One of the most important techniques used for analyzing biological materials is UV-visible range microspectroscopy. In this example, analysis of red blood cells (RBCs) was performed using a combination of UV range microspectroscopy and ultraviolet microscopy.
Non-destructive Analysis of Biological Materials
CRAIC Technologies offers the 20/30 PV™ microspectrophotometer, which enables quick, easy and non-destructive analysis of biological materials. For this experiment series, the 20/30 PV™ microspectrophotometer was configured for UV range absorbance microscopy and spectroscopy of individual RBCs. It was fitted with permanently calibrated variable apertures in order to best measure different sized samples under controlled conditions.
Analysis of RBC samples fixed to a calcium fluoride substrate is performed by means of UV transmission micro-imaging and absorption microspectroscopy. Certain cells included ~1 μm diameter dark spots situated in the middle of the cells.
Cell spectra with and without dark spots were measured and compared. For each measurement, 20 scans were averaged for absorption microspectral analysis; the spectral range was limited to 250 to 850 nm. For imaging and spectroscopy, broadband UV-visible-NIR light was utilized.
For reference, blank areas on the sample substrate were utilized. Apertures used were A4 (6.5 μm x 6.5 μm), A5 (3.2 μm x 3.2 μm), and A6 (1.5 μm x 1.5 μm). In addition, a 40X quartz objective was also utilized.
Four 10 nm FWHM bandpass filters centered at 232 nm, 267 nm, 281 nm, and 415 nm in wavelength were used for imaging analysis.
Red Blood Cells with Dark Spots
Figure 1. RBCs with dark spots
Figure 1 shows a magnified image of a collection of RBCs from the sample slide. The cells highlighted with square boxes were considered as RBCs with dark spots. The sampling area is the black square within the central box and in this case measures 1.5 x 1.5 μm (A6).
At the dark spots of several cells, the spectra below the image were measured. The spectra display absorption peaks at different wavelengths above 500 nm.
Red Blood Cells without Dark Spots
Figure 2. RBCs without dark spots
Figure 2 shows that RBCs marked with square boxes were believed to be cells without the dark spots. The cells’ spectra have absorption spectral peaks at 417 nm and 275 nm in wavelength. An apparent spectral shoulder is demonstrated at 480~540 nm in wavelength.
Microspectra Taken with Different Aperture Sizes
Figure 3. Microspectra with different aperture sizes
Figure 3 shows the next series of spectra with localization of compounds within the RBC. Apertures A4 (6.5 μm x 6.5 μm, lower left hand corner image), A5 (3.2 μm x 3.2 μm, upper center image), and A6 (1.5 μm x 1.5 μm, upper right hand corner image) were utilized to obtain spectra on RBCs with dark spots only.
The central area of the cell is covered by A6, while the whole cell is covered by A4. The same absorption peaks are exhibited by the three spectra. The spectrum with A6 displays an increase above 600nm in wavelength when compared to those with A4 and A5. The difference is possibly due to the edges of the cells.
Microspectra Taken in the Center and Edge of Red Blood Cells
Figure 4. Spectra at the center of the RBCs
Figure 5. Spectra at the edge of the RBCs
Figures 4 and 5 show that spectra collected at the center and edges of the RBCs display the compounds’ distinction in the cells. The aperture used was the A6 aperture.
- RBC with dark spots – the central area’s spectrum depicts an absorption peak at 417nm and 275nm in wavelength. The spectra have different curves above 500nm.
- RBC without dark spots - the central area’s spectrum displays an absorption peak at 417nm in wavelength while that on the edge does not.
Figures 6a - 6d were obtained at the same location of the sample slide. Imaging wavelengths were white light, 415nm, 281nm, 267nm, and 232nm, respectively.
Results and Discussion
Absorbance spectra were taken on RBCs with and without dark spots. It can be seen that both sets of spectra have absorption peaks at 417nm and 275nm in wavelength. Cells’ spectra with dark spots also display absorption peaks at different locations above 500nm, which are missing in cells without dark spots.
Furthermore, the difference between peak amplitudes at 417nm and 275nm for cells without dark spots is considerably larger when compared to cells having dark spots. In addition, central area spectra of the cells with dark spots display an increase in absorbance above 500nm, while edge spectra decrease in absorbance. For cells lacking dark spots, the central area’s spectrum displays an absorption peak at 417nm in wavelength while that on the edge does not.
The RBCs were also analyzed using UV micro-imaging. At 415nm wavelength, cell contrast enhancement is primarily due to the heme group, which reflects the concentration and localization of the hemoglobin in the RBC. Light is absorbed by proteins at 280nm, making UV imaging a quick and precise technique for mapping distribution of proteins. It is also possible to use UV micro-imaging to monitor nucleic acids as they exhibit an optical absorption maximum near 260nm.
Thus, both UV range microspetroscopy and ultraviolet microscopy provide a suitable and effective solution for analysis of biological materials.
Produced from articles authored by Dr. Paul Martin, CRAIC Technologies, Inc.
About CRAIC Technologies
CRAIC Technologies™ specializes in developing superior UV-visible-NIR microanalysis solutions: we build instrument to collect spectra and images of sample features ranging from sub-micron to hundreds of microns. CRAIC Technologies products include UV and NIR microscopes, UV-visible-NIR microspectrophotometers, instruments to measure thin film thickness and colorimetry on the microscopic scale, Raman microspectrometers, automation solutions, traceable standards and more.
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