Search PubMed⌕ Search

Biomedical subjects

B Christoffer Lagerholm

Publications and source records attributed to B Christoffer Lagerholm.

7 recordsLinked to original sources
PubMed2006

Analysis method for measuring submicroscopic distances with blinking quantum dots.

A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment.

Biotinylation↗
PubMed2006

Methods to measure the lateral diffusion of membrane lipids and proteins.

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.

Biochemistry↗
PubMed2005

Detecting microdomains in intact cell membranes.

Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

Biomarkers↗
PubMed2005

Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays.

Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating.

Acrylic Resins↗
PubMed2003

Internet-based image analysis quantifies contractile behavior of individual fibroblasts inside model tissue.

In a cell-populated collagen gel, intrinsic fiber structure visible in differential interference contrast images can provide markers for an in situ strain gauge to quantify cell-gel mechanics, while optical sections of fluorescent protein distribution capture cytoskeletal kinematics. Mechanics quantification can be derived automatically from timelapse differential interference contrast images using a Deformation Quantification and Analysis software package accessible online at http://dqa.web.cmu.edu. In our studies, fibroblast contractile machinery was observed to function entirely within pseudopods, while GFP-alpha-actinin concentrated in pseudopod tips and cortex. Complex strain patterns around individual cells showed instances of both elastic and inelastic strain transmission, suggesting a role in observed long-range alignment of cells.

3T3 Cells↗
PubMedDate not supplied

Noninvasive imaging of quantum dots in mice.

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.

Animals↗