Microspectrophotometer Operation

The microscope spectrometer and microspectrometer work like this: a lamp on the microscope emits white light which is focused onto the sample.  The sample absorbs some wavelengths of light better than others...it all depends upon the sample chromophore's chemical structure and environment.  The light not absorbed is collected by the microspectrophotometer objective and focused onto the entrance aperture of the spectrophotometer.  As the aperture is mirrored, the majority of light is reflected into a digital imaging system.  This allows you to see the spectrophotometer aperture overlaid on the sample and makes it very easy to align the system and take spectra.  The figure of fiber to the left shows the entrance aperture as a black square.  Simply move the stage so that the square is over the sample to make measurements. 

The light that is not reflected into the digital imaging system will pass through the aperture into the spectrophotometer.  The light is separated into component  wavelengths by an optical grating and each component's intensity is measured by a pixel on a Charge Coupled Device or CCD detector.  The computer stores this information and the result is an optical spectrum.  This spectrum is plotted as an XY chart that shows the optical energy at each wavelength (see Figure of spectra to left). 

Different types of microspectroscopy are accomplished by different lighting techniques.  These techniques are determined by the samples themselves.  For example, incident or reflectance illumination is used for opaque samples whereas transmitted light is used for transparent samples.  A microspectrophotometer can be configured to measure transmission, absorbance, reflectance and emission spectra.

In operation, a measurement is straightforward.  One first takes a dark scan to measure the dark counts of the system  The spectrum from a reference material is then collected.  The reference spectrum contains the spectral characteristics of the reference material itself, the light sources, the optics and the CCD.  The spectrum from the sample is then acquired and an algorithm is used to calculate the appropriate spectra for that lighting condition i.e. reflectance spectra when measuring incident illumination.  The algorithm is automatically applied by the computer and the result is displayed as a spectrum.