ResolutionResolution

What can be expected ?What can be expected ? What affects it ?What affects it ? How can it be improved ?How can it be improved ?

What is Resolution ?What is Resolution ?

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

What is “good” resolution ?What is “good” resolution ?

What are the specifications for What are the specifications for Bio-RadBio-Rad Confocals?Confocals?

Lateral (XY) ResolutionLateral (XY) Resolution

How closely can two points in the XY plane How closely can two points in the XY plane approach and still be distinguished ?approach and still be distinguished ?

In reflection, the specification is In reflection, the specification is 100100 nm. nm. In fluorescence, the specification is In fluorescence, the specification is 200200 nm. nm.

Axial (Z) ResolutionAxial (Z) Resolution

What is the thinnest sample you can see ?What is the thinnest sample you can see ? In reflection, the specification is In reflection, the specification is 500500 nm. nm. In fluorescence, the specification is In fluorescence, the specification is 800800 nm. nm.

What Affects Resolution ?What Affects Resolution ?

The Sample Makes a DifferenceThe Sample Makes a Difference

Resolution for reflection is different to Resolution for reflection is different to resolution for fluorescence.resolution for fluorescence.

Something which fluoresces Something which fluoresces will appear bigger than when will appear bigger than when it reflects.it reflects.

Other Important FactorsOther Important Factors

The light wavelength has a fundamental The light wavelength has a fundamental role to play.role to play.

Vibration destroys resolutionVibration destroys resolution Optical alignment is crucialOptical alignment is crucial The condition of the laserThe condition of the laser The cleanliness of the microscope.The cleanliness of the microscope.

How can we Improve Resolution ?

How can we Improve Resolution ?

Here are some HintsHere are some Hints

Avoid Refractive Index Mismatch

Avoid Refractive Index Mismatch

Ensure you use the correct immersion Ensure you use the correct immersion medium for the sample and lens.medium for the sample and lens.

Do NOT mix manufacturers.Do NOT mix manufacturers. Keep the optics cleanKeep the optics clean

Keep the System AlignedKeep the System Aligned

Regular alignment of the system is well Regular alignment of the system is well worth the trouble.worth the trouble.

Regular tuning of the laser is essentialRegular tuning of the laser is essential

Avoid VibrationAvoid Vibration

Use an anti-vibration table if possible.Use an anti-vibration table if possible. If not, use a sturdy table, isolated from the If not, use a sturdy table, isolated from the

room walls.room walls. Keep vibration sources away from the Keep vibration sources away from the

microscope table.microscope table. Vibration sources include the laser fan, the Vibration sources include the laser fan, the

computer, the epi-fluorescence power supply computer, the epi-fluorescence power supply and printers.and printers.

Use Good LensesUse Good Lenses

The best investment is in top quality lenses.The best investment is in top quality lenses. A cheap lens is not good economy as it will A cheap lens is not good economy as it will

never give satisfactory results.never give satisfactory results. Plan apo-chromat lenses are the best to use.Plan apo-chromat lenses are the best to use.

Pay some Attention to the RoomPay some Attention to the Room

Ensuring the room has adequately Ensuring the room has adequately controlled lighting and is away from drafts controlled lighting and is away from drafts and noise is just good sense.and noise is just good sense.

Extract the laser cooling air from the room.Extract the laser cooling air from the room. Restrict the use of the room to microscopy. Restrict the use of the room to microscopy.

General purpose rooms are never properly General purpose rooms are never properly functional.functional.

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

Pay my bill.Pay my bill.