Based upon a collection of compact LEDs (light-emitting diodes) and a compact photodiode, we
have developed a calibration tool for fluorescence microscopes that are used as digital imaging
devices. The entire device (excluding a USB connector) measures 25 mm × 80 mm × 12 mm.
Virtually all commonly-used fluorophores can be simulated with one of the six LEDs. An LED is
chosen from the host computer and its current range is selected (digitally) so as to provide a test of
the complete dynamic range of the imaging system. Thus by varying the current through an LED in a
controlled way, a controlled amount of "emission" light can be produced, transmitted through the
chosen optical path of the microscope, and measured by the image sensor. The digitized intensity
can then be determined as a function of the LED current. Any other (fluorescence) intensity
measured through the same electro-optical path can then be characterized (and thus calibrated) by an
equivalent electrical current.
The excitation light is calibrated by a photodiode which has a dynamic range of 10^5:1 and thus is
suitable for a variety of light sources: mercury lamps, lasers, LEDs, etc. The integration time of the
photodiode as well as its gain can be digitally selected from the host computer. Further, using a
Spectralon® reflector, the inherent non-linearity of the LED emission versus current can be measured
by the photodiode and used to provide a look-up table compensation independent of the image
sensor used in the fluorescence microscope system.
Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular
interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal
with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been
tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We
further report on the derivation and use of several physical parameters and how these parameters change under differing
While fluorescence microscope systems remains an essential tool in modern biology and medical work, no compact instrumentation has been developed for the rapid calibration of such systems. Almost invariably results are presented in terms of the [AU], "arbitrary units". To remedy this situation we have developed a small, portable instrument - the size of a microscope slide - that uses low-power LEDs at different wavelengths to produce calibrated amounts of light. A computer controls the instrument--through a USB connector--so that the current to the selected LED can be swept through an increasing range of values. The amount of light measured by the microscope's total imaging system (lenses, filters, EO sensor, and digitizer) is then recorded to provide a "current in, digital value out" calibration. Further, the current can be translated easily to optical power and thus photons per second at the chosen LED wavelength. We have built and programmed such a system, tested it for accuracy and precision, and used it to calibrate several microscopes and microscope/lens combinations. The results will be presented.
Quantitative analysis in combination with fluorescence microscopy calls for innovative digital image measurement tools. We have developed a three-dimensional tool for segmenting and analyzing FISH stained telomeres in interphase nuclei. After deconvolution of the images, we segment the individual telomeres and measure a distribution parameter we call ρT. This parameter describes if the telomeres are distributed in a sphere-like volume (ρT ≈ 1) or in a disk-like volume (ρT >> 1). Because of the statistical nature of this parameter, we have to correct for the fact that we do not have an infinite number of telomeres to calculate this parameter. In this study we show a way to do this correction. After sorting mouse lymphocytes and calculating ρT and using the correction introduced in this paper we show a significant difference between nuclei in G2 and nuclei in either G0/G1 or S phase. The mean values of ρT for G0/G1, S and G2 are 1.03, 1.02 and 13 respectively.