Adjustment of the Diode laser beam
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Sarah Kefayati |
Adjustment of the Diode laser beam
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Search the CONFOCAL archive at
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Dear list,
I have faced a problem recently and I appreciate if you could help me with that.
For our microscope light sources,we have an arrangement and we switch between diode and Ti-sapphire lasers and they go to the scanner unit (Fluoview 300).
For some reason the arrangement for the diode laser is messed up and we should do that again.The beam comes from the diode laser passes through the anamorphic pair of prisms and hits the mirror that has 3 nobs making it possible for 3 different moving directions: up and down,side to side,and cross movement.Then beam goes through the hole in front of the scanner and hits the galvanometers mirrors.
To make sure that I am adjusting the beam correctly I use this protocol:
1)I loosen up the mirror and shine the beam some where far and make it parallel to the table by using the up-and-down nob.
2) then I tighten the mirror stage and send the beam through the scanner vie a scanner pinhole while making sure that beam passes through the center of the first hole when it enters the scanner
3) while not scanning,I make sure that it also hits the center of the shutter inside the scanner
4)I use a webcam on the corner of the slide holder while it is facing up and showing the pinhole on the top part of the microscope unit( objectives are downward)
5)letting it scan,I zoom in to the last one(10x) to have the beam spot as small as possible,while watching the beam spot and the microscope pinhole on the camera,I try to adjust it in a way that it scan across the pinhole on the top of the microscope unit.
6) I use the Tetraspeck bead slide to check on my adjustment I start imaging with the without the confocal detection first just to find the focal plane,and once I find the beads I decrease the size of the pinhole
As I go step by step,every adjustment may mess up the last step and I should make sure that beam is centred through all the pinholes which I found it very hard.The last time that I tried and I arranged it very close to expected arrangement, I had a very weak signal on the biggest pinhole(#4 on Fluoview300 scanner).Because of space limit,I can't set up the laser in a different arrangement.
I appreciate if you give me your ideas and your suggestions on this problem.
Regards
Sarah Kefayati
Biophysics Master's student
Brock University
St. Catharines,Canada
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John Oreopoulos |
Re: Adjustment of the Diode laser beam
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Sarah, I've had some experience steering laser beams into modified microscopes so I might be able to help. It wasn't clear to me from your description however what exactly is the problem. Is it just that you are getting weak signal and you suspect the reason is a misalignment of the laser beam somewhere? Would you be able to snap some digital photos of key locations on the optical train? You said you had a webcam photo of the beam at the stage too. If you show us these photos, someone might be able to identify where the problem is. It sounds like you've got a pretty straightforward alignment protocol already, but there might be something else going on. Is this an upright or an inverted microscope? What objective are you using? What is the wavelength of the diode laser? How do you switch between the diode laser and the Ti-sapphire laser? Has the diode laser worked before in the past? Does the laser beam fill the back focal plane of the objective you are using? (you can check this by removing the objective and holding a white card near the nosepiece in a dark room to see the diameter of the beam, assuming the diode laser emits at a visible or UV wavelength). John Oreopoulos Quoting Sarah Kefayati <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear list, > > I have faced a problem recently and I appreciate if you could help me with > that. > > For our microscope light sources,we have an arrangement and we switch > between diode and Ti-sapphire lasers and they go to the scanner unit > (Fluoview 300). > For some reason the arrangement for the diode laser is messed up and we > should do that again.The beam comes from the diode laser passes through the > anamorphic pair of prisms and hits the mirror that has 3 nobs making it > possible for 3 different moving directions: up and down,side to side,and > cross movement.Then beam goes through the hole in front of the scanner and > hits the galvanometers mirrors. > > To make sure that I am adjusting the beam correctly I use this protocol: > 1)I loosen up the mirror and shine the beam some where far and make it > parallel to the table by using the up-and-down nob. > 2) then I tighten the mirror stage and send the beam through the scanner vie > a scanner pinhole while making sure that beam passes through the center of > the first hole when it enters the scanner > 3) while not scanning,I make sure that it also hits the center of the > shutter inside the scanner > 4)I use a webcam on the corner of the slide holder while it is facing up and > showing the pinhole on the top part of the microscope unit( objectives are > downward) > 5)letting it scan,I zoom in to the last one(10x) to have the beam spot as > small as possible,while watching the beam spot and the microscope pinhole on > the camera,I try to adjust it in a way that it scan across the pinhole on > the top of the microscope unit. > 6) I use the Tetraspeck bead slide to check on my adjustment I start imaging > with the without the confocal detection first just to find the focal > plane,and once I find the beads I decrease the size of the pinhole > > As I go step by step,every adjustment may mess up the last step and I should > make sure that beam is centred through all the pinholes which I found it > very hard.The last time that I tried and I arranged it very close to > expected arrangement, I had a very weak signal on the biggest pinhole(#4 on > Fluoview300 scanner).Because of space limit,I can't set up the laser in a > different arrangement. > > I appreciate if you give me your ideas and your suggestions on this problem. > > Regards > > Sarah Kefayati > Biophysics Master's student > Brock University > St. Catharines,Canada > |
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Sarah Kefayati |
Re: Adjustment of the Diode laser beam
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi John,Thanks for you reply.
My problem is I am not able to steer the beam at the center of the scanner hole and shutter and at the same time having it scan exactly across the hole that I mentioned (on the top of the microscope unit).
It was working very well about 2 weeks ago with very resealable SNR,we just had to arrange it again for some reason and it doesn't seem that it's gonna work!
it's an inverted microscope
I use UplanApo/IR 60x/1.2 W
Wl=407 nm
I flip the mirror (the one that I mentioned has 3 nobs) down so that infrared can go through the scanner
Yes,I do the same and I make sure that the beam fills the lens.
Sarah kefayati
Biophysics Master's student
Brock University
St. Cathahrines,Canada
On Sat, Jun 14, 2008 at 8:47 PM, <[hidden email]> wrote: Hi Sarah, I've had some experience steering laser beams into modified microscopes so I might be able to help. It wasn't clear to me from your description however what exactly is the problem. Is it just that you are getting weak signal and you suspect the reason is a misalignment of the laser beam somewhere? Would you be able to snap some digital photos of key locations on the optical train? You said you had a webcam photo of the beam at the stage too. If you show us these photos, someone might be able to identify where the problem is. It sounds like you've got a pretty straightforward alignment protocol already, but there might be something else going on. |
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Christian Müller-11 |
Re: Adjustment of the Diode laser beam
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Sarah, if I got the right interpretation of your beam arrangement (John is right in asking for a photograph of it), it sounds to me as if your diode-beam does not hit the mirror at the exact center position where the Ti-Sa-beam should pass(virtually). Maybe a second aperture less than halfway between your mirror and the opening of your miscroscope helps to reposition the diode-laser or your mirror to get both beams in parallel before entering the microscope. If the 2P-signal is perfect, than chances are high that a fully parallel diode-beam works as well. Good Luck, Christian PS: I agree with John that your strategy sounds right... Sarah Kefayati schrieb: > we switch between diode and Ti-sapphire lasers and they go to the > scanner unit (Fluoview 300). > For some reason the arrangement for the diode laser is messed up and > we should do that again. > > To make sure that I am adjusting the beam correctly I use this protocol: -- ...................................................................... Dr. Christian M. Müller Clinical Neuroanatomy JWG University D-60528 Frankfurt/M., Germany |
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Kevin Braeckmans |
Re: Adjustment of the Diode laser beam
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
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Hi, I am not sure if this is a solution to your problem, but you
need two mirrors for beam steering (you are mentioning only one). With one
mirror you can only adjust the angle of the beam, while two mirrors are needed
to adjust both angle and position of the beam in space (translational
movement). The first mirror should be adjusted such that the laser beam enters
the first pinhole, the second mirror is used to direct the beam through the
furthest pinhole. Alignment of a laser beam is then a simple iterative process involving
sequential adjustment of both mirrors. You could easily practice this by
putting two diaphragms (same height) separated by some distance on the optical
table and trying to direct the laser beam through them using two mirrors. So, if I understood correctly your setup and problem
description, my advice would be to insert a second mirror into your setup for
proper beam steering. Good luck and best regards, Kevin Kevin Braeckmans, Ph.D. Lab. General Biochemistry and Physical Pharmacy Ghent University Harelbekestraat 72 9000 Ghent Belgium Tel: +32 (0)9 264.80.78 Fax: +32 (0)9 264.81.89 Van: Confocal Microscopy
List [mailto:[hidden email]] Namens Sarah Kefayati Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,Thanks for you reply. My problem is I am not able to steer the beam at the center
of the scanner hole and shutter and at the same time having it scan
exactly across the hole that I mentioned (on the top of the microscope
unit). It was working very well about 2 weeks ago with very
resealable SNR,we just had to arrange it again for some reason and it doesn't
seem that it's gonna work! it's an inverted microscope I use UplanApo/IR 60x/1.2 W Wl=407 nm I flip the mirror (the one that I mentioned has 3 nobs) down
so that infrared can go through the scanner Yes,I do the same and I make sure that the beam
fills the lens. Sarah kefayati Biophysics Master's student Brock University St. Cathahrines,Canada On Sat, Jun 14, 2008 at 8:47 PM, <[hidden email]>
wrote: Hi Sarah, I've had some
experience steering laser beams into modified microscopes so I might be able to
help. It wasn't clear to me from your description however what exactly is the
problem. Is it just that you are getting weak signal and you suspect the reason
is a misalignment of the laser beam somewhere? Would you be able to snap some
digital photos of key locations on the optical train? You said you had a webcam
photo of the beam at the stage too. If you show us these photos, someone might
be able to identify where the problem is. It sounds like you've got a pretty
straightforward alignment protocol already, but there might be something else
going on. Search the CONFOCAL archive at
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In reply to this post by Sarah Kefayati
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Sarah,
I too have an FV300. With regards to your problem I
typically use this protocol. It's similar to yours although it calls for an
extra pinhole as mentioned in the replies.
1. Place a pinhole at the entrance port where my
NIR beam goes in.
2. Place another pinhole just after the
shutter
3. I use 2 mirrors on the outside so that steering
is easier
4. use external mirrors to steer beam such that the
beam passes straight and level
5. Check beam profile at the turret with the
objectives removed (just to make sure it isn't getting truncated
somewhere)
6. Replace objective, close the shutter/field stop
just before the filter holders in the transmission path (if you use an
inverted)
7. I use the point scan function on the Fluoview
software (default scan position is centred)
8, Make sure that the beam on the field/stop is
centred ( i.e after goinf through objectives and condensor)
9. Repeat ad nauseam
10. I sometimes use the diode lasers that have been
prealigned by Olympus to help with the initial alignment but I find they don't
do it very well.
Hope to hear if this helps, or if it doesn't. If
anyone has comments on any errors in this, do let me know!
Elijah
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John Oreopoulos |
Re: Adjustment of the Diode laser beam
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I would add to this that a nice way to check initial alignment at the step where the objective is removed (step 5) is to place a flat mirror on the stage and observe the beam retracing it's path backwards during the field centered point-scan mode- you'll see the light going through the backside the pinhole apertures if everything is centered to the microscope port axis (you might have to do this in a dark room with the laser power increased to maximum to see the back-reflected beam. Remember to wear laser safety goggles). Stray backward reflections that do not retrace the path can give you hints if there's some kind of misalignment further down in the optical train inside the confocal scan unit or the microscope frame where you can't easily place alignment pinholes.
Also, this may not matter for you, but most opto-mechanical mirror mounts are so-called "kinematic" mounts that in addition to tilting the mirror cause a bit of translational motion as well that might be messing up your alignment if you don't know it's there and if you don't have a second mirror. You could buy a true "gimbal" mirror mount that removes the translational aspect of the other type of mount (these a a bit more expensive). See this link: John Oreopoulos On 17-Jun-08, at 11:04 PM, Elijah wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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