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Apotome guide Queensland Brain Ins tute Microscopy ApoTome Users Guide Op cal Sec oning with ApoTome ApoTome uses a moving grid to change how the slide is illuminated By taking several images with the grid in di ?erent posi ons the ApoTome so ware is able

Queensland Brain Ins tute Microscopy ApoTome Users Guide Op cal Sec oning with ApoTome ApoTome uses a moving grid to change how the slide is illuminated By taking several images with the grid in di ?erent posi ons the ApoTome so ware is able to generate a single image which excludes a large amount of the out ? of ? focus light typically present in uorescent images The resul ng images look crisper than the smoother standard uorescent image which allows ?ner structures and details to be seen clearly but they are also dimmer as there is less light reaching the camera GFP GFP with ApoTome Calibra ng ApoTome Calibra on must be carried separately for each objec ve and ?lter set Insert the correct grid into the ApoTome slider ? Axio Imager ? x x and x objec ve L ? grid ? x x and x H ? grid ? Axio Observer ? x and x L ? grid ? x D ? grid ? x and x H ? grid Unlock and remove the ApoTome slider from the side of the microscope Use the tweezers to gently remove and insert the appropriate grid ? When inser ng match up the white dot on the grid with the one on the slider ? The grid is held in magne cally so if its not siXng level move it around slightly with the tweezers un l it clicks into place ? Once in place check it is secure by li ing the slider and gently tapping it over the palm of your hand Return the ApoTome slider back into the microscope ? There are two stop posi ons All the way in ? st click ? for using ApoTome grid or the click before ?? nd click ? for normal imaging iris ? When you ?nish using ApoTome remember to pull the slider back out to the ? nd click ? iris posi CCalibra ng ApoTome Phase CalibraAon This calibra on requires the mirrored calibraAon slide Focus on the cross ? hair at the centre of the silver square on mirrored calibra on slide ? Ensure you are using the BF RL brighEield re ected light ?lter set and the HXP lamp ? The light shining on the slide should appear yellow ? green and down the eyepieces the cross ? hair should appear black Once you have found the cross ? hair switch to camera mode and ensure the cross ? hair is focused as best you can ? If the cross ? hair is out of focus you will not calibrate ApoTome correctly Out of Focus Correctly Focused In the top menu go to AcquisiAon ApoTome Phase CalibraAon In the window that appears make sure you have the right objec ve selected the correct grid size selected and that you are using the BF RL ?lter Click Next Now adjust the exposure of the camera for imaging the cross ? hair It should adjust automa cally by