News Column

"Methods and Apparatus for Imaging Molecules in Living Systems" in Patent Application Approval Process

June 17, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Grunwald, David (Worcester, MA); Singer, Robert H. (New York, NY), filed on September 13, 2011, was made available online on June 5, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Albert Einstein College of Medicine of Yeshiva University).

This patent application is assigned to Albert Einstein College of Medicine of Yeshiva University.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

"The present invention addresses the need of imaging highly transient molecular interactions in living cells, which can occur over distances smaller than the optical resolution of conventional light microscopes. In addition, the classical use of co-localization in fluorescence microscopy suffers from possible misinterpretations concerning the actual proximity of interrogated components due to intrinsic errors in registration. The present invention allows investigations of molecular interactions in living cells at high resolution, low light levels and high acquisition speeds."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "The present invention provides methods for imaging molecules, where the methods comprise providing a multi channel marker that can be detected by multiple detection areas; labeling one or more types of molecules with a fluorescent marker, wherein different types of molecules are labeled with spectrally distinguishable fluorescent markers; spatially registering the multiple detection areas; recording a registration signal from the multi channel marker on the multiple detection areas; imaging the labeled molecules; evaluating the registration signal to obtain a transformation matrix for each pair of detection areas; and applying the transformation matrix to imaging data recorded on multiple detection areas to thereby image the molecules.

"The invention also provides virtual fiducial markers for imaging comprising either a non-transparent mask containing one or more openings through which light can pass or a mask that is partially transparent and can generate a virtual signal suitable for sub-diffraction registration of multiple detection areas, wherein the mask is held in a translation stage that allows movement of the mask in x and y directions or an optical installation is used to move an image of the mask if it is not mounted in a stage; a first lens system on one side of the mask to deliver light onto the mask; and a second lens system on the opposite side of the mask from the first lens system to project an image of the mask into a sample to be imaged, thereby acting as a virtual fiducial marker.


"FIG. 1A-1O. Super-Registration Precision and Detection of Nuclear mRNA. (A-G) The registration precision achieved in this experiment was based on imaging nuclear pores on two cameras immediately before data acquisition (SI). Data from both cameras (A) red, (B) green, merged image (C) after registration. A filtered merged image (D) with 21 nuclear pores, white circles outlined (E). (F) Coregistration precision between the best aligned 6 (black bars and black line in inset), 10 (light grey) and 15 (dark grey) nuclear pores. Fit (inset), Gaussian fit to the '15 pore' data set: registration=10.+-.1 nm, 13.+-.1 nm FWHM. (G) Distances between pores in (E). Peak=7.5 nm. (H) mRNAs interacted with nuclear pores infrequently and not all interactions resulted in export of mRNAs from the nucleus. (I) Full length traces (H); first (dark box), last (light box). (J) Intensity trace (grey), tracked mRNA, background (black). (K) slow export images. (L) fast export. (M) Distances between mRNA and pore from (L) colocalization precision, 26 nm total (SI). Nucleoplasmic mRNA (+) cytoplasmic mRNA (-). (N) Intensity mRNA signal (grey) vs. background (black). (O) mRNA positions (gray boxes) and pores (circle) overlaid on nuclear pore from (L). Bars=2 .mu.m, 'n/c'=nucleus/cytoplasm, 'max'=maximum intensity projection, (I) & (O) axis pixels (=64 nm). (H-O) LoG filtered (ImageJ, D. Sage).

"FIG. 2A-2B. Dwell times of .beta.-actin mRNA at the NPC. mRNA co-localized with NPCs, no. frames as milliseconds. Histogram=observed mRNAs per time bin of 20 ms. (A) Fit of dwell time of cumulative trace length distribution [23] (black circles). First bin =total number of observed traces. Fast transport events (B) Data from (A) (black circles) replotted as trace duration histogram (black bars). Cut-off (adjacent averaging width=5 bins). Inset=unprocessed raw data. Two-step convolution model (black line) reveals two kinetic rates [24], dwell times ms and k.sub.slow=139.+-.10 ms. Identifying export=two observations=40 ms. Result consistent with multistep process containing at least two rate constants, total time=180 ms.

"FIG. 3A-3C. 'Binding Sites' of mRNAs at Nuclear Pores. Distances between mRNA and POM121-tdT (zero position) bin widths=25 nm. (-)=cytoplasmic C, (+)=nucleoplasmic position N. Red lines are global fits, dark grey line is fit to cytoplasmic binding distribution, light grey line is fit to nucleoplasmic binding distribution. (A) Histogram of all observed transport events at NPCs (B+C). (B) Histogram for fast transported mRNAs (90% translocation). (C) Histogram for slow mRNAs, observed for extended times at NPC.

"FIG. 4A-4B. NPC Topography of mRNA Export. Results from FIGS. 3B and 3C (hatched & open bars) plotted (A) to scale with known NPC dimensions (B) [3]. mRNA export timescale (black=k.sub.slow; along NPC axis combined with single molecule data (grey bars) of Nup358 [23], import factors [25] and import release site [26]. Nuclear peak position of slow transporting mRNAs located between binding sites for import factors and import release site. Length of grey bars =FWHM of binding site distributions.

"FIG. 5A-5F. Experimental Setup for Export Time. (A) A genetically altered mouse was derived whereby endogenous .beta.-actin mRNA was labeled using the 24.times.MS2 stem loop cassette inserted into the 3' UTR of the .beta.-actin gene by homologous recombination in ES cells. MS2 coat proteins (MCP), fused to YFP, bind the RNA stem loops as dimers (inset) further multiplexing the label. (B) NPCs were labeled with POM121-tdTomato using viral infection of immortalized fibroblasts from the .beta.-actin-24 MBS mouse. (C) Optical Setup. Light from a 514.5 nm and a 561 nm laser was delivered by a single mode fiber F and imaged to the specimen plane S by an objective O. An iris I is used to adjust excitation for the field of view. Two dichroic mirrors are used to separate excitation and emission signals DC and split red and green signals DC-1 towards two cameras CCD 1 and CCD2. A mirror M is used to reflect the light out of the microscope stand. Notch filters N are used to block scattered light from the lasers. A minimum number of lenses L is used to optimize detection efficiency by reducing the amount of surfaces in the light path. 'Super-registration' is achieved for each individual data set by post experimental determination of transition matrices between both channels based on nuclear pore signals imaged onto both cameras immediately prior to tracking data acquisition. Dichroic 1 (a z543rdc from Chroma) has a broadband anti-reflective coating. However, it is possible to image front- and back-surface reflections of that mirror on the highly sensitive cameras. (D) A laser beam (658 nm) was placed directly along the optical axis of the microscope and passed through DC-1. Low amounts of light are reflected onto CCD-1 (green channel). (E, F) Using excitation with only 561 nm light the same effect can be produced for nuclear pores labeled by POM121-tdTomato. These signals are used to 'super-register' the two CCD cameras.

"FIG. 6. Example of setup to achieve super registration using a virtual fiducial marker. The key piece of the design is a Mask that has one or multiple openings through which light can travel. This way it can be used for either negative or positive contrast. This mask can either be transluminant or non-transluminate with or without additional structures being added to shape the intensity profile of the mask in the sample. The mask can also be a micro mirror array. This mask can be held in a translation stage that allows the mask to move in x and y directions with a step width small enough to allow sub-diffraction displacements of the image of the mask in the sample. Alternatively, an image of the mask can be moved by optical means to achieve displacement in the sample. An excitation source (Exc.) provides light. The light from that source is delivered by a first lens system (LS1) onto the mask. An image of the mask is projected into the sample acting as a virtual fiducial marker (VFM) by lens system 2 (LS2).

"FIG. 7A-7F. Chromatic corrected Super-registration Approach. Using a dye that emits with a long tail up to the .about.700 nm range a cellular structure (here DNA) was stained. The dye is excitable at 405 nm. A) Emission of the dye in the green channel (527 to 555 nm detection with emission band pass). B) Emission of the dye in the red channel (570 to 620 nm detection with emission band pass). C) Overlay of A) & B) after preforming super-registration. The registration matrix was applied to register the images in D) & E). D) mRNA signals in the center plane of a mammalian cell nucleus, the green signal is coming from a YFP-MS2 tag on the mRNA. E) Nuclear Pores in the same image plane super-registered onto the mRNA signal. D) and E) are showing that the dye is not excited by 515 or 561 nm excitation and does not contribute background in the corresponding channels if not specifically excited. F) Overlay of D) and E) showing a few mRNAs located to nuclear pores, while the majority is roaming the nuclear volume."

URL and more information on this patent application, see: Grunwald, David; Singer, Robert H. Methods and Apparatus for Imaging Molecules in Living Systems. Filed September 13, 2011 and posted June 5, 2014. Patent URL:

Keywords for this news article include: Cytoplasm, Cell Nucleus, Nuclear Pore, Nuclear Envelope, Cellular Structures, Intracellular Space, Albert Einstein College of Medicine of Yeshiva University.

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Source: Life Science Weekly