Memoirs – Optical calibration – William S. Stark

 

I learned about measuring light levels in my undergraduate research project. Tony Wright, then at Columbia College in New York, helped me measure light levels for my Drosophila phototaxis project with his spectral radiometer. In graduate school at the University of Wisconsin, my mentor Jerry Wasserman had me run his interference filters and neutral density filters through a spectrophotometer. Also, it was my job to go to the engineering school to borrow the thermopile and the digital voltmeter. The DVM was pinned, so to measure light levels I needed to crank up the room temperature to 85. When I set up my own lab at Johns Hopkins, I enlisted the help of Howard Seliger who suggested I buy a Schottky barrier photodiode from United Detector which we cross-calibrated with a thermopile. I was enamored by presenting my data in absolute intensity units, and I liked quanta per cm2 per second. I was delighted to compare Reinhard Schumperliıs absolute spectral sensitivities with mine (Hu & Stark, J. Comp. Physiol., 121, 241-252, 1977) after he sent me calibrations that were not in his paper (and after I did a lot of calculating). A few years later, Bob DeVoe, an expert in spider vision asked me if my Drosophila spectral sensitivities were changing (they were) because his were. He had seen an ad for a diode that lacked ³UV hysteresis,² and we worried that our calibrations (UV to visible ratio) had been changing, not the spectral sensitivities of our animals. We bought EG & G HUV photodiodes and did a cross-calibration experiment. When I determined light level in log quanta per cm2 per second, determining the area of the light was dubious, so I shined the light through a pinhole aperture and carefully measured the size.  How do you know if your monochromator is accurate? (There are several ways.) Using Drosophila, I made contributions to a growing literature on light-induced retinal degeneration. Intensity times time (quanta per cm2) was the relevant measurement. I compared the intensities for ultraviolet (UV) and blue light-induced damage in Drosophila to Hamıs and Sperlingıs respective models in monkeys (Stark & Carlson, Current Eye Research, 3, 1441-1454, 1984). Hereıs a trick I learned from Richard Cone who did visual science at the time. I was working on his spectrophotometer when he pointed to the ³home for the holmium.² A filter with holmium oxide has several sharp and precise spectral absorbance peaks (361, 419 and 537 nm). In the laboratory of Doekele Stavenga at Rijksuniversiteit Groningen (the Netherlands), we obtained some interesting excitation and emission spectra (Stark et al., Nature 280, 581-583, 1979) for which it was necessary to determine the integral of the area under the spectral transmission filters of the interference filters we were using. We also had an adventure in which we used a pulse dye laser to determine the speed of visual pigment conversions, and I remember that the oscilloscope was not fast enough so we had to high pass filter it. I eventually purchased a photomultiplier tube to make photometric measurements on my microscope, and carefully cross-calibrated it with my other devices. I undertook a collaboration on the study of spectral excitation and emission of lipofuscin, the aging pigment (Eldred et al., Science 216, 757-759, 1982). My contribution (Stark et al., Methods in Enzymology 105, 341-347, 1984) was to show that huge errors had been made in previous spectrofluorometry because of poor calibration. I was told of a conversation between several colleagues who had advised us (Camillo Ghiron and Beryl Ortwerth), ³You know, Stark and Eldred got a Science paper out of the notorious insensitivity of spectrofluorometers to long wavelengths.²  I went up to Montreal to work with Charles White, then at Concordia University, to study human visual sensitivity to ultraviolet light. There, we cross-calibrated diodes. I have half a file cabinet drawer of folders of calibration data and prided myself on papers with replete methods on optical calibrations (Stark et al., Curr. Eye Res, 4, 1059-1075, 1985; Stark, Curr. Eye Res., 6, 631-638, 1987).

 

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This page was last revised July 18, 2006