(NOTE) Do not forget, it's [[https://c4science.ch/w/bioimaging_and_optics_platform_biop/teaching/probes/ | A Matter of Scale]] = Rule of Thumb = {F5928403, size=full} = Example = {F5928405, size=full} = Limit = (IMPORTANT) If your object size is below the limit of diffraction (see [[ https://en.wikipedia.org/wiki/Diffraction-limited_system | Abbe diffraction limit ]]) , i.e. roughly 250 nm, there is no need to acquire images with pixel size of 2 Å ! (NOTE) Please use this [[ https://svi.nl/NyquistCalculator | Nyquist calculator ]] to obtain the smallest meaningful pixel (voxel) size that matches your microscope's capabilities. If needed, you can lower the obtained value by 1.5 (in XY) and still obtain high quality images. = Object size and intensity measurement = (IMPORTANT) With an **inadequate pixel size**, the measured **size** and **intensity** of your object will be **bias**. In the example below, you can see that a translation of only **half a pixel** would make the object twice as large and twice as dim in the case of an inadequate pixel size (left), and be without consequence with optimal sampling (right) {F5929467, size=full} = Range = {F5928589, size=full} (IMPORTANT) What is true for XY, is also true for Z. PLease do not make confocal acquisition, with a pixel size of 40nm and a z-step of 2um !

(NOTE) Do not forget, it's [[https://c4science.ch/w/bioimaging_and_optics_platform_biop/teaching/probes/ | A Matter of Scale]] = Rule of Thumb = {F5928403, size=full} = Example = {F5928405, size=full} = Limit = (IMPORTANT) If your object size is below the limit of diffraction (see [[ https://en.wikipedia.org/wiki/Diffraction-limited_system | Abbe diffraction limit ]]) , i.e. roughly 250 nm, there is no need to acquire images with pixel size of 2 Å ! (NOTE) Please use this [[ https://svi.nl/NyquistCalculator | Nyquist calculator ]] to obtain the smallest meaningful pixel (voxel) size that matches your microscope's capabilities. If needed, you can lower the obtained value by 1.5 (in XY) and still obtain high quality images. = Object size and intensity measurement = (IMPORTANT) With an **inadequate pixel size**, the measured **size** and **intensity** of your object will be **bias**. In the example below, you can see that a translation of only **half a pixel** would make the object twice as large and twice as dim in the case of an inadequate pixel size (left), and be without consequence with optimal sampling (right) {F5929467, size=full} = Range = {F5928589, size=full} (IMPORTANT) What is true for XY, is also true for Z. Please do not make z-stack acquisition, with a pixel size of 40nm and a z-step of 2um!

(NOTE) Do not forget, it's [[https://c4science.ch/w/bioimaging_and_optics_platform_biop/teaching/probes/ | A Matter of Scale]] = Rule of Thumb = {F5928403, size=full} = Example = {F5928405, size=full} = Limit = (IMPORTANT) If your object size is below the limit of diffraction (see [[ https://en.wikipedia.org/wiki/Diffraction-limited_system | Abbe diffraction limit ]]) , i.e. roughly 250 nm, there is no need to acquire images with pixel size of 2 Å ! (NOTE) Please use this [[ https://svi.nl/NyquistCalculator | Nyquist calculator ]] to obtain the smallest meaningful pixel (voxel) size that matches your microscope's capabilities. If needed, you can lower the obtained value by 1.5 (in XY) and still obtain high quality images. = Object size and intensity measurement = (IMPORTANT) With an **inadequate pixel size**, the measured **size** and **intensity** of your object will be **bias**. In the example below, you can see that a translation of only **half a pixel** would make the object twice as large and twice as dim in the case of an inadequate pixel size (left), and be without consequence with optimal sampling (right) {F5929467, size=full} = Range = {F5928589, size=full} (IMPORTANT) What is true for XY, is also true for Z. PL Please do not make confocalz-stack acquisition, with a pixel size of 40nm and a z-step of 2um !