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.”