Tel: 01223 337859
> Office: H0.03
>
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Fri, 9 Jun 2017 14:15:14 +0000
> From: "Weber, Michael" <Michael_Weber at hms.harvard.edu>
> To: "OpenSPIM at openspim.org" <OpenSPIM at openspim.org>
> Subject: Re: [OpenSPIM] distance telescope illumination objective
> Message-ID:
>         <BLUPR0701MB17450275AAC2BBB12FDF1062DDCE0 at BLUPR0701MB1745.
> namprd07.prod.outlook.com>
>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Stephanie,
>
>
>
> Every distance after the cylindrical lens is critical. Moving the sample
> chamber with respect to the telescope will shift the position of the light
> sheet waist. If you want to increase the distance between telescope and
> chamber you could build the telescope out of two lenses with longer focal
> lengths but the same ratio.
>
>
>
> Best,
>
> Michael
>
>
>
> From: Dr S. Hoehn<mailto:S.Hoehn at damtp.cam.ac.uk>
> Sent: Friday, June 9, 2017 9:43 AM
> To: OpenSPIM at openspim.org<mailto:OpenSPIM at openspim.org>
> Subject: [OpenSPIM] distance telescope illumination objective
>
>
>
> Dear all,
>
> do I understand correctly that there has to be a specific distance
> between the last telescope lens and the illumination objective?
>
> So I could NOT move the sample chamber further away from the telescope?
> I was thinking about doing this since we have a wider bread board and a
> longer path would make it easier to align the laser.
>
> Thank you,
>
> Stephanie
>
>
> Dr. Stephanie Hoehn
> DAMTP, Biological Physics
> Centre for Mathematical Sciences
> Wilberforce Road
> Cambridge
> CB3 0WA
> United Kingdom
> Office Tel: 01223 337859
> Office: H0.03
>
>
>
> _______________________________________________
> OpenSPIM mailing list
> OpenSPIM at openspim.org
> http://openspim.org/mailman/listinfo/openspim
> -------------- next part --------------
> An HTML attachment was scrubbed...
> URL: <http://openspim.org/pipermail/openspim/
> attachments/20170609/e5eae151/attachment-0001.html>
>
> ------------------------------
>
> Subject: Digest Footer
>
> _______________________________________________
> OpenSPIM mailing list
> OpenSPIM at openspim.org
> http://openspim.org/mailman/listinfo/openspim
>
>
> ------------------------------
>
> End of OpenSPIM Digest, Vol 46, Issue 2
> ***************************************
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://openspim.org/pipermail/openspim/attachments/20170610/cdd3d8f0/attachment.html>