Measurement of the Refractive Index of Cytoplasmic Inclusions in Living Cells by the Interference Microscope

Nature ◽  
1954 ◽  
Vol 174 (4435) ◽  
pp. 836-837 ◽  
Author(s):  
K. F. A. ROSS
Keyword(s):  
Refractive Index ◽  
Living Cells ◽  
Interference Microscope ◽  
Cytoplasmic Inclusions

The Use of the Interference Microscope to Determine Dry Mass in Living Cells and as a Quantitative Cytochemical Method

1954 ◽  
Vol s3-95 (31) ◽  
pp. 271-304
Author(s):  
H. G. DAVIES ◽  
M. H. F. WILKINS ◽  
J. CHAYEN ◽  
L. F. LA COUR
Keyword(s):  
Refractive Index ◽  
Pollen Grains ◽  
Dry Mass ◽  
Living Cells ◽  
Mature Pollen ◽  
Interference Microscopy ◽  
Interference Microscope ◽  
Ram Sperm ◽  
Dry Substance ◽  
In Cells

1. The total mass M of substances other than water (the dry mass) in the living cell can be obtained from the expression M = φA/χ, where φ is the optical path difference (o.p.d.) due to the cell and A its projected area. The method makes use of the fact that the refractive increments α(χ = ioocα) of most substances in cells are approximately the same, and independent of concentration. Values for χ have been tabulated. Inaccuracies in the measurement of dry mass due to variations in χ (using λ average = 0.18) will be less than ± 10 per cent, in cells containing nucleic acids, proteins, and lipoproteins. When appreciable quantities of other substances are present the inaccuracy may be somewhat greater. When the total dry mass of living cells is determined in a medium other than water (e.g. isotonic solution), a correction term involving the thickness must be determined; this correction is often small. 3. The total dry masses and, in some cases, the concentrations of dry substance in a variety of biological objects including Amoebae, pollen grains at various stages of development, nuclei of cells in tissue culture, and sperm heads have been determined. In Tradescantia bracteata, during development from the microspore to the mature pollen grains, the dry mass increases by about tenfold. The dry masses of mature pollen grains were measured before and after successive digestion with ribonuclease, which removed about 4 to 14 per cent, of the dry mass, and with trypsin, after which about 40 per cent, of the original dry mass remained. In living ram sperm heads the ratio of deoxyribose nucleic acid to total dry mass determined by ultra-violet and interference microscopy respectively is 40 per cent. This is in good agreement with the value 45 per cent, obtained by bulk biochemical methods. 4. The interference microscope has been used to measure the refractive index of cells and, hence, the concentrations of dry substances in them, by immersing them in media of different known refractive indices. The application of this method to fixed cells is discussed theoretically. In experiments on fixed ram sperm heads the expected linear relationship between o.p.d. and refractive index of the immersion medium was obtained. Data on the average concentration of dry substance in ram sperm heads, the localized refractive index, and concentration in the denatured submicroscopic particles in the head, the percentage of the head volume occupied by them, and the geometrical thickness of the head were obtained. 5. Factors affecting the accuracy of the measurements of o.p.d., such as glare in the microscope, light scatter or absorption by the object, &c, are outlined.

Simultaneous characterization of the shape and refractive index of transparent living cells by an optical aperture

2010 ◽  
Vol 49 (33) ◽  
pp. 6416 ◽  
Author(s):  
Xiaodong Zhou ◽  
Kai Yu Liu ◽  
Nan Zhang ◽  
Christina Tan
Keyword(s):  
Refractive Index ◽  
Living Cells ◽  
Optical Aperture

Detection of refractive index changes in individual living cells by means of surface plasmon resonance imaging

2010 ◽  
Vol 26 (2) ◽  
pp. 674-681 ◽  
Author(s):  
Yuhki Yanase ◽  
Takaaki Hiragun ◽  
Sakae Kaneko ◽  
Hannah J. Gould ◽  
Malcolm W. Greaves ◽  
...  
Keyword(s):  
Refractive Index ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Living Cells ◽  
Surface Plasmon Resonance Imaging ◽  
Resonance Imaging ◽  
Index Changes

scholarly journals Measurement of the integral refractive index and dynamic cell morphometry of living cells with digital holographic microscopy

2005 ◽  
Vol 13 (23) ◽  
pp. 9361 ◽  
Author(s):  
Benjamin Rappaz ◽  
Pierre Marquet ◽  
Etienne Cuche ◽  
Yves Emery ◽  
Christian Depeursinge ◽  
...  
Keyword(s):  
Refractive Index ◽  
Living Cells ◽  
Digital Holographic Microscopy ◽  
Dynamic Cell ◽  
Holographic Microscopy

Application of fluorescence-interference microscope for monitoring of dry weight dynamics and drug distribution within living cells

2001 ◽  
Author(s):  
Gennady G. Levin ◽  
Theodor V. Bulygin ◽  
Eugene Kalinin ◽  
Gennady N. Vishnyakov ◽  
Irene V. Goryainova
Keyword(s):  
Drug Distribution ◽  
Dry Weight ◽  
Living Cells ◽  
Interference Microscope

Shearing Interference Microscope with Decoding of Differential Phase Patterns of Living Cells Using the Phase Stepping Method

2016 ◽  
Vol 58 (11) ◽  
pp. 1238-1243 ◽  
Author(s):  
M. I. Latushko ◽  
G. N. Vishnyakov ◽  
G. G. Levin
Keyword(s):  
Living Cells ◽  
Interference Microscope ◽  
Differential Phase ◽  
Phase Stepping ◽  
Phase Stepping Method ◽  
Shearing Interference

Application of computerized interference microscope for monitoring oscillations of dry cell weight and morphology of living cells

2001 ◽  
Author(s):  
Gennady G. Levin ◽  
Theodor V. Bulygin ◽  
Eugene Kalinin ◽  
Gennady N. Vishnyakov
Keyword(s):  
Living Cells ◽  
Interference Microscope ◽  
Cell Weight ◽  
Dry Cell Weight ◽  
Dry Cell

The Size of Living Bacteria

1957 ◽  
Vol s3-98 (44) ◽  
pp. 435-454
Author(s):  
K.F. A. ROSS
Keyword(s):  
Refractive Index ◽  
Phase Change ◽  
Direct Measurement ◽  
Lactobacillus Bulgaricus ◽  
Refractive Indices ◽  
Interference Microscope ◽  
The Mean ◽  
Different Cultures ◽  
Thickness Measurements ◽  
Living Bacteria

The difficulties involved in the direct measurement, by eyepiece-micrometer or from photomicrographs, of small microscopic objects such as living bacteria are discussed. The accuracy with which this can be done is limited by the numerical aperture of the optical system and the wavelength of light used. With visible light it is scarcely possible to determine the dimensions of an object more accurately than to the nearest 0.4µ. It also seems probable, from the nature of the diffraction pattern at the edges of images of objects of circular cross-section such as bacteria, that direct measurement of the width of the image will tend to give an underestimate of the true width of the object. An interference microscope enables thickness measurements to be made that are not subject to these particular limitations, because with it, the phase-change in the light passing through the middle of a bacterium can be measured very accurately. This phase-change is proportional to the product of the refractive index of the bacterium minus that of the mounting medium, and its true thickness. Two methods were used to determine the mean thickness of the living bacilli in a number of different cultures of Lactobacillus bulgaricus. With the first, the mean refractive index of the bacilli was measured directly by the method of immersion refractometry first used by Barer and Ross (1952), and phase-change measurements were made on the bacilli mounted in dilute saline. Their mean thickness was calculated from these measurements. With the second method, phase-change measurements were made on the bacilli mounted in saline and also mounted in protein solutions with refractive indices ranging from 1.365 to 1.376; and, from these, both their mean thickness and their mean refractive index were calculated. The phase-change measurements were made with a Smith interference microscope and half-shade eyepiece (manufactured by Messrs. Charles Baker). The values for the mean thickness of the living L. bulgaricus from 14 different cultures obtained by the first method ranged from 1·13 µ to 1·23 µ; and those from 9 different cultures obtained by the second method ranged from 1·02 µ to 1·14 µ. The mean refractive indices of the latter calculated by the second method agreed very closely with that obtained by immersion refractometry, and differed by a maximum of 0.009 in all the cultures measured. It therefore seems unlikely that the mean thickness measurements obtained by either method are wrong by more than about ±0.1 µ.

Sign in / Sign up

Export Citation Format

Share Document