Evos                               (first draft of webpage)
ACLSW 421

Sign up online https://nyumc.ilab.agilent.com/account/login

Always sign in the paper logbook in the room too!

Want to read the whole manual? Here it is.

All users must be trained by Microscopy Laboratory staff. No more intralab training!!
New person in your lab needs to use Evos? You may show them what they need to do, but they may not use the system unassisted until checked out by Microscopy Lab staff.

Instrument calibrated for samples that have coverslips on them. Images from thicker bottom dishes may look ok at 10X and 4X, but lenses really designed for #1.5 coverslips.

One of the selling points of the Evos is the ability to use in a fully lighted room.  If you need to keep the sample dark, put the Evos supplied box over the sample or put black cloth or cardboard over sample.  Other people in the room may need the lights on and this is acceptable.

Please always put stage plates in the plastic bin with the other stage plates. Do not leave stage plates on table etc.


Instrument must be left in standard configuration with buttons in this order:
GFP
RFP
Cy5
Brightfield
Phase

There are Dapi & YFP blocks available. Please contact Microscopy Lab staff if you need to use them. (Most YFP imaging will work ok with the GFP block.)

 

Tiling / Mosaics
When done, the stitched composite may be rescaled smaller before being automatically saved. This means that if you used a 20X or 40X lens, the resolutioni s lost.
To solve this problem, go tot he Export tab and save the image as High resolution TIFF.

 

WARNING: Images may not be saved with proper (or any) scaling information embedded in them.
Always put lens magnification in filename &/or folder name!

Based on measurements 20170308, each pixel with each lens is: (We will confirm these numbers and repost any changes if needed.)

Magnification N.A. XY color camera

YX grayscale camera

4X   2.25 um um
10X   0.9 um 0.9 um
20X   0.45 um um
40X   0.22 um um

If need a scalebar in ImageJ, Image > Properties and type in the X and Y for Pixel width and Pixel height respectively and then Analyze > Tools > Scalebar...

For a precise scale bar, save the appropriate picture below and open it with the image you need a scalebar for. You can draw a box over this and then move over your image or measure in ImageJ and set scale.



 

DO NOT TOUCH CALIBRATION SETTINGS IN TAB ON FAR RIGHT!!
When microscope calibrated correctly (as of today 8 Mar 2017), you can focus at 4X (image at left) and then click on the 40X lens and be in the same location needing only a tweak of the focus (image at right).


4X lens

40X lens (original size image 5X pixels linearly in each X and Y)

 

 

 

 

What about the corners being dark and slight gradient from top to bottom? We'll check with Evos, but these are probably unavoidable. Unless you crop them off. Or postprocess using flatfield correction.

 


Two answers to an EVOS color fluorescence question:

Question:

I have taken some pictures of my cells (DAPI and AF488 stain). I got picture 3 pictures as TIFF, 2 as single colors for DAPI and AF488, and one merged. However, when I use ImageJ to split the merged picture in its single colors, the AF488 seems to be contamined by some signal from the DAPI. Do you have an idea about which could be the problem? Am I setting the machine wrongly during the acquisition phase?

Answers:

There should be a setting to save each channel individually.

I believe “Save Underlying Channels” in http://microscopynotes.com/evos/evos_fl_auto_manual-v4.pdf does this. 

Also see the first paragraph of 3. Edit and Analyze Images

And keep in mind that whereas the Evos records images okay, a better microscope, such at the AxioObserver, will give you superior pictures.

other answer:

There is a second issue here:  the color lookup table (LUT).

The blue LUT has a green component in it.  If you choose a pure blue LUT instead of the one that adds some green to the blue and you choose a pure green LUT for the green (looks like you already have), then when you split the RGB image the channels will be exclusive. 

You could do this instead of saving each channel as a separate file if you are satisfied with 8 bits per channel.


 

When there is a problem, questions, suggestions, etc, please tell us michael.cammer@med.nyu.edu

< back