Absolute Photon Calibration of Zeiss Observer Z1 Microscope
Shawn MillerDepartment of Optical Sciences
University of Arizona, Tucson, AZ 85721
Optics 521 Presentation
December 8, 2008
Absolute Photon Calibration
• Detectors give relative light intensity values.
• Must provide reference to actual photon numbers for the detector’s value.
• “Standard” lamp which has been calibrated to national standards.
Standard Lamp
• Tungsten ribbon filament lamp.– Temperature is
calibrated for several currents.
• Stable high current power supply.
• High wattage resistor. • Photon emission is
given by Plank’s curve.
Lamp Calibration Form
• Calibrated at hottest (most central) area of the filament.
• Calibrated in the vertical position.
• 20% variation in lamp emission. – Area of filament
viewed by optical setup is very important.
Plank’s Curve
• Absolute temperature.– 1/ Ta = 1/ Tb + (λ/C2)*[ln(ε(λ)) +
ln(τ(λ))]• Ta = Absolute temperature• Tb = Brightness temperature • λ = Wavelength of radiation• C2 = Plank’s second constant
= hc/Kb
• ε(λ) = Emissivity at emitting wavelength
• τ(λ) = Transmission of pyrex envelope.
• L(λ) = ε(λ,Ta)*τ(λ)*[2hc2/λ5] / (ehc/λK
bT
a – 1).– Integrate over wavelength
region being observed.
Collecting the Lamp’s Light
• Φ = ∫L(λ)dλ*A*Ω.– Φ = Flux of light.
• Watts, or ergs/second.
– ∫L(λ)dλ = Radiance.• W/m2*Sr
– A = Area of filament.• m2
– Ω = Solid Angle.• Sr
Zeiss Observer Z1 Microscope
• EMCCD camera.• Three objectives.• 8 wavelength filters.• 6 gain settings.
– Detector’s response is different for each setting.
A Simple Design
• Small central area of the filament imaged onto the focal plane the objective lens.
• Lamp stand, two lenses, mirror, and stop.
Design Details
• First lens mount.– Barrel mount.– Axially adjustable by
threading the mount into the lamp housing.
– About one focal length from filament.
– Off Axis alignment is a one time adjustment of setscrews.
Design Details
• Mirror mount / aperture stop.– Adjustable kinematic
mirror mounted above aperture at 45°
– One time adjustable height.
– Base for aperture slide.
– Connection to microscope.
Design Details
• Second lens mount.– Adjustable height.– One time off axis
adjustment.• Fixed in place with
epoxy.
• Microscope’s stage acts as fine system adjustment when removing and replacing system.
System Alignment
• Set lamp height.• Adjust first lens.• Adjust mirror height.• Set mirror alignment.• Center second lens.
– Epoxy in place.
• Adjust second lens height.
635.2 µm
316.25 µm
Measurements
• Calculate first lens to filament distance. (z1)– Measure width of filament spot on wall and distance to wall.– z1 = (-Wf/Ww)*(DL1w).
• Aperture radius measured with 10x objective. (Rs)• Distance from first lens to stop and to the second lens
measured with caliper. (DL1S, DL1L2)
Solid Angle
• 1/z1’ = 1/z1 + 1/f1.
• RL1 = Rs +
[Rs/(z1’–(DL1s))]*(DL1s).
– RL1 = Radius of light spot on first lens.
• Ω = π RL12/z1
2
Area of Filament / Magnification
• Radius of the filament being imaged onto the objective lens focal plane. (Rfo)– Rfo = z1*(Rs/(DL1s).
• Radius of the filament image in the objective lens focal plane. (Ro)
• m = -Ro / Rfo.• 100x and 40x objective.
– Wf = Wfov / m.– Af = (Wfov / m)2.
• Area of the filament viewed by the microscope.
635.2 µm
316.25 µm
Number of Photons
• Φ = L*A*Ω*TL*Rm.– TL = Transmission of lenses.– Rm = Reflectivity of mirror.
• Theoretical # Photons = (Φ*t*λ) / (h*c).– t = Exposure time.– λ = Central wavelength.– h = Plank’s constant.– c = Speed of light.
• Calibration factor = #Pt / Detector reading.
References• Kowalski, Brian. Absolute calibration of a spectrometer through the
ultraviolet. Department of Physics Thesis, 1993.• Bickel, William. Absolute intensity calibration of a spectrometer
using a blackbody radiation source. Short paper, September 2001.• Merchant, John. Blackbody calibration sources function as
standards. Laser focus world, April 1995. • Stair, Ralph. Standard of Spectral Radiance for the Region of 0.25
to 2.6 microns. Journal of Research of the National Bureau of Standards, Physics and Chemistry Vol. 64A, No.4, July-August 1960.
• G.A.W., Rutgers. “Relation between brightness, temperature, true temperature and color temperature of tungsten. Luminance of tungsten.”
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