Although the optical systems found in modern microscopes may be capable of producing high resolution images at high magnifications, such a capability is worthless without sufficient contrast in the image. Contrast is not an inherent property of the specimen, but is dependent upon interaction of the specimen with light and the efficiency of the optical system coupled to its ability to reliably record this image information with the detector. Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector.
Contrast, resolution, pixelation, dynamic range and signal-to-noise ratio: Fundamental limits to resolution in fluorescence light microscopy. Journal of Microscopy 189: 15-24 (1998). An excellent foundation for understanding the basis for contrast in optical microscopy from a perspective of resolution and other significant contributing factors.
Refractometry of living cells. Part I. Basic principles. Quarterly Journal of Microscopical Science 95: 399-423 (1954). First in a two-part series on specimen contrast. The authors discuss the theory of refraction, refractive index and density, as well as the effects of temperature, wavelength, concentration, and imaging medium on contrast.
Refractometry of living cells. Part II. The immersion medium. Quarterly Journal of Microscopical Science 96: 1-27 (1955). A continuation of the discussion initiated in Part I, the authors discuss basic requirements of immersion media, permeability of cells, various media, osmotic properties of immersion fluids, and searching for evidence of non-toxicity.
Refractometry and interferometry of living cells. Journal of the Optical Society of America 47: 545-556 (1957). An extensive discussion on the origins of contrast in living cells with emphasis on the refractive index of internal components. The author also discuses osmotic reactions, contrast variations, interference microscopy, and refractometry.
Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity. Journal of Biomedical Optics 1: 200-211 (1996). The authors discuss optical properties of thin tissue sections when subjected to changes in refractive index and osmolarity as the bathing medium is altered in composition. Included is a discussion of the origins of contrast in biological specimens.
Curl, C. L., Bellair, C. J., Harris, P. J., Allman, B. E., Roberts, A., Nugent, K. A. and Delbridge, L.
Quantitative phase microscopy: A new tool for investigating the structure and function of unstained live cells. Clinical and Experimental Pharmacology and Physiology 31: 896-901 (2004). Among the most significant points about this report is the extensive discussion about specimen contrast in transparent unstained cells. The authors discuss how phase maps can be used to emulate contrast imaging modes.
Light scattering from cells: Finite-difference time-domain simulations and goniometric measurements. Applied Optics 38: 3651-3661 (1999). The authors examine the light-scattering properties of inhomogeneous biological cells through a combination of theoretical simulations and goniometric measurements. These simulations provide insight into the scattering and contrast problems associated with imaging living cells.
Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics. Applied Optics 37: 3586-3593 (1998). Optical properties of mammalian cell suspensions were investigated to provide a basis for interpreting the optical properties of tissues in vivo. Isolated organelles revealed that these tiny structures were responsible for light scattering at large angles.
Light scattering from coated spheres: Model for biological cells. Applied Optics 11: 675-680 (1972). A simulation of the cytoplasm and nuclei of living cells using light scattering from non-absorbing, spherically symmetrical spheres. The authors demonstrate that their model is a suitable candidate for examining contrast in live cells.
Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns. New Journal of Physics 9: 363-14 (2007). An interesting method of generating contrast in transparent specimens using a projector that functions as both a light source and mask generator. Filters are used to project illumination patterns in the microscope principal planes.