Notes From digital microscopy----Recommended by John Murray

Microscope basics and optics of microscope Image formation

1. Finite tube-lenghth microscope and infinity optics microscopes

   Difference: infinity optics micrsocope will project the image of the speciman to infinity and do not form a real image of the speciman while the former one forms a magnified real image of the specimen at the intermediate image pane( thus it has a working distance )
   

2. Objective basices

   Plan achromats: color corrected for two wavelengths: red and blue; corrected for spherical aberration in the green ; good for monochromatic applications, fewer lense elements, important for polarization and differential interference contrast microscopy

   plan apochromats: highly corrected, four wavelengths: red,green,blue,and violet ; spherical aberration correction, high degree of flatfield correction; more lens elements than the previous one, handling with great care; not as well suited for polarization or differential interference applications as achromast in monochromatic applications

   plan fluroite lenses: as to optical performance and cost, fluorite lense occupy a niche between the achromats and the apochromats; corrected for chromatic and spherical aberrations at three wavelengths

   Note: infinity-corrected optics shoud not be used on finite tube-length microscopes, same for finity-corrected optics
   

3. Coverslip selection: all objectives, be they finite tube length or infinity corrected, are designed to be used with 1.5 coverglasses that are 0.17 mm thick; This holds true even for oil, glycerol, and water immersion objective; too thin or too thick a coverslip is willintroduce sperical aberration that will noticeably degrade image contrast

4. Video cameras on the microscope

   for higher numerical aperture lenses, the detector will undersamples the image; also pixel spacing of the camera will limit the image quality. Thus it is often of great importance to introduce additional magnification or empty magnification to the image formed on the photo-detecting surface .

5. at a given speciment field size, the larger pixel array cameras will capture an image with greater resolution.

   
   

Optics of microscope image information

1. Basic characteristics of physical optics

   Superposition of wave

   Huygen's principle to explain the propagatin of a plane wave

   Tomas Young's experiment

   Airy disk and the issue of microscope resolution: How interference affects microscope images? -------------Why is it important to know? All objects can be represented as the sum of an infinite set of point sources, if we know how the microscope treats a point object, then through the point spread function we know how it treats an object.

   Fourier or reciprocal space: as resolution also relates to the amount of contrast and the sharpness of edges; Fourier's transform of step function;  Microscopes always act as low-pass filters with some maximum spatial frequency making it through and this is the basis of resolution in the microscope.

2. Resolution of micriscope

   physical systems tend to pass only a certain set of spatial frequencies and the highest frequencies passed becomes a measure of resolution.  in the Fourier transform of biological specimens, there is a cutoff and higher orders fail to enter the objective, the larger the collection angle(the NA) of the objective, the higher the cut off and the higher the resolution.

3. contrast and resolution: Airy disk can be used as an example to view the relationship between contrast and resolution. Resolution: distinguish between two airy disks;  the distance between two airy disks can be viewed as spatial frequency. Say if the the distance between two airy disk become closer and this corresponds to the higher spacial frequency, and the contrast between these two disks then decreases.

Conclusion:  1. microscope's resolution is related to the highest spatial frequency that a microscope                                can collect and this increase with NA; NA depends on wavelength, thus resolution                                      increases with decreasing wavelength

                         2. the resolution is also depend on contrast and that the higher the contrast, the higher                                 the resolution