Microspectrography - A Short Review

Patil S.B

The analysis of pharmaceutical formulations or different materials can be done with the help of different Analytical instruments such as U.V.Spectrophotometer, HPLC, HPTLC and Colorimeter.

The objective of this review is to explore a new method which includes Spectrography i.e. measurement of fluorescence excitation and emission spectra of fluorochromes and a fluorescence microscope to give “Microspectrography”. The microscope has automatic light shutter for the different light path. The instrument is equipped with monochromators and photomultiplier to record the results.

Introduction:

Spectrography is used to record fluorescence excitation and emission spectra of dyes and fluorochromes. A spectrofluorometer is generally used for this purpose. Fluorescing dyes can show a different spectral behavior in the unbound (in solution in a cuvette) and bound (to cellular components or natively fluorescing cellular constituents) state. To determine the spectral characteristics of bound dyes, for example fluorescing cell components, a microscope is combined with a spectrofluorometer¹.

Instrumentation:

The instrument consist of fluorescence microscope with epi-illumination, equipped with two monochromators, one for varying the excitation (absorption) wavelength and one for varying the emission wavelength. The emission light is recorded with photomultiplier (PM) and visualized on a graphic recorder, oscilloscope or TV monitor. For convenience, the monochromator are generally equipped with stepping motors working under computer control. The microscope has automatic light shutter for the different light path. These are also under computer control². The resulting instrument, the microspectrograph is shown in figure 1.

Measuring fluorescence spectra:

Excitation spectra:

To measure excitation spectra, the fluorescence intensity is measured at a fixed wavelength. The monochromator is used to vary the excitation wavelength. The relative fluorescence intensity values can then be plotted as the excitation spectrum. A number of errors must first be corrected for, however. The excitation light source has different light intensities at different wavelength, and the optical system has different transmission for each wavelength.

This can be corrected by measuring the responses of the system simultaneously with a photodiode with known response. The ratio between measured photodiode response and known response then give the correction factor for the excitation spectrum which must be measured. This correction factor can be computed on line by computer.

It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue – induced fluorescence emitted by yolk granules was analyzed Microspectrographically³.

Emission spectra:

These spectra are recorded by exciting the specimen at a fixed wavelength and analyzing the emission at various wavelengths using a monochromator and PM. The obtained spectra must be corrected for different sensitivity of PM to different wavelengths and also for the varying transmission behavior of the optics. To correct for these influences, light from the tungsten lamp which passes through exactly same optical path as the specimen, is measured.

Since the lamp has a current stabilized power supply, the relative light intensities for each wavelength are known (color temperature) and can be stored in computer memory. The ratio between measured and theoretical color temperature is calculated for each wavelength and used as a correction for the measured fluorescence emission spectrum. An example of such a correction is given in figure 2.

Another method of correction is to measure the fluorescence emission of cuvette with a rhodamine solution. The ratio of this emission to the theoretical rhodamine spectrum can then be used to correct spectra of other fluorescing dyes.

For registration of excitation and emission spectra, it is important that the fluorescence microscope has facilities for measuring very small area of fluorescing objects (a new square micrometer for example in cells and tissues). This is in order to be able to measure characteristics of just one particular fluorochrome in a specific area of the object. This is accomplished with prism optics for varying size and shape of the measuring diaphragm.

Excitation and emission spectra are measured to determine in an exact way the correct filter combination for particular fluorochrome. Especially if two fluorochromes with overlapping colors must be measured separately, a micro spectrograph is helpful in determining the best filter combinations. Beside this, spectral analysis can be performed for the identification of fluorescing cellular components. Examples are certain neurotransmitter compound which show minor differences in emission characteristics. These differences are too small for accurate visual identification, but can be identified reproducibly and accurately by Microspectrography ⁴.

References: -

1. G. E Pearse, F. W. D. Rost; A microspectrofluorometer with epi-illumination and photon counting. J. microscopy 1968, 89, 321.
2. J. S. Ploem, J. A. de Sterke, J. Bonnet, H. Wasmund; A microspectrofluorometer with epi-illumination under computer control. Journal of Histo- and Cytochemistry 1974, 22, 668-677.
3. F. Harrison, M.Callebaut, and L.Vakaet; Microspectrographic analysis of Trypan blue induced fluorescence in oocytes of the Japanese quail. Journal of Histochemistry and Cell Biology 1981, 72(4), 563-578.
4. M. M. Jotz, J. E. Gill, D. T. Davis; A new optical multichannel microspectrofluorometer. Journal of Histo- and Cytochemistry 1976, 24, 91-99.

Figure 1: Schematic diagram of Microspectrograph.

L = Light source, PM = Photomultiplier.

Figure 2: Diagram representing correction for emission spectra.

The four bar figure from left to right represent fluorescent intensities. From left to right (1, 2, 3 and 4) they are: measured emission spectrum of sample, measured tungsten spectrum, known tungsten spectrum and corrected sample spectrum.

About Authors:

Naikwade N.S, Magdum C.S

Appasaheb Birnale College of Pharmacy, South Shivaji Nagar, Sangli-Miraj Road, Sangli-416416.

Sandeep Balvant Patil

Appasaheb Birnale College of Pharmacy, South Shivaji Nagar, Sangli-Miraj Road, Sangli-416416.

Sonawane L.V

Kondawar M.S.