Fluorescent microspheres for 405/440 lasers PSF

[Ahmed S. Hassan]

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Hi everyone,
I am unable to find suitable fluorescent beads to measure the PSF of confocal microscope that uses 405 laser line. Few have suggested the popular TetrSpeck but these are rather exc. @ 365nm and they did not work for the systems I am using (LSM 710 and SP8) with rather low NA objectives (0.45-1). The measured PSF will be used to deconvolve the images in addition to check the quality of the microscopes.
 
Additionally, we want to evaluate the chromatic aberration of the system If the sample is excited at 405, and 561 so our plan is to image fluorescent reference samples, e.g., fluorescent beads with relatively large diameter that are stained with multiple fluorophores. I would appreciate any suggestion for products that would fit these purposes.

Best,
Ahmed

[Alison J. North-2]

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Hi Ahmed,

The Oxford Micron facility group tested out a whole bunch of blue beads for acquiring PSFs for the OTFs on their 3D-SIM.  One that they recommended was the 170 nm blue PS-Speck beads from Molecular Probes/Life Technologies (P7220 for the kit of 4 different colours), and those are what I still use to acquire blue PSFs with the 405 line.  I mount them in glycerol and they don't even bleach horribly.

I use 200 nm Tetraspecks for measuring chromatic aberration, to give that extra bit of brightness over the 100 nm ones.

But I'll be interested to hear if others have found better, more recent options!
Good luck!
Alison

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From: Confocal Microscopy List <[hidden email]> on behalf of Ahmed S. Hassan <[hidden email]>
Sent: Friday, April 23, 2021 9:33 AM
To: [hidden email] <[hidden email]>
Subject: Fluorescent microspheres for 405/440 lasers PSF

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Hi everyone,
I am unable to find suitable fluorescent beads to measure the PSF of confocal microscope that uses 405 laser line. Few have suggested the popular TetrSpeck but these are rather exc. @ 365nm and they did not work for the systems I am using (LSM 710 and SP8) with rather low NA objectives (0.45-1). The measured PSF will be used to deconvolve the images in addition to check the quality of the microscopes.

Additionally, we want to evaluate the chromatic aberration of the system If the sample is excited at 405, and 561 so our plan is to image fluorescent reference samples, e.g., fluorescent beads with relatively large diameter that are stained with multiple fluorophores. I would appreciate any suggestion for products that would fit these purposes.

Best,
Ahmed

[Michael Doube-2]

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Hi Ahmed,

On the LSM 710 at least you can put the microscope into reflection mode and bounce the laser off a suitably tiny and inexpensive thing like gold nanoparticles to get the PSF at the laser's wavelength. IIRC 80 nm particles worked OK, and the reflection mode setting was not obvious (there is a little 'comb' inside the scanhead that blocks the laser lines from hitting the spectral detector - you have to find the setting that pulls the comb out of the way). It has been nearly a decade since I last did this so my memory is a bit rusty!

Good luck!

Michael


On 23/04/2021 21:33, Ahmed S. Hassan wrote:

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*****

Hi everyone,
I am unable to find suitable fluorescent beads to measure the PSF of confocal microscope that uses 405 laser line. Few have suggested the popular TetrSpeck but these are rather exc. @ 365nm and they did not work for the systems I am using (LSM 710 and SP8) with rather low NA objectives (0.45-1). The measured PSF will be used to deconvolve the images in addition to check the quality of the microscopes.

Additionally, we want to evaluate the chromatic aberration of the system If the sample is excited at 405, and 561 so our plan is to image fluorescent reference samples, e.g., fluorescent beads with relatively large diameter that are stained with multiple fluorophores. I would appreciate any suggestion for products that would fit these purposes.

Best,
Ahmed


--
[Jockey Club College of Veterinary Medicine and                      Life Sciences - City University of Hong Kong]



Dr. Michael Doube
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