CYTOPHOTOMETRY
- 2. Cytophotometry/ Microdensitometry The absorption of a chromophore in monochromatic light is proportional to the quantity of the reacted substance, provided that the amount of chromophore taken up is proportional to the amount of original substance present. Quantitative cytophotometry depends on two laws of physics: Lambert's law Beer's law, (Beer's law) = which state that the amount of monochromatic light absorbed by a material is related to both its concentration (Lambert's law) = its thickness or path length. That is to say, microdensitometry (cytophotometry) is the measurement of the light absorption of objects under a microscope and can be used to determine the amounts of a histochemical staining reaction in individual nuclei to estimate the cellular content of substances such as DNA
- 4. Quantification of estimates of genome size (the C value) depends on having a reliable method to determine that the amount of Feulgen stain bound to the chromatin of a nucleus is directly proportional to the amount of DNA present. Microdensitometry is concerned with the precise measurement of light-absorbing components in microscopic preparations. Measurement relies on the interaction between photons at specific wavelengths and the chromophores (i.e., chemical substances such as the Feulgen-DNA dye complex that is contained within the nucleus of a cell). Loss of photon energy (absorption) or optical density (OD) results from the interaction of the insoluble stain reaction product with incident light. The transmittance (T) of the nonabsorbed incident light to a photosensing element in the system is used to translate the difference between the intensity of incident light entering the object and that of the light leaving the object. In fact, it is only possible to measure the transmittance of an object.
- 5. The conversion of the transmission values (T) into absorbance (A) is not direct but is expressed as A = log (1/T). This nonlinear transform is the basis of the relative photometric error (RPE) as a function of specimen transmittance in cytophotometry. The magnitude of the error can be computed as shown in Figure. The percent error rises sharply as transmittance is increased (= low-density
- 6. With this particular method, although two scans are necessary for each measurement, it is possible to work with rapidity because a precise "0" density background setting is not required for scanning individual nuclei in a given field. This method has the additional advantage of enabling a stray light electronic compensation to be included at the start of measuring, which will further assist in the reduction of electronic noise. The major difficulty encountered may be the location of a clear background area as large as the mask used for the specimen nucleus. Therefore, the method is most suitable for smears, squashes, or hemolymph droplets. All values of density of background and object background are registered and shown as digital values on the display panels of the instrument. IOD values and the relative area of absorbing chromophore are stored in keyed files within the computer on disks and can be rendered as hard copy via a suitable interface to a dot matrix or other type of printer (12,65,66). Several major components used in the following procedure for actual measuring of individual nuclei. Once again, it important to emphasize optic and specimen cleanliness, which, in cytophotometry, verges on sanctity.
- 8. STEPS 1. Turn the masking control knob to "Set" and carefully adjust the Kohler illuminator; place the specimen slide on the stage of the viewing microscope. 2. Select the correct wavelength, bandwidth, and spot size and scanning frame. 3. With the mask and/or specimen-stage controls, place the object within a suitable size of mask centered within the scan frame (reticle in left eyepiece). Then, move to a nearby clear (background) area somewhat larger than the mask size. 4. With the spot manual controls, place the scanning spot in a clear background area within the centered mask and focus carefully to get as sharp an image of the scanning spot as possible. Then, use the fine focus of the microscope to bring the object plane of the specimen to coincide with that of the scanning spot image. 5. Turn the masking control knob to "Scan."
- 9. 6. Set 0 on the density meter (with photomultiplier blocked) and set background density to 0.05 with the "set" zero control. Set the gating meter to the green line. 7. Switch scanner unit to "Automatic.“ 8. Press "Integrate" button to scan. Record displays for density and area. 9. Turn the masking control knob to "Set.“ 10. With specimen-stage controls, move the object from the mask and substitute a clear background area.
- 10. 11. Turn the masking control knob to "Scan.“ 12. Press "Integrate" button to scan. Record displays for density and area. 13. Subtract value obtained at step 12 from value obtained at step 8.
- 11. APPLICATIONS Precision of the information concerning quantities of constituents is high. Morphologic identification by a trained operator can be used advantageously to classify any given cell interactively. Carefully performed specimen-adapted cell preparation procedures in which each step is strictly supervised, highly standardized staining methods, and internal controls are prerequisites in order to obtain reliable cytochemical results. In a number of human tumors with well-controlled cytopathologic and/or histopathologic and clinical data, quantitative cytochemical analysis has been demonstrated to provide diagnostic and prognostic information complementary to that obtained by conventional clinical and morphologic methods.
- 12. REFERENCES Beer Lambert's Law Equation - Bing images FeulgenDNA Cytophotometry - Drosophila Melanogaster (barnardhealth.us) beer lambert law - Bing images