In a confocal microscope, 488 nm laser line is used as the light source, and an
ID: 253145 • Letter: I
Question
In a confocal microscope, 488 nm laser line is used as the light source, and an oil immersion objective with NA = 1.4 is used to image 100 micron thick tissue. The tissue is a frozen section embedded in Gelvatol with a refractive index of 1.46. a) Please estimate the image resolutions of confocal microscope in both lateral and axial direction. b) To obtain a three-dimensional (3D) image stack, you need to obtain a series of confocal images at different z (axial) positions. The smallest step size the objective can move is 20 nm. Please estimate the z-position step size you will use to obtain the 3D image stack. Please justify your choice of the step size.
Explanation / Answer
Image resolution along the lateral direction (dlateral) according to the formula, dlateral= 0.8 ? / 2 NA is (0.8 X 488) / (2 X 1.4) nm or 139.42 nm. Similarly image resolution along the axial direction (daxial) according to the formula, daxial = 2?? / NA2 is (2 X 488 X 1.46) / (1.4)2 or 727.02 nm.
In confocal microscopy, images of different optical sections can be taken by changing focus and create three dimensional representations of a specimen without actually cutting or sectioning the specimen. A confocal image is not continuous in the way that a wide field image is. Rather, it consists of a number of discrete measurements of light intensity that, taken together, represent the image. If an object is to be resolved by this method it must occupy more than one sample. This type of sampling has been determined by the Nyquist sampling theorem which says that to resolve an object, we must sample that object 2 times more frequently than its dimensions. In practice, this is done 2.3 times. Another way to say this is our voxel size needs to be a minimum of approximately (the dimensions of that object) / 2.3 or 0.4 x the dimensions of that object. So, if the axial direction resolution limit is 727.02 nm, then our best z step should be 727.02 nm x 0.4 = 290.8 nm.
In practice, the step size for Z stacks is kept between half to 0.4 of the axial direction resolution limit, also considered as optical section thickness.