[OpenSPIM] Out of focus signal and optics questions

Mon Apr 11 09:00:48 CDT 2016

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_Drawing_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