Re: DAPI on a 405 laser and 150x 1.45 objective

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/3D-volume-rendering-and-measurement-tp3647068p3700979.html

Stephen: I agree, technically 405 is 'violet' not UV.  At best you
might call it 'near UV' in the same way a Ti:Saph laser is 'near IR'.
Most objectives that can handle 'blue' can usually handle 'violet', in
terms of raw power transmission through the objective.  Your mileage
may vary for chromatic correction, however.

Craig


On Tue, Sep 22, 2009 at 10:41 PM, Stephen Cody <[hidden email]> wrote:

> 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: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara
>
>
> --
> Stephen H. Cody
>