Re: DAPI on a 405 laser and 150x 1.45 objective
Posted by
Stephen Cody-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/3D-volume-rendering-and-measurement-tp3647068p3703991.html
Sorry List my previous email was a little confusing. It should have read:
When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is often used for excitation and certainly in order to transmitt "340nm" efficiently a UV objective should be used. But if no UV transmitting objectives are available it is possible to use 360nm and 380nm combination instead. You should not have problems with Visible Violet transmission at 405nm with any objective.
2009/9/23 Stephen Cody
<[hidden email]>
G'day Renato,
I'm not sure I would describe 405nm as UV, I tend to call it visible violet. 405nm should be transmitted very well by most objectives. Transmission curves for Olympus objectives can be found at
http://microscope.olympus.com/uis2/ . I could not find any objective listed there with poor transmission at 405nm.
When doing Ca2+ imaging with Fura-2 a combination of 340nm and 380nm is often used for excitation and certainly in order to transmitt 405nm efficiently a UV objective should be used. But if no UV transmitting objectives are available it ispossible to use 360nm and 380nm combination instead. You should not have problems with Visible Violet transmission at 405nm with any objective.
Having said that some objectives are chormatically corrected at 405nm, which can improve imaging especially when using confocal.
Cheers
Stephen Cody
2009/9/22 Renato Mortara
<[hidden email]>
Hello,
I wonder if I could get some feedback on how two doubts;
1) How well does the solid-state 405 laser performs with DAPI-labeled samples (Cells, tissues, etc.) with not-so-UV-transparent objectives ?
I currently run an old BioRad 1024UV and with the Coherent 622 Inova laser (351/363 lines) even with not-so-UV-transparent objectives such as a 63x PlanApo and a 100X 1.4 PlanApo (both from Zeiss), by using DAPI at a relatively high concentration (20-50 uM) we get very nice signal.
2) Has anyone tried the 150x 1.45 NA Olympus objective (presumably designed for TIRF) with conventional/confocal fluorescence ? Is the small WD a problem ?
Any input will be greatly appreciated,
Best regards,
Renato
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: 55 11 5579-8306
Fax: 55 11 5571-1095
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Stephen H. Cody
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Stephen H. Cody