Andor Revolution X1 Spinning Disk confocal inverted microscope

Olympus Imaging Suite
Location: S910A

Andor Revolution X1 Spinning Disk confocal inverted microscope.

* Andor 4 laser stack (405, 488, 561, 640 nm).

* Olympus IX81 inverted microscope

* yokogawa CSU-X1 spinning disk scanhead.

* MetaMorph acquisition.

* Andor TuCam image splitter, onto 2 cameras (i.e. green and red).

* Two Andor iXon 888 EMCCD cameras. Note: user is responsible for optimizing the EM gain settings (will typically NOT be identical).


TuCam device infromation 

http://www.andor.com/scientific-cameras/multi-wavelength-imaging/tucam

TuCam cubes ... easiest way to see which set is in the TuCam is to find the cases for the cubes, whichever is empty is in the TuCam.

TuCam filter sets

"1E" ... "Red / NIR" ... Cy3/Cy5, AF568/AF647

dichroic T605 LPXR

Camera 1 emission filter:  575/50 ...    550-600 nm

Camera 2 emission filter:  675/50M ... 650-700 nm

"2B" ... "FITC/TRITC" ... "GFP / RFP"

dichroic FF580-FDi01 x36

Camera 1 emission filter:  525/50 ... 500-550 nm

Camera 2 emission filter:  617/73 ... 580-635 nm"3C"

"3C"    CFP/YFP

dichroic FF509-FDi01 x36

Camera 1 emission filter:  475/28 ... 461-489 nm

Camera 2 emission filter:  550/49 ... 525-575 nm

Please note: 405 nm is not optimal for exciting "CFPs" and 488 nm is not optimal for exciting "YFPs".

April 27, 2018 idea: Potential future filter set we would like your opinion of:

Quad filters, to enable two sequential scans -> four fluorophores.

this would be similar to how many point scanning confocal microscopes achieve "low bleedthrough 4plex".

Laser line pairs: 405 and 561 ("blue and red") and 488 and 640 ("green and NIR")

Microscope: Quad cube, i.e. DAPI/Green/Red/NIR

Sutter emission filter wheel: pair of dual emission filters,

Filter "A": blue, red.

Filter "B": geen, NIR..

TuCam dichroic: "blue and red" toward camera 1, "green and NIR" toward camera 2.

Emission filter for camera 1: dual "blue and red".

Emission filter for camera 2: dual "green and NIR".

Fluorescence onto cameras depends on laser lines and emission wheel.

April 27, 2018 idea #2: confocal fluorescence anisotropy imaging microscopy (FAIM).

Andor offers a polarizing beamsplitter & clean up filters for tghe TuCam, not clear whether the lasers are linearly polarized, nor if the whole light path is 'clean' with respect to POL.

Equally or bigger question: do users really need this capability?

Dix and Verkman 1990 publkished a nice paper ( https://www.ncbi.nlm.nih.gov/pubmed/2317548 ) measuring cytoplasmic viscosity by FAIM, and recently Ralph Weissleder has published several MP-FAIM articles, reviewsnd protocol papers. See:

   https://www.ncbi.nlm.nih.gov/pubmed/29410158

   Fluorescence anisotropy imaging in drug discovery.

   Vinegoni C, Feruglio PF, Gryczynski I, Mazitschek R, Weissleder R.

    Adv Drug Deliv Rev. 2018 Feb 2. pii: S0169-409X(18)30027-9.

    doi: 10.1016/j.addr.2018.01.019. 


 

A simple way to get light to the TuCam is to set Sutter filter wheel "A" to "Quad" (the empty position also works), and sutter "A" shutter to open.

Key operating point: there is a shutter button on the Yokogawa spinning disk unit. I have not seen a Metamorph illumination control for it (yet), so user's responsibility to open the shutter by pushing a button on the Yokogawa X1 scanhead (the MetaMorph Acquire - Special tab has a 'camera shutter - always open' option that may or not do what you need).

 

tip: as a startinng point for optimizing the cameras (getting the settings optimized is user's responsibility), MetaMorph - Acquire - special tab screen shot:

MM Acquire Special - TuCam dual EMCCD

 

You want more dialogs? Sure:

big screen shot


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