Microspectrophotometer System Research Articles

Laser-induced autofluorescence microscopy of normal and tumor human colonic tissue

Laser-induced autofluorescence (LIAF) spectroscopy has been found to be a promising tool for early cancer diagnosis in various organs, but the reasons responsible for the spectral differences between normal and diseased tissue are still not well understood. In this study, a microspectrophotometer (MSP) system was used to identify the microscopic origins of tissue autofluorescence in the colon under the excitation of a helium-cadmium laser at 442 nm. Colonic tissue samples (normal: n=8, adenocarcinoma: n=10) were obtained from 12 patients with known or suspected malignancies of the colon. The intrinsic fluorescence spectra and images of fresh tissue sections prepared from normal and tumor colonic tissue were measured by the MSP system. Three distinct tissue layers of the colon were found for fluorescence, the mucosa, the submucosa and the muscularis propria, with submucosa being the most fluorescent. Differences in the spectral shape and intensity of the intrinsic fluorescence originating from different colonic layers indicate that fundamentally different fluorophores may be present in the respective tissue layers. There was no significant difference in the intrinsic fluorescence features of the submucosa between normal and tumor colonic tissue, but the fluorescence intensity of the submucosa in tumor tissue was significantly reduced due to the infiltration of tumor cells into the submucosa. The intrinsic fluorescence spectrum peaking at about 520 nm for tumor stroma appeared more evident than that of normal lamina propria. Limited areas of the lamina propria layer in some adenocarcinoma colon exhibited an emission band at about 635 nm, which was attributed to endogenous porphyrins in tumor. Autofluorescence microscopy revealed that differences in the clinically measured autofluorescence spectra between normal and tumor tissue were mainly due to thickening of the tumor mucosa resulting in a reduced submucosa fluorescence contribution, as well as the increased hemoglobin absorption in tumor tissue. Therefore, investigation of the microscopic origins of tissue autofluorescence and images can provide new insights into morphological structures and biochemical components of tissues, which are vital to improve the implementation of the LIAF technique for non-invasive in vivo tissue diagnostics.

Effects of pH on structure and function of single living erythrocyte

The spontaneous changes in cell shape and size, the membrane bending elastic modulus, and the structure and functions of intracellular hemoglobin of single human living erythrocyte incubated in different suspension pH were studied. The non-invasive,in situ, real time measurements on the parameters at the three levels of intracellular molecule, membrane and cell morphology were performed simultaneously by using a fast micro-image analyzing technology and a multi-channel micro-spectrophotometer system. The result shows that the cell shape and size, the mechanical property of the cell membrane, and the structure and function of intracellular hemoglobin are correlated and affected by each other with pH.

Microspectrophotometric measurements of vertebrate photoreceptors using CCD-based detection technology.

We have developed a charge-coupled-device (CCD)-based microspectrophotometer (MSP) system and provide the first report on the successful employment of this technology to measure the spectral absorbance properties of vertebrate photoreceptors. The principal difference between the CCD-based MSP system and wavelength-scanning MSP systems, commonly used in vision biology, is that a short duration (800-1200 ms), broad-spectrum flash is employed rather than ascending and descending wavelength scanning. Data acquisition is thus significantly faster, with the added possible advantages of less variance due to movement of target photoreceptors during measurement, reduced spectral distortion due to photoproduct interference and an ability to measure fast, transient changes in absorbance as bleaching proceeds. Rainbow trout photoreceptors, previously measured with a wavelength-scanning MSP system, were again measured using the CCD-based MSP system. Our analysis of optical recordings from 102 photoreceptors corroborated data obtained previously with rainbow trout photoreceptors on lambda(max) (wavelength of maximum absorbance), A(max) (maximum absorbance) and half maximum bandwidth (HBW) of ultraviolet-, blue-, green- and red-sensitive cones and rods. There were slight differences in lambda(max) and half-maximum bandwidth of the ultraviolet-, blue- and green-sensitive cone classes, but this was most probably due to variation in the A(1):A(2) visual pigment ratio of the trout used in the two different studies. However, we were capable of resolving the A(1) and A(2) visual pigment spectra in the red-sensitive cones and the rods.

Microspectrophotometric analysis of respiratory pigments using a novel fibre optic dip probe in microsamples

A microspectrophotometer system to monitor the reduction of mitochondrial respiratory pigments in cell extracts and permeabilized cells has been developed. The novel optical fibre set-up uses visible spectrophotometry to measure the reduction of mitochondrial electron carriers. The basis of the system is an Ocean Optics S1000 spectrometer, a broadband tungsten based light source, input and output coupling fibre optics and a fibre optic dip-probe which requires less than 20 µl of sample for analysis. The spectral range of the system is from 250 to 850 nm with a spectral resolution of 0.5 nm. Data are presented for the reduction of purified cytochrome c by the reducing agent sodium dithionite and the reduction of cytochrome c by isolated mitochondria using sodium succinate as substrate. Reduction of cytochrome c by digitonin permeabilized cultured mouse cells, C2C12, is also shown. The effect of temperature on cytochrome c reduction in these assays is also demonstrated. The optical design of the probe system is optimized to maintain maximum light throughput and spectral resolution. The key features of the system are small sample size, front-end adaptability, high sensitivity and fast multispectral acquisition which are essential for observing these biological reactions in vivo.

Determination of myoglobin saturation of frozen specimens using a reflecting cryospectrophotometer.

This report describes a method and instrumentation for determining myoglobin (Mb) oxygen saturation in skeletal muscle. Canine gracilis is frozen in situ using a liquid N2-cooled copper block. Transverse section surfaces of frozen unstained muscle are observed at -110 degrees C using a microspectrophotometric system. The Mb saturation is determined using epi-illumination and a four-wavelength optical method. A special aperture permits illumination of a 20-microns-square area, and the radius of the catchment volume is estimated to be approximately 60 microns, with the strongest signal arising from the central region. The equibestic wavelengths used were 546.6, 570.5, and 584.1 nm. The method was validated using the nonlinear multicomponent analysis method of Lübbers. End-point (0 and 100% saturation) calibration was set using ischemic and adenosine-treated highly oxygenated muscles, respectively. The effects of hemoglobin (Hb) and metmyoglobin (metMb) signal contamination were evaluated experimentally and by computer-mixing simulations. Mb saturation determinations adjacent to large vessels are to be avoided. MetMb and capillary Hb do not interfere with the determination. The reproducibility of the method is estimated to be +/- 5%.

Design of an instrument for far-infrared microspectroscopy using a synchrotron radiation source

A design of a microspectrophotometric system using a synchrotron radiation (SR) source is described. The system covers a wide spectral range of 50–13000 cm−1, being under construction at the UVSOR BL6B beamline in the Institute for Molecular Science. Preliminary experiments in the mid-infrared region (500–5000 cm−1) have qualitatively confirmed the theoretical calculation that the synchrotron radiation is more intense than a blackbody (T=1200 K) when a microspectrophotomeric technique is applied, which is due to natural collimation and high brilliance of SR source. The SR as an infrared source exhibits its advantage on measuring the spectra of small single crystals especially in the far-infrared region.

Determination of red blood cell oxygenation in vivo by dual video densitometric image analysis

We have developed a new video microspectrophotometric system for the in vivo determination of oxygen saturation in red blood cells in striated muscle capillaries. This method allows one to quantify changes in the oxygenation of small groups of red blood cells as they traverse the capillary. Simultaneous images of a single microscopic field are recorded using two silicon-intensified target cameras and high-resolution video recorders. One image is recorded at an oxygen-dependent wave-length (431 nm) and the other at an isosbestic wavelength (420 nm). Light intensities from 10 adjacent pixels aligned along the axis of the capillary from identical 10-s segments of the video-tapes are digitized once per frame. Both sets of data are redisplayed simultaneously as two-dimensional images (10 pixels high x 300 frames wide) using a graphics system. These images show alternating bright and dark bands corresponding to plasma gaps and red blood cells. Light intensities in the presence and absence of red blood cells are determined by positioning a window over the appropriate region of the graphics image. Optical densities of single red blood cells at the two wavelengths, OD431 and OD420, are computed as is their ratio (OD431/OD420), which is linearly related to oxygen saturation. In vivo calibration studies in capillaries of the hamster retractor muscle indicate that the error in measuring oxygen saturation with this technique is approximately 2.7% saturation for a group of 10 cells.

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells.

The cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomyl distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.