Re: 405 laser intensity at the objective is 0.15% of actually intensity ... is this normal?

Posted by lgelman on
URL: http://confocal-microscopy-list.275.s1.nabble.com/405-laser-intensity-at-the-objective-is-0-15-of-actually-intensity-is-this-normal-tp7587264p7587295.html

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Hi Emmanuel,

These are the values we get at our 10x/0.45 objective mounted on a Zeiss Axioimager equipped with a Yokogawa W1 (Note that laser power is set to 10%):
Laser 488 at 10% with 10x/0.45 = 1.7 mW
Laser 561 at 10% with 10x/0.45 = 0.227 mW
Laser 405 at 10% with 10x/0.45 = 0.201 mW
Our lasers must be rather in the 150-200mW range.
As the others mentioned already, I would try to realign the lasers in the combiner and/or to by a new fiber.
Best regards,
Laurent.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Emmanuel Levy
Sent: Wednesday, September 6, 2017 11:54
To: [hidden email]
Subject: 405 laser intensity at the objective is 0.15% of actually intensity ... is this normal?

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Dear All,

We are experiencing problems to detect fluorescent proteins, in particular with blue fluorescence. BFPs are generally weak and so I initially thought it was normal, but today we measured the laser intensities at the fiber exit or "entrance to the microscope" as well as at the objective and we loose a lot of intensity on the path.

We have three lasers:
405 - 120mW
488 - 100mW
561 - 75mW

The lasers are combined inside a combiner, enter a multi-mode fiber that is attached to a spinning disk microscope (Yokogawa W1), itself coupled to an Olympus X83.

The intensities measured at the fiber exit are:
405 - 21mW
488 - 34.5 mW
561 - 32.4mW

The intensities measured at the objective (measured after removing the
objective) are:

405 - 0.18 mW
488 - 1.18 mW
561 - 2.16 mW

Does this look reasonable to you? I guess that the 488 and 561 lines are somewhat OK, but for the 405 laser, the intensity at the objective is ~700x lower compared to the original intensity.

I would be keen to hear about typical values you are used to see with spinning disk systems.

Thank you for your feedback,
Best wishes,

Emmanuel