(1967). Internal Reflection Spectroscopy. Applied Spectroscopy Reviews: Vol. 1, No. 1, pp. 99-130.

Internal Reflection Spectroscopy

The previous chapter described how the confocal approach developed from conventional light microscopy and outlined the basic advantages gained by the use of confocal sampling techniques, primarily that the exclusion of light by the confocal pinhole makes it possible to record data from optical sections. This chapter will discuss the fundamental considerations that limit the performance of all confocal microscopes. Though at present no commercially available equipment approaches these limits, some simple tests will be described to help the user assess how well a given instrument performs. Additional information to help the user to operate the confocal microscope in an optimal manner can be found in Chapter 35, “ATutorial on Confocal Microscopy,” and Chapter 36, “Practical Confocal Microscopy.” These also include methods for measuring resolution and other useful parameters.

Fundamental Limits in Confocal Microscopy

By means of a photomultiplier tube, a Si-photodiode, and a Si-avalanche photodiode, the absolute scintillation yield of recently developed LaBr3:Ce, LaCl3:Ce, and (Lu,Y)2SiO5:Ce scintillators and traditional Lu2SiO5:Ce, Bi4Ge3O12, NaI:Tl, CsI:Tl, and CsI:Na scintillators were determined. These are all well known scintillators that cover emission wavelengths from 250 nm to 750 nm. By comparing the scintillation yield independently measured with the three different photon detectors reliable yield values are obtained.

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Advances in Yield Calibration of Scintillators

The confocal imaging geometry provides a dramatic optical advantage for fluorescence microscopy by discriminating against out-of-focus background with minimal loss of image-forming signal. Significant enhancement of both axial and lateral imaging resolution is also available but only with substantial signal loss. Because of these optical advantages, the confocal laser scanning microscope (CLSM) can clearly image thin optical sections from within thick fluorescence-labeled living specimens. A stack of optical sections is easily combined to reveal three-dimensional (3D) fluorescent marker distributions with diffraction-limited spatial resolution. When bright stable fluorophores are available, cellular dynamics can be measured by recording a time series of CLSM images.

Quantitative Fluorescence Confocal Laser Scanning Microscopy (CLSM)

The absolute light yield of four different scintillation materials measured with two photomultiplier tubes and two avalanche photodiodes is presented. Commonly, the photoelectron yields and electron–hole pair yields are corrected for the quantum efficiency of the detector, as specified by the manufacturer, to obtain the absolute photon yield. However, the effective quantum efficiency under scintillation measurement conditions is substantially higher due to back reflection. Only when back reflection is properly accounted for, the absolute photon yields obtained with the photomultipliers agree with those obtained with the photodiodes. The effect of optical coupling between the scintillator and detector is also discussed.

Measuring the absolute light yield of scintillators

Maximum quantum efficiencies attainable with commercially available semitransparent photocathodes are near 30% (for blue light). Work in the Instrumentation Division at Ames Research Center, NASA, has achieved quantum efficiencies as high as 58% with these same photocathodes through the use of optical enhancement. Improvement ratios in the red and near ir are even larger, but of course, the original sensitivities are smaller at these wavelengths. In addition to simply improving sensitivity, effort is also directed toward extending the applicability of the technique. A new class of devices recently conceived at Ames Research Center will allow application of optical enhancement to TV camera tubes, image intensifiers, and other imaging detectors. This paper describes the optical enhancement work in detail with major emphasis on results in the areas mentioned above.

Optical devices to increase photocathode quantum efficiency.

Photomultiplier tube sensitivity enhancement with very thin semitransparent photocathode and total internal reflection

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Enhancement of photomultiplier sensitivity by total internal reflection.

High absolute photocathode sensitivity.

A laser velocimeter (12) has a laser beam generator (18) for generating first and second laser beams (20, 22) having a waist (40) of reduced cross-section area. A first focusing lens (28) has a first focal length F1 for focusing the first and second laser beams (20, 22) so that their waists focused by the lens (28) intersect at a focal point (26) at which a measurement is to be made. A second focusing lens (30) is positioned between the laser beam generator (18) and the first focusing lens (28). The second focusing lens (30) has a second focal length F2. The second focusing lens (30) is positioned from the first focusing lens (28) at a distance corresponding to a sum of the first and the second focal lengths F1 and F2. A third focusing lens (36) having a third focal length F3 is positioned between the laser beam generator (18) and the second lens (30). The third lens (36) is positioned from the second lens (30) a distance corresponding to the sum of the second and third focal lengths F3 and F2. The third focusing lens (36) is positioned in front of the beam waists (40) formed by the laser beam generator (18) a distance corresponding to the third focal length F3. The focusing lenses (28, 30 and 36) coact to focus the laser beams (20, 22) so that their waists intersect at a first focus spot of size W1. The second and third lenses (30, 36) are interchangeable in position by pivoting about a point between them to focus the laser beams (20, 22) so tat their waists intersect at a second focus spot size W2 different than the first focus spot size W1. Photodetector (31) is connected to computer (41), which processes signals derived from scattered light by the photodetector (31) to give measurements of the scattered light.

Dual mode laser velocimeter

Near IR sensitivity enhancement by external field assisted photoemission from semitransparent photocathode

William D. Gunter

Papers

Optical devices to increase photocathode quantum efficiency.

Enhancement of photomultiplier sensitivity by total internal reflection.

High absolute photocathode sensitivity.

Dual mode laser velocimeter

Dual enhancement of photocathode sensitivity in the near infrared.