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Hang on, the requirement was for a sample which would measure the
RELATIVE intensities of 505 and 488nm lasers. Mild bleaching shouldn't
affect that. If a bit is seriously bleached, use another spot. A quick
scan at very low power will reveal that. You're doing pretty well if
you can bleach an entire slide in a lifetime - and it you do, the
replacement is free. I've never succeeded in bleaching any part but I
have managed to laser-ablate spots on the surface! As for RI mismatch -
well of course there is. I'll bet there is with uranyl glass too (but
in the opposite direction). But the killer with the U glass is that you
can't actually see it very well in a scanned image. If you really want
a solid-state unbleachable sample get a slice of emerald. I've never
actually tried it at 405nm but it's so bright at 488 I'm sure it would
work. Unless you have a very generous lab budget I would recommend the
synthetic version over the natural one!
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Cammer, Michael
Sent: Friday, 3 June 2011 9:00 PM
To: [log in to unmask]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines
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Depending on your tolerance, they may or may not be stable. I use these
all the time for aligning the microscope because they are great for
this, although there may be an issue with refractive index mismatch
especially with the newer TIRF objectives, but I'm not so sure as a
reproducible sample.
Please see
http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm and
http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachin
gtest/index.htm
for two examples of bleaching.
-Michael
_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208 Cell: (914) 309-3270
________________________________________
From: Confocal Microscopy List [[log in to unmask]] On
Behalf Of Guy Cox [[log in to unmask]]
Sent: Thursday, June 02, 2011 11:32 PM
To: [log in to unmask]
Subject: Re: Fluorophore excitable with 405nm and 488nm lines
I just checked out a Chroma green fluorescent plastic slide - it seemed
to fluoresce pretty well with both uv and blue LEDs so surely that
should work well enough. Doesn't have the ultra-long lifetime problem
of uranyl glass. Reproducible, robust and best of all free!
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
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