Java程序辅导

Lab1 Due date: 01/24/2013 
 
Virtual microscopy LAB Part 1 
http://micro.magnet.fsu.edu/primer/virtual/virtual.html 
 
I. Go to Translational Microscopy 
http://micro.magnet.fsu.edu/primer/virtual/translational/index.html 
Choose a sample: Pentium Microprocessor 
1. Bring sample to a sharp focus 
2. Adjust intensity of illumination 
3. Explore the sample by translating stage 
4. Zoom on different features 
Question 1. Does zooming improve the resolution of the image? What does it change? 
Go BACK TO VIRTUAL MICROSCOPY (bottom of the page) 
 
II. Follow the link to Magnifying Microscopy 
http://micro.magnet.fsu.edu/primer/virtual/magnifying/index.html 
1. Click the sample “Onion root mitosis” 
2. Change magnification from the lowest 25x to the highest 1000x 
Question 2. What determines the total magnification of a microscope? 
Question 3. What is the major difference between the functions of the zooming lens vs. use of 
objectives with different magnification? 
 
III. Go to Numerical Aperture and Resolution 
http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html 
Scroll down to first Java tutorial Numerical Aperture light cones. Change numerical aperture. 
Note relationship between N.A. and magnification of objectives. Read the text. 
Question 4. What is an angular aperture? What major factors affect the value of a numerical 
aperture? 
Using “Back” button, return to a previous page. Scroll down to the second Java tutorial 
“Immersion oil and n.a.” Follow the tutorial. 
Question 5. Calculate the highest practical numerical aperture for an oil immersion objective. 
 
IV. Follow the link for 
Diffraction Effects on Image Contrast 
http://micro.magnet.fsu.edu/primer/java/mtf/spatialvariation/index.html 
(Another way to get there: Type ‘MTF” in the search window Scroll down to 
tutorial #10) Run the script. 
Scroll down the page and return to MTF 
http://micro.magnet.fsu.edu/primer/anatomy/mtfhome.html 
 
Follow the link to 
Cutoff Frequency and Airy Disk Size 
http://micro.magnet.fsu.edu/primer/java/mtf/airydisksize/index.html 
Due Date: Monday 1 18/2016
Run the tutorial. 
Question 6. What is the cutoff frequency and what it is related to? 
 
Go to: 
Airy Disks, Numerical Aperture, and Resolution 
http://micro.magnet.fsu.edu/primer/java/microscopy/airydiscs/index.html 
Run the tutorial. 
Question 7. What is the best resolution achievable in this tutorial? What would you change to 
improve it? What resolution you’d be able to achieve? 
 
 
Virtual Microscopy LAB Part 2 
Specimen Contrast in Optical Microscopy 
http://www.microscopyu.com/articles/formulas/specimencontrast.html 
This is your reference page. 
 
Question 1. What major contrast-forming microscopy techniques can you name? 
(list at least two diascopic and two episcopic techniques). 
 
Go to Phase Contrast microscopy 
http://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phaseindex.html 
Scroll down the page to Phase Plate/Ring alignment 
http://micro.magnet.fsu.edu/primer/java/phasecontrast/phasemicroscope/index.html 
1. Pick the sample “Chinese Hamster Ovary” 
2. Use lowest possible magnification (4x) 
3. Try to focus image and Illuminate properly (Voltage) 
4. Phase telescope “In” 
5. Using phase ring positioner, align phase ring 
6. Focus 
7. Phase telescope “Out” 
8. Adjust illumination (Voltage) 
9. Magnify image (10x, 20x, 40x) 
10. Check ring alignment, illumination 
11. Repeat with another sample of your choice 
Question 2: 
a) What properties of phase object utilized in phase contrast microscopy? 
b) What are the limitations of Phase Contrast microscopy? 
 
 
Go to Positive and Negative Phase Contrast tutorial at 
http://www.microscopyu.com/tutorials/java/phasecontrast/positivenegative/index.html 
From the pull–down menu pick Tissue Culture Cells specimen Observe differences in 
negative and positive phase contrast, and brightfield images. 
tran mittedList at l st t nsmitted and two reflected techni )
Question 3: What is the main difference between Positive and Negative Phase 
Contrast techniques? 
 
Go to Inverted Microscope Light Pathways 
http://micro.magnet.fsu.edu/primer/java/lightpaths/ix70fluorescence/index.html 
Choose Filter cube with green (550 nm) excitation. Explore! 
Question 4. 
a) What is a major role of a filter cube? 
b) How to choose the components in the filter cube? (hint: consider how to split the 
excitation beam and emission beam) 
 
Go to Laser Scanning Confocal Microscopy 
http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html 
1. Pick Mouse Intestine sample 
2. Pick small pinhole aperture size 
3. Scan line speed Fast 
4. Both PMT at 25% gain 
5. Scan through the depth of a sample by changing Z-axis position 
6. Pick a plane near the center 
7. Change scan speed to Slow, then Medium 
8. Change aperture size to Medium; Large; go back to Small 
9. Increase/decrease gain on 
a) Green PMT 
b) Red PMT 
10. Change focus and brightness for Widefield image (disengage Focus lock) 
11. Repeat steps 1 to 7 using Rat Hypotalamus sample. 
12. Change Gain on Red, than Green PMT. 
Question 6. Explain effects caused by changing the scanning speed from fast to slow. 
Any positive effects? Would there be any negative side effects also? 
 
Go to 
http://www.olympusconfocal.com/java/confocalsimulator/index.html 
1. Choose Raccoon Uterus Cell Culture 
2. Explore the panel 
3. Change Scanning speed 
4. Change Z-position 
5. Turn lasers on and off 
6. Look at the spectral characteristics of the fluorophores (press VBF button) 
7. Change emission filters band’s position. Observe changes in image 
8. Change settings on all three PMTs (HV, Gain, Offset) 
9. Turn off fluorescecent channels and observe transmittance on the fourth 
channel (TD1) 
 
For Multiphoton Microscopy go to 
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphoto 
nhome.html 
 
Proceed to Multiphoton Jablonski Diagram 
http://micro.magnet.fsu.edu/primer/java/multiphoton/jablonski/index.html 
Start with NIR wavelength. 
Question 7. Change excitation delay from shortest to a longest one. What happens? 
Unlike the case for single-photon absorption, the probability that a given 
fluorophore will simultaneously absorb two photons is a function of both the spatial 
and temporal overlap between the incident photons. 
Question 8. 
a) What determines the time constraint on arrival of a second photon for a 
successful two-photon excitation? Give an order of magnitude. 
Change wavelength to visible. 
b) What happens? Change wavelength to IR (above 900nm). Choose shortest 
excitation delay. 
c) What process do you observe now?