A method to remove the spectral components of illumination energy from a sample spectrum without the use of optical barrier filters, the method comprising: obtaining from an empty reference chamber and from an empty sample chamber a first set of illumination spectra when illumination energy is provided simultaneously to said reference and empty sample chambers, said reference chamber and said sample chamber are separate chambers having the same size, shape and reflectivity properties; determining the intensity differences between the illumination spectrum from said reference chamber and the illumination spectrum from said empty sample chamber at each wavelength across the illumination wavelength range; obtaining from said reference chamber and sample chamber a second set of spectra wherein the spectrum from the reference chamber contains only illumination spectral components and the spectrum from said sample chamber contains both illumination and sample spectral components when illumination energy is provided simultaneously to said reference and sample chambers; adjusting the intensities of the illumination spectrum from said reference chamber of the second set of spectra across the wavelength range of the illumination by subtracting or adding the previoulsy determined intensity difference at each corresponding wavelength to the intensities of said illumination spectrum wavelength from said reference chamber of the second set of spectra to make the illumination intensities from said chambers equal across the illumination wavelength range; and subtracting the said adjusted illumination spectrum of the said second set of spectra from the spectrum from said sample chamber of the second set of spectra that contains both illumination and sample components, effectively removing the spectral component of illumination energy from a sample spectrum. obtaining from an empty reference chamber and from an empty sample chamber a first set of illumination spectra when illumination energy is provided simultaneously to said reference and empty sample chambers, said reference chamber and said sample chamber are separate chambers having the same size, shape and reflectivity properties; determining the intensity differences between the illumination spectrum from said reference chamber and the illumination spectrum from said empty sample chamber at each wavelength across the illumination wavelength range; obtaining from said reference chamber and sample chamber a second set of spectra wherein the spectrum from the reference chamber contains only illumination spectral components and the spectrum from said sample chamber contains both illumination and sample spectral components when illumination energy is provided simultaneously to said reference and sample chambers; adjusting the intensities of the illumination spectrum from said reference chamber of the second set of spectra across the wavelength range of the illumination by subtracting or adding the previoulsy determined intensity difference at each corresponding wavelength to the intensities of said illumination spectrum wavelength from said reference chamber of the second set of spectra to make the illumination intensities from said chambers equal across the illumination wavelength range; and subtracting the said adjusted illumination spectrum of the said second set of spectra from the spectrum from said sample chamber of the second set of spectra that contains both illumination and sample components, effectively removing the spectral component of illumination energy from a sample spectrum.