Question

This is a confocal microscopy class. So please answer clear and detail according to the subject. Thank you

1.Explain how light diffraction causes the lateral point spread function of light as shown by Airy. Illustrate with a diagram of the PSF in the lateral direction.

2.What is the relative size of the point spread function in the vertical compared to lateral direction and how does this affect the shape of the voxels we record in a optimal resolution z-stack.

3.Is the point spread function wider or narrower with large aperture lenses? Explain why.

4.Does a larger aperture lens have higher resolution (more detail) or lower resolution (poor detail)? Or is aperture size unrelated to resolution?

5.Definedynamicrange.Explaintheimportanceofadjustingthedynamicrangeof the light intensity sensors (PMTs) and how the confocal encodes intensity into numbers.

6.Explain the advantage and disadvantage of frame-averaging.

6b.Explain how to optimize the resolution of your scanned image to avoid oversampling and minimize file size.

7.Make a list of the most functionally-important parts inside of the scan head box and briefly explain what each part does. Hint1: there are 5 kinds of things, and the AOTFs & lasers are not in the scan head box. Hint2: Without these 5 things, it would not be a laser scanning confocal microscope.

8.Explain why immersion oil is used for some lenses. Explain how it works.

9.Explain the relationship between the point-spread function and super-resolution.

10. What is chromatic aberration?

11. How does one detect cross-talk and bleed-through? How can you avoid it, and how can you correct for it if you could not avoid it?

12. Describe the three main ways to manage autofluorescence.

Answer 1

Answer-

1. how light diffraction causes the lateral point spread function of light:

While we observe images in digital camera, in the digital camera, diffraction effects interact with the effects of the regular pixel grid. The combined effect of the different parts of an optical system is determined by the convolution of the point spread functions (PSF). The point spread function of a diffraction limited lens is simply the Airy disk and he intensity distribution of an Airy disk in three dimensions is referred to as apoint-spread function and completely describes the diffraction pattern of a point source of light (such as a single fluorophore) in the lateral (x,y) and axial (z) dimensions as modified by a diffraction-limited optical microscope.

2)

In optimal resolution Z-stack- two objectives would result in a point-spread function that is symmetrical in the axial and lateral dimensions.

3)

The PSF varies depending on the wavelength of the light we are viewing: shorter wavelengths of light (such as blue light, 450nm) result in a smaller PSF, while longer wavelengths (such as red light, 650nm) result in a larger PSF and therefore worse resolution. Also, the Numerical Aperture (NA) of the objective lens that we used.

4)

The higher aperture, the more potential resolution in the system. The size of the aperture is not the only factor, as the distance that the aperture is from the focus plane also determines the resolution.

5)

The dynamic range of a charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS) image sensor is typically specified as the maximum achievable signal divided by the camera noise, where the signal strength is determined by the full-well capacity. Photomultiplier tubes (PMTs) in current mode is used as the sensing elements, because of their high sensitivity at low light levels and reasonable dynamic range..

6)

Frame imaging is a process in which , the digital image processor can eliminate electronic noise by a process called frame averaging which is a mathematical process. After a certain number of frames are averaged, it is difficult to see any advantage in increasing the number of frames, and inspection slows.

The benefit is that the beam moves more quickly over the sample, avoiding artifacts that may occur.

7)

Immersion oil is used in the imaging and oil immersion is a technique used to increase the resolution of a microscope. This can be accomplished by submerging the objective lens and the specimen in a transparent oil of high refractive index, increasing the numerical orifice of the objective lens.

9)

A feature of super-resolution techniques is that the PSF must accurately be known for the results

10) Chromatic aberration (CA) is an effect resulting from dispersion in which there is a failure of a lens to focus all colors to the same convergence point. It is also called chromatic distortion and spherochromatism.

11.

a) The usage of a negative control to determine the level background fluorescence.

b) Multiple short washing and increased blocking time will be good

3) Antigen retrieval