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.

The analysis of mitoses in single living cells by interference microscopy

1961 ◽  
Vol 153 (952) ◽  
pp. 357-366 ◽  
Keyword(s):  
Cell Plate ◽  
Dry Mass ◽  
Living Cells ◽  
Interference Microscopy ◽  
Tissue Cell ◽  
Interference Microscope ◽  
Continuous Record ◽  
Plate Area ◽  
Single Living Cells ◽  
Single Living

A new method of using the interference microscope is described which enables a continuous record to be obtained of changes in dry mass and concentration during the growth of a single tissue cell. The method has been applied to the analysis of changes of dry mass in the chromosomes, spindle and cell-plate area during mitosis in endosperm. The results of these observations have been compared with earlier studies of birefringence during mitosis. It is shown that birefringence develops under conditions of progressively increasing concentration of macro-molecular material.

Probing nucleus-enriched proteins in single living cells via subcellular-resolved plasmonic immunosandwich assay

The Analyst ◽  
2021 ◽  
Author(s):  
Jia Liu ◽  
Dan Xie ◽  
Zhen Liu
Keyword(s):  
Living Cells ◽  
Nuclear Proteins ◽  
Biological Functions ◽  
Abnormal Expression ◽  
Single Living Cells ◽  
Single Living ◽  
In Cells

Nuclear proteins are crucial in cells and are greatly linked to various biological functions. Abnormal expression of nuclear proteins is associated with many diseases ranging from inflammation to cancer. However,...

scholarly journals Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux

PLoS Biology ◽  
2019 ◽  
Vol 17 (12) ◽  
pp. e3000553 ◽  
Author(s):  
Patrick A. Sandoz ◽  
Christopher Tremblay ◽  
F. Gisou van der Goot ◽  
Mathieu Frechin
Keyword(s):  
Refractive Index ◽  
Mass Flux ◽  
Mammalian Cells ◽  
Dry Mass ◽  
Label Free ◽  
Index Maps

scholarly journals Transformation of Seed Non-Transmissible Hop Viroids in Nicotiana benthamiana Causes Distortions in Male Gametophyte Development

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2398
Author(s):  
Lenka Steinbachová ◽  
Jaroslav Matoušek ◽  
Gerhard Steger ◽  
Helena Matoušková ◽  
Sebastjan Radišek ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Male Gametophyte ◽  
Pollen Grains ◽  
Potato Spindle Tuber Viroid ◽  
Apple Fruit ◽  
Mature Pollen ◽  
Rare Occurrence ◽  
Gametophyte Development ◽  
Male Gametophyte Development ◽  
Marker Proteins

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that “forcing” overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.

scholarly journals Effect of temperature on dry mass of polytene nuclei in Drosophila

1979 ◽  
Vol 38 (1) ◽  
pp. 405-416
Author(s):  
I.J. Hartmann-Goldstein ◽  
D.J. Goldstein
Keyword(s):  
Salivary Glands ◽  
Dry Mass ◽  
Nuclear Volume ◽  
Interference Microscopy ◽  
Effect Of Temperature ◽  
Percentage Loss ◽  
Mass Group ◽  
The Mean ◽  
Polytene Nuclei ◽  
Aqueous Detergent

Nuclei were isolated by an aqueous detergent method from Drosophila prepupal salivary glands, and measured by integrating interference microscopy. There was a highly significant correlation between nuclear volume and dry mass. Dry masses fell into 2, 3 or 4 distinct groups corresponding to polytene replication classes; the mean of a given dry mass group was between 8 and 30% less than twice that of the group below, indicating that the ratio of DNA:dry mass increases during polytenic growth. The proportion of nuclei in the higher mass groups, the mean dry mass of nuclei within a given mass group, and the percentage loss of nuclear dry mass in the first hour after isolation were all higher when animals were reared at 15 degrees instead of 25 degrees C. Nuclear dry mass in prepupae was affected by the temperature during both the embryonic and larval periods, and also to some extent by the nutrition and degree of crowding of the cultures.

scholarly journals On the Mature Pollen Grains in Angiosperms

1937 ◽  
Vol 51 (606) ◽  
pp. 524-529 ◽  
Author(s):  
Nobuhide Suita
Keyword(s):  
Pollen Grains ◽  
Mature Pollen

On the Identity of the ‘Neurofibrils’, ‘Nissl complex’, ‘Golgi Apparatus’, and ‘Trophospongium’ in the Neurones of Vertebrates

1961 ◽  
Vol s3-102 (60) ◽  
pp. 481-493
Author(s):  
G. B. DAVID ◽  
A. W. BROWN ◽  
K. B. MALLION
Keyword(s):  
Angular Velocity ◽  
Golgi Apparatus ◽  
Golgi Method ◽  
Interference Microscopy ◽  
Normal Adult ◽  
Interference Microscope ◽  
Clear Space ◽  
Fixed Cell

1. Classical histological methods demonstrate 4 cytoplasmic networks in fixed vertebrate neurones: ‘neurofibrils’, ‘Nissl complex’, ‘Golgi apparatus’, and ‘trophospongium’. The work described in this paper was undertaken to find out whether the 4 networks of classical histology correspond to 4 structures recognizable as such in the living neurone, or to only one structure, which may be coloured in characteristic ways by the classical methods. 2. A single continuous network, comprising features traditionally associated with the four classical networks of the fixed cell, can be isolated by micro-dissection and detected by interference microscopy in living vertebrate neurones. 3. When living neurones are centrifuged at a moderate angular velocity, a single continuous network remains visible under the interference microscope. There does not appear to be enough clear space left for 3 other voluminous structures. 4. When living neurones are centrifuged at a high angular velocity, a single continuous network is pushed to the centrifugal pole of the cell. The remainder of the cell then contains only small separate objects. 5. A single continuous network can be detected by interference microscopy in the cytoplasm of fixed, unstained neurones. When the same cells are dyed by a Nissl method, the cytoplasmic network seen in the unstained cells becomes deeply coloured. When the same cells are bleached and then silvered by a Golgi method, the objects that before had bound the dye now are blackened by the reduced silver. The same effect is obtained when the Nissl method is used after silvering. 6. It is inferred from the results of these experiments that there is only one cytoplasmic network in living normal adult vertebrate neurones. This network is demonstrated in fixed neurones of the same type with varying degrees of faithfulness by the classical methods. It is suggested that the terms ‘neurofibrils’, ‘Nissl complex’, ‘Golgi apparatus’, and ‘trophospongium’ be abandoned.

The Concentrations of Dry Matter in Mitotic Apparatuses in Vivo and After Isolation From Sea-Urchin Zygotes

1972 ◽  
Vol 10 (2) ◽  
pp. 387-418
Author(s):  
A. FORER ◽  
R. D. GOLDMAN
Keyword(s):  
Sea Urchin ◽  
Dry Matter ◽  
Mass Medium ◽  
Dry Mass ◽  
Interference Microscopy ◽  
High Mass ◽  
Isolation Medium ◽  
High Ph ◽  
Cytoplasmic Material

We have measured the concentrations of dry matter in mitotic apparatuses (MA) in vivo and after isolation from the same cell type. The isolation medium was hexylene glycol plus buffer. The MA were from sea-urchin zygotes (Echinus esculentus Linn. and Psammechinus miliaris Gmelin), and measurements were made using interference microscopy. MA as isolated have much lower concentrations of dry matter than do MA in vivo. The dry mass concentrations of isolated MA vary with the pH of the isolation medium, ranging from about 20 % of the in vivo concentration (at pH 7.3) to about 60 % of the in vivo concentration (at pH 5.3). The isolated MA were further characterized. Evidence is presented which suggests that non-specific cytoplasmic material adsorbs to MA, and thus that at least some of the material in isolated MA is not derived from in vivo MA. Some MA components are apparently changed during the isolation procedure: MA lysed in low pH (high mass) medium and quickly transferred to high pH (low mass) medium have higher concentrations of dry matter than do MA lysed in high pH medium. The isolation media as generally used do not have enough buffering capacity: the pH changes after the isolation. These data suggest that the isolation procedures need be improved before studies of isolated MA can give data relevant to the chemistry of in vivo MA. We discuss the problem of obtaining functional isolated MA, and also the relevance of our data to previous work on MA from other species.

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