Reflection microscopy

Fresnel reflection measurements are convenient for certain types of microsamples because essentially no sample preparation is required. Ideally, only radiation reflected from the front surface of the sample is measured at the detector in this type of measurement, so that the absorption spectrum may be calculated by the Kramers-Kronig transform, as described in Chapter 13. However, for scattering samples, diffusely reflected radiation (see Chapter 16) also contributes to the signal measured by the detector. When both mechanisms contribute significantly to the measured spectrum, no amount of data manipulation will allow an undistorted absorption spectrum to be calculated. 

Vibrational microspectrometry will undoubtedly be applied to medical diagnosis in the near future. One particularly important application of microspectrometry is for the characterization of tissue samples. Tissue samples can be mounted on a water-insoluble infrared-transparent window such as ZnSe, but these windows are expensive and not conducive to visual examination (e.g., after staining of the tissue). A convenient alternative to transmission spectrometry is the measurement of the transflectance spectrum (see Section 13.5) of tissue samples mounted on low-emissivity glass slides [4]. These slides are transparent to visible light but highly reflective to mid-infrared radiation. 

Attenuated total reflection (ATR) spectrometry is becoming an increasingly important sampling technique for infrared microspectrometry using both single-element and array detectors. This topic is covered in Chapter 15. 

---

A point light source is imaged onto the specimen by the objective and the transmitted light collected by the collector lens and detected by a broad-area detector in the case of reflection microscopy, the objective lens also serves simultaneously as a collector (see figure Bl.18.10. The resolution is solely detennined by the objective lens, because the collector has no imaging fimction and only collects the transmitted light. The... 

Lasentech Focused Beam Reflectance Microscopy (FBRM)... 

Cell/substrate contacts can be located by a nonfluorescence technique completely distinct from TIRF, known as internal reflection microscopy (IRM).(1 9) Using conventional illumination sources, IRM visualizes cell/substrate contacts as dark regions. IRM has the advantage that it does not require the cells to be labeled, but the disadvantages that it contains no information about biochemical specificities in the contact regions and that it is less sensitive to changes in contact distance (relative to TIRF) within the critical first lOOnm from the surface. 

I. Todd, J. S. Mellor, and D. Gingell, Mapping cell-glass contacts ofDictyostelium amoebae by total internal reflection aqueous fluorescence overcomes a basic ambiguity of interference reflection microscopy, J. Cell Sci. 89, 107-114 (1988). 

D. Gingell and I. Todd, Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement, Biophys. J. 26, 507-526 (1979). 

D.C. Prieve and N.A. Frej Total Internal Reflection Microscopy A Quantitative Tool for the Measurement of Colloidal Forces. Langmuir 6, 396 (1990). 

Curtis ASG (1964) The mechanism of adhesion of cells to glass a study by interference reflection microscopy. J Cell Biol 20 199-215... 

In transmission microscopy of specimens with properties not too different from those of water, Rayleigh waves may safely be disregarded. But in reflection microscopy of specimens of higher stiffness, Rayleigh waves generally play a dominant role. This is recognized explicitly in the ray theory treatment. 

Figure 12.12 Images of the motility of the actin/Au-wire/actin filaments on a glass surface modified with myosin, upon the addition of ATP. Images were recorded by reflectance microscopy (a), (b), (c), and (d) correspond to the same imaged frame at time intervals of 5 s. Adapted with permission from Ref. 56. Copyright Nature Publishing Group, 2004.

Examples are Laser Differential Microanemometry (LMA) and Total Reflection Microscopy (TMA) (8). Both LMA and TMA measure the velocity profile of the fluid in tube flow. However, such optical techniques are generally not suitable for opaque and/or heterogeneous substances such as foods. Acoustic velocimetry seems to be more promising for determining the velocity profiles of opaque substances. Such an acoustic technique has been applied by Brunn et al (19) as an on-line viscometer for flow of mayonnaises in pipes. 

A portion of the exiting stream of the molten blend is diverted into the Flow Cell , where Nomarsky reflection microscopy is carried out in a thin slit, the bottom plate of which is reflective polished steel and the top is a quartz window. The microscope, the rapid image data acquisition device, and analyzer are capable of producing dispersion data down to sizes of one micrometer. The TSMEE is shown schematically for both the (M-M) and DMM) modes in Fig. 11.31 (119-121). 

Until fairly recently, the theories described in Secs. II and III for particle-surface interactions could not be verified by direct measurement, although plate-plate interactions could be studied by using the surface forces apparatus (SFA) [61,62]. However, in the past decade two techniques have been developed that specifically allow one to examine particles near surfaces, those being total internal reflection microscopy (TIRM) and an adapted version of atomic force microscopy (AFM). These two methods are, in a sense, complementary. In TIRM, one measures the position of a force-and torque-free, colloidal particle approximately 7-15 fim in dimension as it interacts with a nearby surface. In the AFM method, a small (3.5-10 jam) sphere is attached to the cantilever tip of an atomic force microscope, and when the tip is placed near a surface, the force measured is exactly the particle-surface interaction force. Hence, in TIRM one measures the position of a force-free particle, while in AFM one measures the force on a particle held at a fixed position. 

Park. K, and Park. H, q>lication of video-mhanced interference reflection microscopy to the study of platelet-surface interactions, Scann. Microscopy. Supplement, 3,137-146 (1989). 

Deformation by intracrystalline slip and dynamic reaystallization. These are important mechanisms for the development of crystallographic preferred orientation (CPO) and property anisotropy. Slip bands associated with intracrystalline flow may easily be seen in optical reflection microscopy of previously flattened and polished surfaces of specimens that are subsequently deformed as described above. CPO developments are less easily demonstrated, because it required the making of thin sections of deformed ice and the use of a simple universal stage to determine the orientation of the crystallographic c axis. 

---