Introduction

Microscopy is an approximation of reality. The point spread function is the better example of this: A single point will appear as a blurry ellipse using photonic microscopy. This transformation depends on the optical components which varies with time.

Quality control monitors this transformation over time.

Download MetroloJ QC from GitHub - MontpellierRessourcesImagerie/MetroloJ_QC

Download iText Library v 5.5.13 from https://repo1.maven.org/maven2/com/itextpdf/itextpdf/5.5.13.2/itextpdf-5.5.13.2.jar

Download ImageJ Download (imagej.net) or FIJI Fiji Downloads (imagej.net)

Open ImageJ or FIJI

Install MetroloJ QC and iTextPDF by dropping the .jar file into ImageJ status bar

Start up the microscope

Follow the set up procedure (load the test sample and focus with the lowest magnification objective).

Adjust to obtain a Kholer illumination

Remove the sample from the microscope

Nikon Ti2 without occular a shadow appears on the upper left corner of the FOV

Take a BF image for Widefield illumination Homogeneity

Analyze the image using MetroloJ QC and Check centering accuracy and total homogeneity

4x

Here the centering is off to the left side and the intensity is falling below 30%.

replace the illumination arm by the application screenlight

removing the objective:

If image is similar then the issue comes from the detection side.

Test all items on the detection path: Objective, filter cubes, lightpath selector, camera

Objective 20x-075

It improve the hopmogeneity but centering is still off

60x1.4 gives a perfect homogeneity

Repeat for each objective

Change objective

Adjust Kholer

Take a BF image for Widefield illumination Homogeneity

If illumination is not homogeneous then 

Conclusion: Camera may be re-aligned to obtain an homogenous illumination in bright field.

5408nm laser line not working used for YFP. Laser is shining outside the fiber but not reachingh the objective. Has the cube been changed?