CytoViva-Elyse

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  • #4369


    CytoViva-Elyse

    Moderator

      Hello! This is normal. This band is caused by a “hot” pixel. If you look at the Z profile spectrum of a pixel along that band, it should have a sharp spike. It does not appear that there is any defect in the camera.

      #4344


      CytoViva-Elyse

      Moderator

        Hi Paula,

        First you should check which gaskets are being used. Are you assembling the chamber with the 250-um gaskets or the 375-um gaskets? If you are using the thicker gaskets, you will not be able to focus properly. The chamber is intended for use with oil immersion objectives.

        Elyse

        #3946


        CytoViva-Elyse

        Moderator

          Hi! Would you mind attaching an image of what you are seeing? Thin vertical bands in a hyperspectral image are normal, but if they are multiple pixels wide, there may be dust on the optics or an issue with the camera (likely the former).

          #3940


          CytoViva-Elyse

          Moderator

            In my experience, a dark room is not necessary for CytoViva fluorescence imaging as long as you’re using an oil-coupled objective. If you are using an air-coupled objective, it would be a good idea to turn the lights off.

            #3913


            CytoViva-Elyse

            Moderator

              Hey Alex,

              Thanks for the image! I will email you directly to discuss the next steps to resolve this issue.

              Thanks!
              Elyse

              #3892


              CytoViva-Elyse

              Moderator

                Hi Alex,

                It all depends on the thickness and the transparency of your fibers. If your fibers are multi-microns thick and opaque, detection may be difficult. It also will depend on the size of your nanocrystals. Definitely worth trying!

                Elyse

                #3734


                CytoViva-Elyse

                Moderator

                  Hi Alex,

                  There is a Lamp Correction routine built into ENVI. It can be accessed from the main toolbar–> CytoViva Analysis–> Calibration and Correction–> Normalize for Lamp Spectrum.

                  Lamp correction/normalization isn’t always necessary, especially for plasmonic NPs, but it can be useful in pulling out features that are otherwise obscured by lamp. The spectra in the app note you attached were not corrected.

                  There is a pre-processing step involved, which requires that you first take a recording of the lamp spectrum. This needs to be done every so often (e.g., once a month for daily use) since the light bulb and light guides change over time. Please see the steps for acquiring a lamp recording below:

                  1. Sample slides are usually pretty dirty, but if you can find a clean area, you can use it to take the lamp scan. Otherwise, I would get a clean slide and mark on it with a permanent marker. This will give you something to focus on.
                  2. Once you have something to focus on, use 100x and adjust until particles, mark, or whatever is in focus.
                  3. Move to a clean area.
                  4. Open up the iris on the 100x objective. If you look through the eyepieces it will be very bright.
                  5. On HSI controls, adjust exposure to 0.005 seconds or so. The same rules for setting exposure apply.
                  6. I usually name the sample “100x lamp DATE”.
                  7. Take a quick or 21 line scan.
                  8. Take a ROI across the width of the lamp scan. It’s easier to choose a rectangle shape to draw with (under ROI_Type).
                  9. Hit Stats. Once this opens, right click the plotPlot Key.
                  10. Right click the plot–>Options–>New Window Blank.
                  11. Drag and drop the Mean into the new window from the plot key.
                  12. In the main toolbar–>Basic Tools–>Spectral Math–>Restore–>Desktop–>Click normalizing.exp–>Highlight–>Click OK
                  13. Highlight the Mean in the next window that pops up–>OK
                  14. In the window with the old spectrum and the new spectrum–>Right click–>Plot Key
                  15. Right click the first Mean–>Remove
                  16. Right click the plot area–>Reset Range
                  17. Save this as a spectral library. I would name it “100x NORM lamp DATE.”

                  Now you can proceed with the actual lamp correction of the image.

                  Lamp correction/normalization often introduces noise on the ends of the spectrum that will need to be eliminated before analysis. Let us know if you need more information on spectral subsetting.

                  Elyse

                  #3622


                  CytoViva-Elyse

                  Moderator

                    Hi Jill!

                    I believe you have the QI695 optical camera. This is the cube-shaped black camera with “CytoViva” on the front.

                    In the Ocular software, the Readout should tell you the Digitization Rate and Output (16-bit). The pixel size of the camera is 4.54 um, therefore at 60x your resolution is 75.7 nm. You can calculate this at any magnification by dividing the camera pixel size by the magnification.

                    Elyse

                    #3599


                    CytoViva-Elyse

                    Moderator

                      Hi Alex,

                      I wouldn’t expect to be able to see sub-10 nm AuNPs. However you should certainly be able to detect the larger AuNPs in the population or aggregates.

                      It’s always hard to say what the appropriate concentration should be, especially with NPs so small, but you may try to increase the concentration.

                      A couple of suggestions to try first:

                      1. Make sure your coverslip is on top when viewing under the microscope.
                      2. Check all areas under the coverslip. Sometimes the AuNPs cluster at one side.

                      Hope this helps,
                      Elyse

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