Статті в журналах: "Interference reflection microscopy (IRM)"

1

Verschueren, H. "Interference reflection microscopy in cell biology: methodology and applications." Journal of Cell Science 75, no. 1 (April 1, 1985): 279–301. http://dx.doi.org/10.1242/jcs.75.1.279.

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Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.

2

Valavanis, Dimitrios, Paolo Ciocci, Gabriel N. Meloni, Peter Morris, Jean-François Lemineur, Ian J. McPherson, Frédéric Kanoufi, and Patrick R. Unwin. "Hybrid scanning electrochemical cell microscopy-interference reflection microscopy (SECCM-IRM): tracking phase formation on surfaces in small volumes." Faraday Discussions 233 (2022): 122–48. http://dx.doi.org/10.1039/d1fd00063b.

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3

Valavanis, Dimitrios, Paolo Ciocci, Gabriel N. Meloni, Peter Morris, Jean-François Lemineur, Ian J. McPherson, Frédéric Kanoufi, and Patrick R. Unwin. "Hybrid scanning electrochemical cell microscopy-interference reflection microscopy (SECCM-IRM): tracking phase formation on surfaces in small volumes." Faraday Discussions 233 (2022): 122–48. http://dx.doi.org/10.1039/d1fd00063b.

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4

Zand, M. S., and G. Albrecht-Buehler. "Long-term observation of cultured cells by interference-reflection microscopy: near infrared illumination and Y-contrast image processing." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 70–71. http://dx.doi.org/10.1017/s0424820100102432.

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Analysis of dynamic changes in cell-substratum adhesion patterns during cell locomotion requires continuous, extended observation of single living cells. To date, interference-reflection microscopy (IRM) is the only method available to visualize cell -substratum adhesions in vitro. This method uses 1% of the incident illumination to produce an IRM image, and so far requires use of a high intensity visible light source (400 - 800 nm). However, light of this intensity and spectral range induces marked changes in fibroblast behavior, including cessation of locomotion. Therefore, we developed a method allowing continuous IRM observation of live cells for up to 8 hours, without any observable changes in normal cell behavior, using near infrared illumination (750-1100 nm). In addition, we use Y-contrast image processing of IRM images to create a 3-dimensional relief of the ventral cell surface.Single locomoting PTK1 cells were observed continuously in IRM with time lapse video recording for periods of up to 8 hours.

5

Lai, Quintin J., Stuart L. Cooper, and Ralph M. Albrecht. "Thrombus formation on artificial surfaces: Correlative microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 3 (August 12, 1990): 840–41. http://dx.doi.org/10.1017/s042482010016176x.

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Thrombus formation and embolization are significant problems for blood-contacting biomedical devices. Two major components of thrombi are blood platelets and the plasma protein, fibrinogen. Previous studies have examined interactions of platelets with polymer surfaces, fibrinogen with platelets, and platelets in suspension with spreading platelets attached to surfaces. Correlative microscopic techniques permit light microscopic observations of labeled living platelets, under static or flow conditions, followed by the observation of identical platelets by electron microscopy. Videoenhanced, differential interference contrast (DIC) light microscopy permits high-resolution, real-time imaging of live platelets and their interactions with surfaces. Interference reflection microscopy (IRM) provides information on the focal adhesion of platelets on surfaces. High voltage, transmission electron microscopy (HVEM) allows observation of platelet cytoskeletal structure of whole mount preparations. Low-voltage, high resolution, scanning electron microscopy allows observation of fine surface detail of platelets. Colloidal gold-labeled fibrinogen, used to identify the Gp Ilb/IIIa membrane receptor for fibrinogen, can be detected in all the above microscopies.

6

Todd, I., J. S. Mellor, and D. Gingell. "Mapping cell-glass contacts of Dictyostelium amoebae by total internal reflection aqueous fluorescence overcomes a basic ambiguity of interference reflection microscopy." Journal of Cell Science 89, no. 1 (January 1, 1988): 107–14. http://dx.doi.org/10.1242/jcs.89.1.107.

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The widespread ability of eukaryotic cells to produce thin cytoplasmic sheets or lamellae 100–200 nm thick can give rise to uncertainties in the interpretation of interference reflection microscopy (IRM) images when cell-substratum topography is the key interest. If allowed to spread upon a poly-L-lysine-coated surface, Dictyostelium discoideum amoebae typically form ultrathin lamellae of approximately equal to 100 nm thickness by cytoplasmic retraction. Whereas the cell body is grey, the lamellae appear very dark under IRM optics. These dark areas could be misinterpreted as stemming from a closer cell-substratum apposition beneath the lamellae than the cell body. This ambiguity can be avoided if the technique of total internal reflection aqueous fluorescence (TIRAF) is used in conjunction with a high refractive index glass (n = 1.83) as substratum. Contributions to the image generated by thin cytoplasm and also variable cytoplasmic refractive index are thereby minimized due to the extremely short range of the ‘illuminating’ evanescent wave. From our comparative IRM and TIRAF study of the ultrathin lamellae of Dictyostelium amoebae it is concluded that the cell-glass gap is relatively uniform beneath the entire cell. We briefly discuss the sensitivity of several cell types to TIRAF, the generation of ultrathin lamellae and the nature of the cell-glass gap.

7

Richter, Ekkehard, Hermine Hitzler, Heiko Zimmermann, Rolf Hagedorn, and G�nter Fuhr. "Trace formation during locomotion of L929 mouse fibroblasts continuously recorded by interference reflection microscopy (IRM)." Cell Motility and the Cytoskeleton 47, no. 1 (2000): 38–47. http://dx.doi.org/10.1002/1097-0169(200009)47:1<38::aid-cm4>3.0.co;2-w.

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8

Singer, I. I., D. M. Kazazis, and S. Scott. "Scanning electron microscopy of focal contacts on the substratum attachment surface of fibroblasts adherent to fibronectin." Journal of Cell Science 93, no. 1 (May 1, 1989): 147–54. http://dx.doi.org/10.1242/jcs.93.1.147.

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We have examined the cell-to-substratum attachment surface of hamster fibroblasts with scanning EM, and describe the surface ultrastructure of focal contacts and microspikes during cellular attachment and spreading on fibronectin. Nil 8 fibroblasts were seeded onto fibronectin-coated glass coverslips in serum-free medium, fixed, and the fibroblast-fibronectin monolayer was separated from the glass and inverted for scanning electron microscopic (EM) analysis. Focal contact development was detected by interference reflection microscopy and correlated with the immunofluorescence microscopic distribution of fibronectin receptor antigens. The cell undersurface appeared smooth and featureless at 0.5 h when focal contacts were undetectable and fibronectin receptors were distributed diffusely. By 1–2 h, undersurface membrane impressions of focal contacts were detected with scanning EM; their size, shape and distribution matched that of focal contacts seen with interference reflection microscopy (IRM). These contacts had smooth external surfaces and were often arranged in chevron-shaped complexes. However, at 4–6 h, the surface texture of focal contacts became fibrous and the contact periphery was delineated with the orifices of membrane-associated vesicles. Development of this filamentous substructure is correlated with the maximum concentration of fibronectin receptors and fibronectin at focal contacts, suggesting that these molecules are involved in the maturation and stabilization of focal contacts.

9

Paddock, S. W. "Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells." Journal of Cell Science 93, no. 1 (May 1, 1989): 143–46. http://dx.doi.org/10.1242/jcs.93.1.143.

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This paper describes two applications of the tandem scanning reflected-light microscope (TSM) for the observation of the structure of individual cells growing in tissue culture. First, the TSM is used as an alternative to interference reflection microscopy (IRM) or total internal reflection aqueous fluorescence microscopy (TIRAF) to observe cell-substratum adhesions in unstained living cells growing on a glass coverslip. Second, the TSM is used to produce improved images of cellular structures in 3T3 cells stained with various protein dyes including Napthol Blue Black (NBB) and Coomassie Brilliant Blue (CBB). More specifically, close contacts and focal contacts are resolved in living 3T3 cells, and features of the nucleus, the cytoskeleton and extracellular matrix are resolved in both NBB- and CBB-stained cells. The focal contacts and associated stress fibres are clearly imaged in NBB-stained cells. The TSM is an improvement over conventional incident light microscopy because of the confocal image excludes information from out-of-focus regions of the cytoplasm, and, unlike the laser-based confocal microscope, the actual colour of the specimen is viewed directly with TSM in almost real-time.

10

Izzard, C. S. "Optical studies on the development of the focal contact." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 120–21. http://dx.doi.org/10.1017/s0424820100102687.

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The focal contact is a localized region of relatively strong adhesion formed between cultured fibroblasts and planar substrates. The contact can be visualized and its formation followed in the live cell by the use of high illuminating-numerical-aperture interference reflection microscopy (IRM). The focal contact is the site into which stress fibers, or bundles of microfilaments, insert at the plasma membrane via a patch of amorphous material, the adhesion plaque. Through the use of immunochemical staining, a number of proteins have been shown to be concentrated at the focal contact/adhesion plaque complex. However, little is known about the sequence of events at the structural and protein levels that result in formation of the focal contact/adhesion plaque complex. We demonstrated previously that the focal contact forms rapidly as a stable adhesion beneath the motile lamellipodium at the cell margin. Parallel IRM and differential interference contrast (DIC)observations further showed that the contact forms specifically beneath a rib-like structure in the lamellipodium, which therefore functions as a precursor of the focal contact.

11

Yuruker, B., and V. Niggli. "Alpha-actinin and vinculin in human neutrophils: reorganization during adhesion and relation to the actin network." Journal of Cell Science 101, no. 2 (February 1, 1992): 403–14. http://dx.doi.org/10.1242/jcs.101.2.403.

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We have studied the reorganization of vinculin and alpha-actinin during the process of adhesion in human neutrophils using immunofluorescence microscopy and interference reflection microscopy (IRM). Neutrophils in contact with uncoated glass formed black IRM areas in the cell periphery, indicative of very close contact with the substratum. Eight to twelve minutes after addition of cells to glass, vinculin was found to become concentrated in small patches at the cell periphery, partially colocalizing with the black IRM areas and with small F-actin-containing adherent protrusions. In contrast, vinculin was not significantly enriched in the less adherent F-actin-rich large pseudopods. alpha-Actinin became enriched during cell adhesion in retraction fibers and, in 40–50% of the inspected cells, also in large less adherent pseudopods where it colocalized with F-actin. The latter finding suggests a continuous dynamic reorganization of pseudopods, with incorporation of alpha-actinin at a certain stage. Disruption of the actin network with cytochalasin D revealed a differential interaction of alpha-actinin and vinculin with the actin network. alpha-Actinin was strongly influenced by cytochalasin D, comparable to F-actin, and both proteins formed colocalizing peripheral caps in 10(−5) M of the drug. Vinculin organization in contrast was not affected by up to 10(−6) M cytochalasin. At 10(−5) M of the drug, however, the patches disappeared completely, vinculin now assuming a diffuse cytoplasmic location. Our results suggest a specialized function of vinculin in adhesion sites of human neutrophils, whereas alpha-actinin may structure the actin network in retraction fibers and in less adherent pseudopods.

12

Owens, N. F., D. Gingell, and A. Trommler. "Cell adhesion to hydroxyl groups of a monolayer film." Journal of Cell Science 91, no. 2 (October 1, 1988): 269–79. http://dx.doi.org/10.1242/jcs.91.2.269.

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We have studied cells on chemically defined monomolecular films of the long-chain alcohol docosanol. Langmuir-Blodgett films of the alcohol were deposited on glass coverslips, previously made hydrophobic with octadecyl groups. This gives films in which the alcohol headgroups face outwards to the water. Molecular orientation and film integrity were shown by a fluorescence adsorption test. Cell contacts on the films were observed in media without proteins by interference reflection microscopy (IRM) and the mechanics of detachment were examined by hydrodynamic shearing in a flow chamber. Cell contact with docosanol was compared with that on an adjacent area of octadecyl glass without a monolayer. Dictyostelium amoebae settled and spread on both docosanol and octadecyl glass, but little or no locomotion was seen on docosanol. On octadecyl glass the amoebae moved actively, forming ultrathin cytoplasmic lamellae, which look dark under IRM, and left distinctive trails of membranous debris. Hydrodynamic shearing showed that the amoebae stuck strongly to both surfaces and could not be removed from either at the maximum attainable wall shear stress of 6Nm-2. Red blood cells also adhered to both surfaces and removal from both occurred between 1 and 3Nm-2. IRM and scanning electron microscopy (SEM) studies indicated that this force leads to a minimal measure of red cell adhesion, since removal often involved the breakage of cytoplasmic tethers. Our results show that alcoholic -OH groups, in a two-dimensional array, provide a surface that is strongly adhesive for cells. No other method has made it possible to demonstrate cell adhesion purely to -OH groups, in a known orientation and density, and in the absence of any other functional groups on the interface.

13

Keating, Blane, Ian McPherson, Dimitrious Valavanis, Aaron-Jerome Agyei, and Patrick Unwin. "Seccm-IRM: A New Tool for Quantitative in Situ Studies of Crystal Growth." ECS Meeting Abstracts MA2022-01, no. 24 (July 7, 2022): 2498. http://dx.doi.org/10.1149/ma2022-01242498mtgabs.

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Scanning electrochemical cell microscopy (SECCM) is a versatile scanning probe imaging technique that allows for simultaneous elucidation of structure activity relationships at the nanoscale in defined electrolyte volumes and provides high resolution (nm length scale) information on the topography of surfaces and interfaces1. Since its inception in 2010 SECCM has improved understanding of model systems such as graphene, graphite, carbon nanotubes, nanoparticles and conductive diamond and provided electrochemists with a tool for true single entity measurements. Electrodeposition of microscale thin films has previously been reported2 but not of crystalline species, which is the focus of this work. SECCM can also be used as a tool for the precise delivery of nano to microscale solution droplets to surfaces. By using optically transparent yet conductive substrates (such as indium tin oxide (ITO), gold or graphene ) we can couple SECCM to highly surface-sensitive imaging modes such as interference reflectance microscopy (IRM)3 or traditional brightfield microscopy, allowing the evaporation and consequent precipitation of solutes to be monitored. The SECCM apparatus allows the deposition of large arrays, under well-defined conditions (deposition time, surface potential), with each droplet representing an individual and independent crystallisation experiment and provides statistical data based on single particle level. The emergence of precipitates within these arrays is tracked to investigate the effect of additives and also surface potential on crystallisation. SECCM can use capacitative response to examine minimally ionised systems, non-aqueous solvents etc allowing organic crystals to be studied. This work focuses on the precipitation of two such model organic systems: l-cystine and 5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile (ROY). l-Cystine is of biological relevance due to its role in the formation of kidney stone’s which are resistant to traditional therapies4. The need therefore arises for l-cystine crystallisation inhibitors making it a prime candidate for testing additive screening procedures. ROY is investigated as a model system5 to identify the merits of SECCM-IRM as a polymorph screen method. We show how sensitive precipitation is to substrate, applied potential and solvent system. References Ebejer, N.; Güell, A. G.; Lai, S. C. S.; McKelvey, K.; Snowden, M. E.; Unwin, P. R., Scanning Electrochemical Cell Microscopy: A Versatile Technique for Nanoscale Electrochemistry and Functional Imaging. Annual Review of Analytical Chemistry 2013, 6 (1), 329-351. Aaronson, B. D. B.; Garoz-Ruiz, J.; Byers, J. C.; Colina, A.; Unwin, P. R., Electrodeposition and Screening of Photoelectrochemical Activity in Conjugated Polymers Using Scanning Electrochemical Cell Microscopy. Langmuir 2015, 31 (46), 12814-12822. Valavanis, D.; Ciocci, P.; Gabriel; Morris, P.; Lemineur, J.-F.; McPherson, I. J.; Kanoufi, F.; Unwin, P. R., Hybrid scanning electrochemical cell microscopy-interference reflection microscopy (SECCM-IRM): tracking phase formation on surfaces in small volumes. Faraday Discussions 2022. Rimer, J. D.; An, Z.; Zhu, Z.; Lee, M. H.; Goldfarb, D. S.; Wesson, J. A.; Ward, M. D., Crystal Growth Inhibitors for the Prevention of l -Cystine Kidney Stones Through Molecular Design. Science 2010, 330 (6002), 337-341. Lévesque, A.; Maris, T.; Wuest, J. D., ROY Reclaims Its Crown: New Ways To Increase Polymorphic Diversity. Journal of the American Chemical Society 2020, 142 (27), 11873-11883.

14

Bailey, J., and D. Gingell. "Contacts of chick fibroblasts on glass: results and limitations of quantitative interferometry." Journal of Cell Science 90, no. 2 (June 1, 1988): 215–24. http://dx.doi.org/10.1242/jcs.90.2.215.

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We have examined the contacts made by explanted chick heart and limb bud fibroblasts after 24–48 h on glass, using quantitative interference reflection microscopy (IRM). Contacts beneath very thin cytoplasmic lamellae were avoided because the images of such contacts depend on the thickness of the lamellae. Plaque-like focal contacts, distinguished on the basis of shape and low irradiance (darkness), are intimate adhesions to the substratum. These images can be interpreted if it is assumed that microfilaments associated with the lower membrane increase the local cytoplasmic refractive index. The range of irradiances measured for focal contacts was found to be rather wide, and our modelling shows that the most likely explanation for this is that the images receive variable contributions from the adjacent cytoskeleton. For this reason it is particularly difficult to assign a characteristic thickness for these contacts from IRM data. Close contacts, seen principally as ‘grey’ regions under migrating cells at the edges of the explants, also show a wide range of irradiances. Unlike focal contacts, it is not necessary to postulate any involvement of the cytoskeleton in their images and they can be modelled as regions where an aqueous glycocalyx zone about 20–30 nm thick separates the membrane bilayer from the glass. Paler grey regions that also look like close contacts are apparently formed where the cell surface has lifted several tens of nanometres from the glass.

15

DePasquale, J. A., and C. S. Izzard. "Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact." Journal of Cell Biology 105, no. 6 (December 1, 1987): 2803–9. http://dx.doi.org/10.1083/jcb.105.6.2803.

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The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F-actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F-actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F-actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear.

16

Woods, A., and J. R. Couchman. "Protein kinase C involvement in focal adhesion formation." Journal of Cell Science 101, no. 2 (February 1, 1992): 277–90. http://dx.doi.org/10.1242/jcs.101.2.277.

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Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

17

Zheng, J., R. E. Buxbaum, and S. R. Heidemann. "Measurements of growth cone adhesion to culture surfaces by micromanipulation." Journal of Cell Biology 127, no. 6 (December 15, 1994): 2049–60. http://dx.doi.org/10.1083/jcb.127.6.2049.

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Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.

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Park, Jeesun, David R. Fooksman, Amitabha Mazumder, and Michael L. Dustin. "Multiple Myeloma Cells Adhere to Netrin-1 Via Heparin-Sulphate Moieties." Blood 120, no. 21 (November 16, 2012): 3952. http://dx.doi.org/10.1182/blood.v120.21.3952.3952.

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Abstract Abstract 3952 A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells. We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50μM), and VCAM-1(50μM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60–90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well. Disclosures: No relevant conflicts of interest to declare.

19

Ramsay, Alan G., Rachel Evans, Lena Svensson, Shahryar Kiaii, Nancy Hogg, and John G. Gribben. "Defective LFA-1 Mediated T Cell Motility In Chronic Lymphocytic Leukemia Is Mediated by Defects In the Rho GTPase Signaling Pathway." Blood 116, no. 21 (November 19, 2010): 914. http://dx.doi.org/10.1182/blood.v116.21.914.914.

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Abstract Abstract 914 T lymphocytes have an essential role in adaptive immunity and rely on tightly regulated signaling through integrin lymphocyte function-associated antigen (LFA)-1 to migrate into lymph nodes and interact with antigen-presenting cells. Malignant cells modify their immune microenvironment to prevent effective host anti-tumor responses, promote tumor progression, and suppress the therapeutic benefit of immunotherapy treatments. Here we assessed LFA-1-mediated cell migration of highly purified T cells from treatment naïve chronic lymphocytic leukemia (CLL) patients compared to age-matched healthy donor T cells using CXCL12 stimulation and immobilized ICAM-1, the principal integrin ligand. Video microscopy with motility tracking analysis identified that both CD4 and CD8 T cells from CLL patients (n=14) exhibited significantly reduced migration rates (P < .01) compared to healthy donor T cells (5.5 ± 0.3 (SEM) μm/min and 4.4 ± 0.2 μm/min compared to 8.2 ± 0.3 μm/min and 7.5 ± 0.3 μm/min respectively). We further identified that direct CLL cell contact, and not soluble factors alone, induced similar T cell motility dysfunction in previously healthy CD3 T cells. Primary co-culture of healthy donor T cells with CLL cells caused a significant decrease in the speed of migration on ICAM-1 compared to coculture with control healthy B cells (6.2 ± 0.3 μm/min versus 9.5 ± 0.6 μm/min) (n=9) (P < .05). Next we sought to repair this T cell defect in CLL using a clinically relevant agent. We identify that treatment of CLL patient T cells (n=9) with lenalidomide restores rapid LFA-1 mediated migration on ICAM-1. Ex vivo treatment of CLL T cells with lenalidomide (1μM for 24 hours) significantly increased the speed of T cell migration compared to untreated patient T cells (7 ± 0.4 μm/min versus 2.5 ± 0.7 μm/min) (P < .05) and the rescued T cell migratory function of lenalidomide exposed patient T cells was comparable to healthy donor T cells treated with or without drug. Interference reflection microscopy (IRM) examining the contact zone between migrating T cells and ICAM-1 identified a significant CLL patient T cell adhesion defect (P < .05) with reduced spreading area and strength of adhesive contacts (pixel density) compared to healthy donor T cells. IRM was further utilized with pharmacological inhibitors to demonstrate that exposure to lenalidomide rescued CLL T cell adhesion by acting on the Rho family GTPases that are dysregulated in cancer patient T cells. Lenalidomide significantly increased (P < .05) levels of active RhoA in CLL patient T cells compared to untreated cells. In addition, untreated CLL patient T cells adhering to ICAM-1 exhibited significantly reduced expression levels of phosphorylated myosin light chain (MLC) compared to healthy donor T cells (P < .05) and this defect was repaired following lenalidomide treatment. MLC is normally phosphorylated by MLC kinase at the T cell leading edge and by the RhoA target, ROCK at the trailing edge, and is an important downstream signaling molecule during LFA-1-mediated T cell motility. Further expression analysis identified that lenalidomide significantly increased (P < .01) ICAM-1-engaged high-affinity LFA-1 in CLL patient T cells to levels comparable to healthy donor T cells. Overall, our results show that T cells in CLL patients have dysfunctional tumor-induced cytoskeletal signaling via the Rho GTPase signaling pathway, and this is reversed by lenalidomide, rescuing dynamic LFA-1 mediated outside-in signalling and migration. Lenalidomide's immunomodulatory activity was highly cancer T cell specific: rescuing defective LFA-1 migration and signaling in CLL T cells, but with no detectable effects on healthy donor T cells. These findings provide important mechanistic insight into the action of lenalidomide, and highlight the potential clinical utility of immunomodulatory drugs to rescue normal immune function in cancer. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

20

Kojima, Seiji. "Interference Fringes in Reflection Acoustic Microscopy." Japanese Journal of Applied Physics 26, S1 (January 1, 1987): 233. http://dx.doi.org/10.7567/jjaps.26s1.233.

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21

Velas, Lukas, Philipp Zelger, Alexander Jesacher, and Gerhard J. Schütz. "Correlating Interference Reflection Microscopy with 3D Superresolution Fluorescence Microscopy." Biophysical Journal 120, no. 3 (February 2021): 180a. http://dx.doi.org/10.1016/j.bpj.2020.11.1254.

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22

Kim, Kipom, and Omar A. Saleh. "Stabilizing method for reflection interference contrast microscopy." Applied Optics 47, no. 12 (April 16, 2008): 2070. http://dx.doi.org/10.1364/ao.47.002070.

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23

Gingell, D., O. S. Heavens, and J. S. Mellor. "General electromagnetic theory of total internal reflection fluorescence: the quantitative basis for mapping cell-substratum topography." Journal of Cell Science 87, no. 5 (June 1, 1987): 677–93. http://dx.doi.org/10.1242/jcs.87.5.677.

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Total internal reflection fluorescence (TIRF) has recently been used to look at the contacts made between cells and a glass surface on which they are spread. Our method utilizes the fluorescence of a water-soluble dye that acts as an extracellular aqueous volume marker. Fluorescence is stimulated by the short-range electric field near the glass surface that exists under conditions of total internal reflection. Since fluorescence is normally generated beneath a spread cell and not beyond it, the fluorescence of the image is related to the size of the cell-glass water gap. The images obtained are remarkable for their detail, contrast and the absence of confusing granularity due to cytoplasmic heterogeneity, which is commonly seen in interference reflection (IRM) images. We here develop a rigorous electromagnetic theory of total internal reflection in layered structures appropriate for cell contacts and apply it to quantitative TIRF. We show that: (1) TIRF, unlike IRM, can report cell-glass gaps in a way that is practically independent of the detailed physical properties of the cell; (2) TIRF is also far more sensitive than IRM for measuring cell-glass water gaps up to approximately equal to 100nm. These striking results explain the image quality seen by TIRF. As the initial step towards verifying our theory we show that measurement of the fluorescence stimulated by total internal reflection at a simple glass-water interface matches theoretical predictions.

24

Chmelik, Christian, and Jörg Kärger. "Unprecedented Wealth of Information on Guest Dynamics in Nanoporous Materials from Transient Concentration Profiles." Defect and Diffusion Forum 309-310 (March 2011): 177–94. http://dx.doi.org/10.4028/www.scientific.net/ddf.309-310.177.

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The application of interference microscopy (IFM) and infrared microscopy (IRM) to monitor the evolution of the concentration of guest molecules in nanoporous host materials opens a new field of diffusion research in condensed matter. It combines the methodical virtues of the profiling methods of solid-state diffusion studies with the benefit of the mobility enhancement in fluids. We are going to illustrate the rich options of diffusion studies provided by this novel experimental approach.

25

Contreras-Naranjo, Jose C., James A. Silas, and Victor M. Ugaz. "Reflection interference contrast microscopy of arbitrary convex surfaces." Applied Optics 49, no. 19 (June 23, 2010): 3701. http://dx.doi.org/10.1364/ao.49.003701.

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26

YOSHIKAWA, Hiroshi Y., and Takahisa MATSUZAKI. "Reflection Interference Microscopy~Noninvasive Nano-Imaging of Soft Interfaces~." Seibutsu Butsuri 57, no. 6 (2017): 318–22. http://dx.doi.org/10.2142/biophys.57.318.

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27

Hillner, P. E., M. Radmacher, and P. K. Hansma. "Combined atomic force and scanning reflection interference contrast microscopy." Scanning 17, no. 3 (December 7, 2006): 144–47. http://dx.doi.org/10.1002/sca.4950170304.

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28

Pahl, Tobias, Johannes Breidenbach, and Peter Lehmann. "Quasi-analytical and rigorous modeling of interference microscopy." EPJ Web of Conferences 266 (2022): 10013. http://dx.doi.org/10.1051/epjconf/202226610013.

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We present an extended vectorial Kirchhoff model of coherence scanning interferometry including several vector rotations occurring in the imagining and scattering process as well as polarization dependent reflection coefficients. For validation simulated results are compared to those of the conventional scalar Kirchhoff model and a rigorous finite element modeling.

29

ICHIKAWA, M., S. MARUNO, S. FUJITA, H. WATANABE, and Y. KUSUMI. "MICROPROBE RHEED/STM COMBINED MICROSCOPY." Surface Review and Letters 04, no. 03 (June 1997): 535–42. http://dx.doi.org/10.1142/s0218625x97000511.

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We have developed microprobe reflection high energy electron diffraction combined with scanning tunneling microscope and molecular beam epitaxy equipment. This combination makes it possible to study and control surface processes in the magnification range from several hundred micrometers to the atomic scale. An electron biprism is also attached to the incident electron beam path, which produces a new kind of scanning electron microscopy called scanning interference electron microscopy. The two coherently divided electron beams created by the biprism produce electron interference fringes. The electron interference fringes are used to form ultrafine periodic structures by electron-stimulated surface reaction and to characterize electromagnetic properties of the surfaces. The formation of periodic carbon grid lines produced by the interference fringes on a GaAs surface and the study of Ge thin film growth on a partially Ga adsorbed Si (111) surface are described for application examples of the microscopy.

30

Saper, Gadiel, and Henry Hess. "Kinesin-propelled label-free microtubules imaged with interference reflection microscopy." New Journal of Physics 22, no. 9 (September 17, 2020): 095002. http://dx.doi.org/10.1088/1367-2630/abb47b.

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31

Monzel, Cornelia, Susanne F. Fenz, Rudolf Merkel, and Kheya Sengupta. "Probing Biomembrane Dynamics by Dual-Wavelength Reflection Interference Contrast Microscopy." ChemPhysChem 10, no. 16 (November 3, 2009): 2828–38. http://dx.doi.org/10.1002/cphc.200900645.

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32

Keith, C. H., and R. Witek. "Enhancement of Reflection-Enhanced Backscatter Confocal Microscopy." Microscopy and Microanalysis 7, S2 (August 2001): 1014–15. http://dx.doi.org/10.1017/s1431927600031147.

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Tremendous advances have been made in the last twenty years in extending the usefulness of the light microscope in visualizing living biological specimens, and it is now possible to visualize most thin tissues in an unstained state at – or near – the limit of optical resolution. in general, however, these techniques have been used successfully on biological specimens in the transmitted light mode; unstained biological specimens generally do not have sufficient albedo or sufficient difference in refractive index from their surroundings to be efficiently visualized in epi-illumination. A number of applications, including studies of the growth of bacteria on metallic substrates, and studies of the studies of the formation of interfaces between living neurons and silicon chips, would benefit from being able to resolve living, unstained biological specimens growing on opaque substratesWe found that by imaging biological specimens on a reflective substrate in the reflection mode of the confocal microscope, we could get images that resemble transmission mode differential interference contrast images.

33

Dubois, Arnaud. "Effects of phase change on reflection in phase-measuring interference microscopy." Applied Optics 43, no. 7 (March 1, 2004): 1503. http://dx.doi.org/10.1364/ao.43.001503.

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34

Stott, D., and C. C. Wylie. "Invasive behaviour of mouse primordial germ cells in vitro." Journal of Cell Science 86, no. 1 (December 1, 1986): 133–44. http://dx.doi.org/10.1242/jcs.86.1.133.

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We have isolated migrating primordial germ cells (PGCs) from 10.5-day mouse embryos and studied their behaviour when cultured on a mouse embryo fibroblast (STO) cell line. Living and fixed PGCs were identified by fluorescent labelling with a monoclonal antibody specific for PGCs in the culture system used. The behaviour of the cells was studied using interference reflexion microscopy (IRM) and time-lapse video cinematography. The IRM pattern displayed by PGCs is typical of highly motile cell types, the cells lack focal contacts and possess large areas of close contacts indicative of weak membrane to substrate interaction. The PGCs exhibit relatively high rates of translocation and lack contact inhibition. They were observed to underlap STO cells in subconfluent monolayers and to penetrate between the cells of confluent monolayers, becoming located between the monolayer and its substrate. These observations support the hypothesis that migrating mouse PGCs are inherently motile and are able transiently to disrupt the adhesion of surrounding cells. These results suggest that PGCs actively migrate to the developing gonad in vivo.

35

Weber, Igor, and Richard Albrecht. "Image processing for combined bright-field and reflection interference contrast video microscopy." Computer Methods and Programs in Biomedicine 53, no. 2 (June 1997): 113–18. http://dx.doi.org/10.1016/s0169-2607(97)01810-5.

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36

CHIU, L. ‐D, L. SU, S. REICHELT, and W. B. AMOS. "Use of a white light supercontinuum laser for confocal interference‐reflection microscopy." Journal of Microscopy 246, no. 2 (March 20, 2012): 153–59. http://dx.doi.org/10.1111/j.1365-2818.2012.03603.x.

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37

HOLT, M. R., Y. CALLE, D. H. SUTTON, D. R. CRITCHLEY, G. E. JONES, and G. A. DUNN. "Quantifying cell-matrix adhesion dynamics in living cells using interference reflection microscopy." Journal of Microscopy 232, no. 1 (October 2008): 73–81. http://dx.doi.org/10.1111/j.1365-2818.2008.02069.x.

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38

Limozin, Laurent, and Kheya Sengupta. "Quantitative Reflection Interference Contrast Microscopy (RICM) in Soft Matter and Cell Adhesion." ChemPhysChem 10, no. 16 (November 3, 2009): 2752–68. http://dx.doi.org/10.1002/cphc.200900601.

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39

Huerre, A., M. C. Jullien, O. Theodoly, and M. P. Valignat. "Absolute 3D reconstruction of thin films topography in microfluidic channels by interference reflection microscopy." Lab on a Chip 16, no. 5 (2016): 911–16. http://dx.doi.org/10.1039/c5lc01417d.

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The topography of thin films in microfluidic channels can be reconstructed at the nanometric scale from interference microscopy imaging by modelling the multiple reflections at the upper and the lower surfaces of the microchannel.

40

Bohannon, Kevin P., Ronald W. Holz, and Daniel Axelrod. "Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy." Microscopy and Microanalysis 23, no. 5 (September 18, 2017): 978–88. http://dx.doi.org/10.1017/s1431927617012570.

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AbstractThe refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

41

Kärger, Jörg, and Rustem Valiullin. "Transport-Optimized Nanoporous Materials for Mass Separation and Conversion as Designed by Microscopic Diffusion Measurement." Diffusion Foundations 19 (November 2018): 96–124. http://dx.doi.org/10.4028/www.scientific.net/df.19.96.

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Nanoporous materials find widespread application in material upgrading by separation (“molecular sieving”) and catalytic conversion. Mass transfer in these materials is a key phenomenon deciding about their technological performance. This chapter deals with the application of measurement techniques which are able to follow the diffusive fluxes of the guest molecules in such materials over “microscopic” distances, including the pulsed field gradient (PFG) technique of Nuclear Magnetic Resonance (NMR) and the techniques of microimaging by interference microscopy (IFM) and by IR microscopy (IRM). Microscopic measurement is a prerequisite for attaining unbiased information about the elementary steps of mass transfer and about their role within the overall process of technological exploitation. We dedicate this treatise to the memory of our dear and highly esteemed colleague Nicolaas Augustinus Stolwijk, notably in recognition of his manifold activities in the field of diffusion, distinguished by their impressively high standard in connecting the message of various techniques of measurement and in combining them to comprehensive views on quite intricate subjects.

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Dupuis, Julia Rentz, James Needham, Emre Özkumur, David A. Bergstein, Bennett B. Goldberg, James R. Engel, David L. Carlson, and M. Selim Ünlü. "Hyperspectral Fourier transform spectrometer for reflection spectroscopy and spectral self-interference fluorescence microscopy." Applied Optics 47, no. 9 (March 17, 2008): 1223. http://dx.doi.org/10.1364/ao.47.001223.

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43

Fang, Ning, Vincent Chan, Kai-Tak Wan, Hai-Quan Mao, and Kam W. Leong. "Colloidal adhesion of phospholipid vesicles: high-resolution reflection interference contrast microscopy and theory." Colloids and Surfaces B: Biointerfaces 25, no. 4 (August 2002): 347–62. http://dx.doi.org/10.1016/s0927-7765(01)00336-8.

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44

Opas, Michał, and Vitauts I. Kalnins. "Multiple Labeling of Cellular Constituents by Combining Surface Reflection Interference and Fluorescence Microscopy." Pathobiology 53, no. 5 (1985): 241–51. http://dx.doi.org/10.1159/000163318.

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45

Terborg, Roland A., Josselin Pello, Ilaria Mannelli, Juan P. Torres, and Valerio Pruneri. "Ultrasensitive interferometric on-chip microscopy of transparent objects." Science Advances 2, no. 6 (June 2016): e1600077. http://dx.doi.org/10.1126/sciadv.1600077.

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Light microscopes can detect objects through several physical processes, such as scattering, absorption, and reflection. In transparent objects, these mechanisms are often too weak, and interference effects are more suitable to observe the tiny refractive index variations that produce phase shifts. We propose an on-chip microscope design that exploits birefringence in an unconventional geometry. It makes use of two sheared and quasi-overlapped illuminating beams experiencing relative phase shifts when going through the object, and a complementary metal-oxide-semiconductor image sensor array to record the resulting interference pattern. Unlike conventional microscopes, the beams are unfocused, leading to a very large field of view (20 mm2) and detection volume (more than 0.5 cm3), at the expense of lateral resolution. The high axial sensitivity (<1 nm) achieved using a novel phase-shifting interferometric operation makes the proposed device ideal for examining transparent substrates and reading microarrays of biomarkers. This is demonstrated by detecting nanometer-thick surface modulations on glass and single and double protein layers.

46

Joshi, N. V., and H. Medina. "Multiple Beam Interference Confocal Microscopy: Tool for Morphological Investigation of a Living Spermatozoon." Microscopy and Microanalysis 6, no. 5 (September 2000): 471–77. http://dx.doi.org/10.1007/s100059910020.

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AbstractThe interference pattern obtained from a multiple internal reflection of a spermatozoon, sandwiched between the glass plate and the cover plate, was focused on the objective of a scanning confocal microscope. According to optical path differences, morphological details were revealed. The combined features, namely improved resolution in z axis, originating from the interference pattern and the optical sectioning of the confocal scanning system, enhanced the resolution and contrast in an impressive manner. These features permitted unprecedented images of the spermatozoon to be obtained at 0.1 μm optical sectioning and reconstruction of three-dimensional (3-D) images. With the help of optical sectioning, it was possible to estimate the thickness and the morphological details of the spermatozoon.

47

Streeter, H. B., and D. A. Rees. "Fibroblast adhesion to RGDS shows novel features compared with fibronectin." Journal of Cell Biology 105, no. 1 (July 1, 1987): 507–15. http://dx.doi.org/10.1083/jcb.105.1.507.

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As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.

48

Stuart, J. K., and V. Hlady. "Reflection Interference Contrast Microscopy Combined with Scanning Force Microscopy Verifies the Nature of Protein-Ligand Interaction Force Measurements." Biophysical Journal 76, no. 1 (January 1999): 500–508. http://dx.doi.org/10.1016/s0006-3495(99)77218-8.

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49

Silk, Ely. "Reflected/Transmitted Nomarsky DIC Lighting." Microscopy Today 6, no. 4 (May 1998): 8–10. http://dx.doi.org/10.1017/s1551929500067195.

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What could be better than reflected or transmitted Nomarski differential interference contrast? Why, combining the best features of both and for very little cost. My intended use of the technique was for Nomarski reflection DIC microscopy. It will work, of course, with other types of reflection microscopy.What this embarrassingly simple artifice accomplishes is to simultaneously add transmission capabilities to reflection observations with the result being improved viewing of delicate details, And, yes, because of the reflective front surface layer, the observer can study details on the bottom side of the specimen which is usually hidden from view. This requires focusing through the specimen and below the point of normal focus.

50

MAHAMDEH, MOHAMMED, STEVE SIMMERT, ANNA LUCHNIAK, ERIK SCHÄFFER, and JONATHON HOWARD. "Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy." Journal of Microscopy 272, no. 1 (July 25, 2018): 60–66. http://dx.doi.org/10.1111/jmi.12744.

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