[OpenSPIM] Out of focus signal and optics questions

Thu Apr 14 17:57:09 CDT 2016

Hi all, thanks for the tips.

I moved things around and realigned to what I now feel is a better focus, where slight movements of the second lens of the second telescope shifts the beam waist left and right on the camera (with the cylindrical lens removed).

Monika, I tried to collimate the beam using the objective as you suggest, but found it hard to see how well the beam was collimated through the water and chamber...  I think it was collimated to some degree.  My assumption here is that you'd need those in place?

Either way, it looks like the beam waist and I've gotten some bead data to share with you as I still feel something is wrong regarding the out of focus light.  I've taken a z-stack of 100nm beads in 1% agarose, including a bright piece of schmutz that highlights the out of focus illumination.  The images are from an ORCA CCD, but I've reduced the bit depth and cropped to a thin column of data to make the files smaller.  The height of the sheet does not completely fill the CCD, I think due to the 0.5x c mount and the fact that the beam width on the Stradus is a little smaller than I need and I might need a little more beam expansion.  The sheet is incident from the right hand side of the images.

Tif stacks:
0.4mm slit
https://drive.google.com/file/d/0BzfJAGNfrgieOUszTGYwZGh1YVE/view?usp=sharing
0.8mm slit
https://drive.google.com/file/d/0BzfJAGNfrgiea21vLUtqZTdsTHc/view?usp=sharing
Orthogonal screen shots:
https://drive.google.com/file/d/0BzfJAGNfrgieekktMnFETEwzd3M/view?usp=sharing
https://drive.google.com/file/d/0BzfJAGNfrgieOExHRVNYYndHVGs/view?usp=sharing

It appears that there's significant difference in the sheet profile for the different slit sizes.  The piece of schmutz is quite large, so this is a guess, but it looks like the thickness of the sheet is 50um or more.  I have the contrast cranked up, and this is over emphasizing the tails of the sheet profile, but I'm a little perplexed.

Thanks in advance for your time.  Please let me know if you need any further details/data.
Neil

-----Original Message-----
From: openspim-bounces at openspim.org [mailto:openspim-bounces at openspim.org] On Behalf Of Monika Pawlowska
Sent: Monday, April 11, 2016 10:01 AM
To: openspim at openspim.org
Subject: Re: [OpenSPIM] Out of focus signal and optics questions

Hi,

I think it would be easier for us to guess the reason of the problem if we had an example image of the "out of focus light". :)

But: there is a trick to find the BFP even when you have no idea where it is (I didn't  :P ). Start with a collimated beam after the 1st telescope , mount the second telescope so that the beam is collimated again, and then take out first lens of second telescope and put the objective in. This way, second lens should focus and objective recollimate the beam.

Afterwards, when everything is in place, rotating cyl. lens 90 degrees lets you see (without fluorescence filter) whether the light sheet is in the centre of FOV (this is similar to Pete's hint, but you don't have to take the cyl. lens out).

Monika

W dniu .04.2016 o 23:03 Anthony, Neil <nantho2 at emory.edu> pisze:

> Hi all,  I'm pleased to say that I have my two colour light sheet
> setup up and running :)
>
> Alas, I have a few teething problems, one of which is that I'm seeing
> a lot of out of focus light during my z-stacks.  I expect to see some
> out of focus blur, but with the selective illumination I thought it
> would be less.  I think things are pretty well aligned, with
> everything in the right place, and wondered if you could help me think
> about a couple of things.  The main two I'm thinking about are the
> function of the slit and the position of the final lens before the
> illumination objective, and how this is affecting my sheet.
>
> I have the first telescope in exactly the correct position, following
> the drawings on the Thorlabs page, with the lens back-to-back such
> that infinity of their design is pointing out at both ends (see below
> link to Thorlabs page; I'm referring to the threaded side as the
> back).  This gives me a really consistent expanded spot size across
> the room.  The slit, cylindrical lens, gimbal corner mirror and first
> lens of the second telescope are all pretty much exactly 50mm spaced.
> The horizontal focused beam is as crisp as I can get it on the gimbal mirror.
>
> The second telescope is where I get confused as we have two focusing
> paths in one.  Viewed from above it's the same as the expanding
> telescope before the slit, but in reverse to reduce the beam, so it
> would be nicer to have the Thorlabs optics back-to-back.  For the side
> view the beam path goes through the telescope in infinity space so
> we'd like the lens to be front-to-front, so to speak.  The preference
> for back-to-back or front-to-front is just for accurately locating the
> optics, and I'm guessing the focus in the opposite direction would be
> just as good, but difficult to know where to measure the focal
> distance from.
>
> In my setup I have both lens pointing the same direction with the
> front towards the illumination lens.  I have a picture of my SPIM
> below (low res; I'll get a better one for the website soon).  My
> confusion comes when I look at the back-focal-plane (BFP) of the
> objective lens, which lies 19mm above the flange, just before the
> yellow line (see image sent from Olympus TAC; they couldn't send a PDF
> so I'm guessing this a clip from their system resources).  I have my second lens of the second
> telescope pair (25mm) right up against the objective to reach the BFP.
> This is not how it looks in the website assembly and pictures, but as
> far as I know, is where the optical setup dictates it should be.
>
> My next question is the function of the slit.  Following the ray path
> diagrams the width of the slit changes the width of the horizontal
> beam focused on the BFP of the objective.  A wider beam would lead to
> a higher NA focused sheet, with the corresponding thinner focus and
> shorter depth of focus.  When I reduce the width of the slit I appear
> to get a thinner and thinner sheet, which doesn't make sense.  I
> should see a thinner beam with a wider beam, right?
>
> I've checked the alignment by removing the cylindrical lens and
> creating the sharpest thinnest beam of light in a dense sample of tiny
> beads.  On inserting the cylindrical lens again it shifts a little,
> but I can get the sheet in focus with a small adjustment.
>
> In writing this email and composing my thoughts I think I have my
> sheet focused either before or after the collection lens axis, such
> that the decrease in sheet thickness is likely coming from the reduced
> width of the triangle of light and not from the tighter focus.
>
> Thanks for taking the time to read this long email.  I hope you have
> some thoughts that might lead me in the right direction.  I'm not sure
> why the original design has such a large gap from the objective given
> the details of the BFP?  Is there a different plane on the back of the
> objective that should be focused on instead?  If not, what would the
> ray diagram look like for the focus prior to the BFP?
>
> The last caveat to my setup is that I opted to make it on the imperial
> spaced table, and as such I've mounted the chamber holder on the same
> rail as that last telescope setup (I thought it might also allow me to
> slide things if I ever change lenses).   I have two cameras that are
> free standing and have been aligned to a mm or so accuracy to be on
> the centre of the axis as needed.  I include this for completeness and
> feel it's not likely to play a roll, as moving things around lets me
> find the edges of my field of view from the detection objective but I
> don't really see much change in the thickness of my sheet in the
> different translations.
>
> Please let me know if you need further details, images, etc.
>
> Neil
>
> Thorlabs lens ray drawing:
> https://www.thorlabs.com/images/TabImages/Achromatic_Doublet_Ray_Drawi
> ng_A3-780.gif
>
> Second telescope optics image:
> https://drive.google.com/file/d/0BzfJAGNfrgieRWpxbk5xckg4R2M/view?usp=
> sharing
>
> BFP location image from Olympus:
> https://drive.google.com/file/d/0BzfJAGNfrgieQXNFRFowd0xxems/view?usp=
> sharing
>
>
>
>
>
>
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--
Dr Monika Pawłowska
Nencki Institute
02-093 Warsaw
Pasteura 3
Poland

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