1. Resolution ä What can be expected ? ä What affects it ? ä How can it be improved ?

2. What is Resolution ? ä How close two points can be and still be seen as two separate points is known as the resolution of an optical system.

3. What is “good” resolution ? ä What are the specifications for Bio-Rad Confocals?

4. Lateral (XY) Resolution ä How closely can two points in the XY plane approach and still be distinguished ? ä In reflection, the specification is 100 nm. ä In fluorescence, the specification is 200 nm.

5. Axial (Z) Resolution ä What is the thinnest sample you can see ? ä In reflection, the specification is 500 nm. ä In fluorescence, the specification is 800 nm.

6. What Affects Resolution ?

7. The Sample Makes a Difference ä Resolution for reflection is different to resolution for fluorescence. ä Something which fluoresces will appear bigger than when it reflects.

8. Other Important Factors ä The light wavelength has a fundamental role to play. ä Vibration destroys resolution ä Optical alignment is crucial ä The condition of the laser ä The cleanliness of the microscope.

9. How can we Improve Resolution ?

10. Here are some Hints

11. Avoid Refractive Index Mismatch ä Ensure you use the correct immersion medium for the sample and lens. ä Do NOT mix manufacturers. ä Keep the optics clean

12. Keep the System Aligned ä Regular alignment of the system is well worth the trouble. ä Regular tuning of the laser is essential

13. Avoid Vibration ä Use an anti-vibration table if possible. ä If not, use a sturdy table, isolated from the room walls. ä Keep vibration sources away from the microscope table. ä Vibration sources include the laser fan, the computer, the epi-fluorescence power supply and printers.

14. Use Good Lenses ä The best investment is in top quality lenses. ä A cheap lens is not good economy as it will never give satisfactory results. ä Plan apo-chromat lenses are the best to use.

15. Pay some Attention to the Room ä Ensuring the room has adequately controlled lighting and is away from drafts and noise is just good sense. ä Extract the laser cooling air from the room. ä Restrict the use of the room to microscopy. General purpose rooms are never properly functional.

16. .........and above All !

17. Pay my bill.