trichroic o several independent filters

[Konstantín Levitskiy]

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Hello dear list.

 

We need to decide about the fluorescent cubes for new equipment which
include: upright microscope with water objectives ELWD; DMD Andor Mosaic 3
for simultaneous illumination (for experiments on Optogenetics, Bleaching,
Uncaging, Photoactivation and Photoconvertion, voltage-sensitive dyes);
illumination LED system (365, 470, 550, 585 nm); illumination LED system
(370, 460, 550nm); digital camera Andor Ixon Ultra 897. The speed of filter
exchange is 0,3 s. Would you recommend us to ask for trichroic filter to
avoid the use of filter changer?

 

Thanks in advance.

 

Best regards,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: [hidden email]

Web: www.ibis-sevilla.es

 

 

AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y
pueden estar protegidos por disposiciones legales. Si Vd. no es el
destinatario del mismo, por favor, notifíquenoslo inmediatamente y destruya
o devuelva el original. No deberá copiar este mensaje ni sus anexos o usarlo
para propósito alguno, ni divulgar su contenido a ninguna persona.

P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir
este mensaje, compruebe que sea necesario hacerlo.

 

[George McNamara]

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Yes.
You might also purchase quad dichroics.
Our local Nikon staff mentioned to me that the thicker glass support
"TIRF" dichroics have better performance than standard (thin) dichroics.
Check with Chroma and Semrock on those.

Photoconversion and some voltage senstive dyes, -- and while you did not
mention it, fluorescence resonance energy transfer (FRET), would benefit
from an additional camera or cameras.

For example, Cairn Research multiCam LS web page has a diagram with for
four cameras,

http://www.cairn-research.co.uk/catalogue/detection/image-splitters/product/multicam

you could purchase for the emission side splitter(s) dichroic and
emission filters to give you optimized adjacent emission bands AND full
field of view for each camera. For example (and ignoring your LEDs
details), for four cameras:

400-490 nm
490-560 nm
560-640 nm
640-800 nm

Even better would be 8 cameras (I've done the modelling in Semrock
Searchlight), which would enable you to image ALL the Brilliant violet
fluorophores and all the Brilliant Ultraviolet fluorophores. Advantage
of 8 cameras is no moving parts, compared to (say) one camera and a fast
emission filter wheel (or two cameras, two fast emission filter wheels).
More important then no moving parts is: "time is money" and "it's the
photon's, brilliant" (which reads better than "photon is money").
Confocal microscopes have been at four and five FL detectors enabling
simultaneous acquisition for a long time. If the 32 PMT detector
confocals ever 'nailed the landing" on spectral unmixing software, they
could do 8plex now. I believe it is time for widefield fluorescence
microscope imaging systems to catch up - and maybe surpass - what
confocal microscopes and flow cytometers&sorters can quantify
simultaneously. I also note that no company has "nailed the landing" on
using the approach of Hoppe et al 2008 to do joint spatial deconvolution
and spectral unmixing to get ~10x better SNR (which in turn could enable
~3x decrease in exposure time), http://www.ncbi.nlm.nih.gov/pubmed/18339754

For summary of BV's and BUV's see Jaimes presentation
https://www.bdbiosciences.com/documents/webinar_071713_multicolor-bv.pdf

page 3 ... BV's emission spectra
page 4 ... BV421 excitation spectra (and use that simultaneously for all
the BV tandems)
page 33 ... BUV395

The Semrock Searchlight graphing web tool

http://searchlight.semrock.com/

has all 8 BV's, 5 BUV's, and also BB515 = 14plex, 3 LEDs sequentially,
no moving parts (Semrock also can graph QDots). The current flow
cytometers and sorters are at 13plex (ex. IntelliCyt iQue PLUS and ACEA
Bioscience Novocyte), Propel-labs YETI has 28 fluorescence detectors, BD
Fortessa X20 can have 18 FL detectors (plus two for scatter),
http://www.propel-labs.com/introducing-the-yeti/
and I believe the Beckman Coulter MoFlo XDP can go to 28 (or is it 32?)
FL channels.

George
p.s. I see Chroma is behind with only two Brilliant's in Chroma Spectra
Viewer.
https://www.chroma.com/spectra-viewer
Should be easy enough for them to catch up.
The BD spectrum viewer is defunct since it uses Java. Beckman Coulter
has one at http://www.astrioseq.com/gb/?page_id=442
Spectra Database at University of Arizona has QDots but not Brilliant's
http://www.spectra.arizona.edu/



On 10/5/2015 5:21 AM, Microscopia-IBIS wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello dear list.
>
>
>
> We need to decide about the fluorescent cubes for new equipment which
> include: upright microscope with water objectives ELWD; DMD Andor Mosaic 3
> for simultaneous illumination (for experiments on Optogenetics, Bleaching,
> Uncaging, Photoactivation and Photoconvertion, voltage-sensitive dyes);
> illumination LED system (365, 470, 550, 585 nm); illumination LED system
> (370, 460, 550nm); digital camera Andor Ixon Ultra 897. The speed of filter
> exchange is 0,3 s. Would you recommend us to ask for trichroic filter to
> avoid the use of filter changer?
>
>
>
> Thanks in advance.
>
>
>
> Best regards,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email: [hidden email]
>
> Web: www.ibis-sevilla.es
>
>
>
>
>
> AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y
> pueden estar protegidos por disposiciones legales. Si Vd. no es el
> destinatario del mismo, por favor, notifíquenoslo inmediatamente y destruya
> o devuelva el original. No deberá copiar este mensaje ni sus anexos o usarlo
> para propósito alguno, ni divulgar su contenido a ninguna persona.
>
> P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir
> este mensaje, compruebe que sea necesario hacerlo.
>
>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Kurt Thorn]

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*****

On 10/5/2015 3:21 AM, Microscopia-IBIS wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello dear list.
>
>  
>
> We need to decide about the fluorescent cubes for new equipment which
> include: upright microscope with water objectives ELWD; DMD Andor Mosaic 3
> for simultaneous illumination (for experiments on Optogenetics, Bleaching,
> Uncaging, Photoactivation and Photoconvertion, voltage-sensitive dyes);
> illumination LED system (365, 470, 550, 585 nm); illumination LED system
> (370, 460, 550nm); digital camera Andor Ixon Ultra 897. The speed of filter
> exchange is 0,3 s. Would you recommend us to ask for trichroic filter to
> avoid the use of filter changer?

I would set up both individual emission filters for each channel and the
trichroic emission filter; that's how I set up most of our laser and
LED-based systems. Not all fluorophores can be imaged by just switching
the excitation source without being cross-excited at the other
excitation wavelengths. Having both the trichroic and the single
emission filters gives you the ability to use the trichroic when you
need speed and the single filters when you need to minimize crosstalk.

Also, you should be able to get filter switching speeds closer to 50 -
100 ms with the use of the right filter wheel and minimizing the weight
of the filters. We typically use Sutter filter wheels and can get
multicolor acquisition with switching filters to run at about 8 fps.

Best,
Kurt

>
>  
>
> Thanks in advance.
>
>  
>
> Best regards,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email: [hidden email]
>
> Web: www.ibis-sevilla.es
>
>  
>
>  
>
> AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y
> pueden estar protegidos por disposiciones legales. Si Vd. no es el
> destinatario del mismo, por favor, notifíquenoslo inmediatamente y destruya
> o devuelva el original. No deberá copiar este mensaje ni sus anexos o usarlo
> para propósito alguno, ni divulgar su contenido a ninguna persona.
>
> P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir
> este mensaje, compruebe que sea necesario hacerlo.
>
>  
>

--
Kurt Thorn
Associate Professor
Director, Nikon Imaging Center
http://thornlab.ucsf.edu/
http://nic.ucsf.edu/blog/