Calibrating and Acquiring images with the OptiGrid

The OptiGrid is a device that you can use with most standard research fluorescence microscopes that will allow you to acquire confocal quality images without using laser scanning technology.  You can find out more about the OptiGrid on the Volocity Grid Confocal page, or the 3DM page.  It is really easy to use and I’m going to run through the procedure here. 

Let’s start by taking a look at the grid controls in Volocity, these feature in the Acquisition controls of the Video Preview.

From left to right they are; Grid Off, Grid Confocal, Grid Wide field.

Grid Off switches the grid off, the grid will remain stationary.

Grid Confocal turns on the grid on, you will see a confocal image in the video preview. If you look down the microscope eyepieces you will see the grid pattern moving around. When Grid Confocal is enabled in the Video Preview you will have the additional controls Grid Gain and Grid Quality.

Grid Wide Field turns the grid on, you will see a wide field image without the grid lines on the Video Preview. If you look down the eyepieces you will see a moving grid pattern.

If you are installing the grid for the first time you will need to calibrate it. Once calibrated you can take a view on how often you choose to recalibrate it in the future.

In order to do the calibration this you will need a uniformly illuminated fluorescent sample with which the grid can be imaged.  You can make your own calibration slide, all you need to do is seal a small amount of fluorescent ink between a slide and a coverslip, a highlighter pen is ideal or you could use some dilute fluorochrome such as fluorescein.

Mount the calibration slide on the microscope and select a fluorescence fllter. For this particular example I will use blue excitation and green emission as this works well with yellow ink.

For best results when calibrating the grid use a low power lens.  The lenses used should have a high degree of spherical and chromatic aberation correction.

Firstly focus on the fluorescent ink, move to a field of view that is uniformly illuminated. Slide in the OptiGrid paddle so that it is engaged in the fluorescence path, the grid will also need to be turned off.

Adjust the aperture diaphram on the microscope so that is between 15-50% open, this will help to reduce any stray light and improve the contrast.  Make sure that all light is directed to the camera, after doing this select auto exposure from the Video Preview controls.

Adjust the position of the OptiGrid by using the hex wrench focusing tool, to gets the best calibration the focus needs to be as sharp as possible.

Let’s take a closer look at the grid lines.

After fine tuning the grid focus go to the 'Video' menu and select 'Calibrate Confocal Grid', the calibration process will take a few minutes.

After completing the calibration it is time to image the specimen, so remove the calibration slide and introduce a slide with your specimen.

Here are some images I acquired with the different grid settings.

Grid Off

Grid Confocal

Grid Wide Field

You can see that my grid image looks a bit dark. You can amplify the signal by increasing the Grid gain.

You can also use a different quality setting by using the Grid Quality control.

Using a higher quality setting reduces the appearance of grid lines in the image but will take longer. If speed is of the essence use a lower quality setting.

The image looks a lot better now.  Try to avoid the temptation to of using camera controls such as exposure and gain to improve the brightness of the grid image as they will not have the desired effect.

For a good Grid Confocal image you need to see the grid lines prominently in the sample with the grid turned off.  If you do not see them adjust the grid focus using the hex wrench. If you are performing multichannel acquisition you may need to adjust the grid focus for each channel, try to find a common focus that works for all of you channels.

Remember this is just an overview on how to use the grid for further details please consult the manual or contact your local support desk.