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    Orange and Blue Doodle Science Project Presentation

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    Noralyn Gunnawa
    This document reviews advances made in single-molecule imaging techniques for studying bacteria over the past 10 years. It discusses how labeling and tracking single proteins in live bacterial cells using fluorescent probes has allowed researchers to image subcellular processes. Combining high-precision single-molecule microscopy with well-studied bacterial model systems like E. coli could provide new insights into fundamental biological questions. While progress has been made, challenges remain such as developing methods applicable to non-model bacteria and correlating multiple targets simultaneously.

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    Orange and Blue Doodle Science Project Presentation

    Uploaded by

    Noralyn Gunnawa
    29 views14 pages
    This document reviews advances made in single-molecule imaging techniques for studying bacteria over the past 10 years. It discusses how labeling and tracking single proteins in live bacterial cells using fluorescent probes has allowed researchers to image subcellular processes. Combining high-precision single-molecule microscopy with well-studied bacterial model systems like E. coli could provide new insights into fundamental biological questions. While progress has been made, challenges remain such as developing methods applicable to non-model bacteria and correlating multiple targets simultaneously.

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    This document reviews advances made in single-molecule imaging techniques for studying bacteria over the past 10 years. It discusses how labeling and tracking single proteins in live bacterial cells using fluorescent probes has allowed researchers to image subcellular processes. Combining high-precision single-molecule microscopy with well-studied bacterial model systems like E. coli could provide new insights into fundamental biological questions. While progress has been made, challenges remain such as developing methods applicable to non-model bacteria and correlating multiple targets simultaneously.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    29 views14 pages

    Orange and Blue Doodle Science Project Presentation

    Uploaded by

    Noralyn Gunnawa
    This document reviews advances made in single-molecule imaging techniques for studying bacteria over the past 10 years. It discusses how labeling and tracking single proteins in live bacterial cells using fluorescent probes has allowed researchers to image subcellular processes. Combining high-precision single-molecule microscopy with well-studied bacterial model systems like E. coli could provide new insights into fundamental biological questions. While progress has been made, challenges remain such as developing methods applicable to non-model bacteria and correlating multiple targets simultaneously.

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    FROM SINGLE

    BACTERIAL CELL
    IMAGING TOWARDS IN
    VIVO

    introduction
    Bacteria, such as Escherichia coli and Caulobacter crescentus, are
    the most studied and perhaps best-understood organisms in biology.
    The advances in understanding of living systems gained from these
    organisms are immense. Application of single-molecule techniques in
    bacteria have presented unique difficulties owing to their small size
    and highly curved form.
    The aim of this review is to show advances made in single-
    molecule imaging in bacteria over the past 10 years, and to look to the
    future where the combination of implementing such high-precision
    techniques in well-characterized and controllable model systems such
    as E. coli could lead to a greater understanding of fundamental
    biological questions inaccessible through classic ensemble methods.
    01
    BASIC
    CONCEPTS
    DEFINITIONS
    MICROBIOLOGY
    Microbiology is the scientific study of microorganisms,
    those being unicellular, multicellular, or acellular.
    Microbiology encompasses numerous sub-disciplines
    including virology, bacteriology, protistology, mycology,
    immunology, and parasitology.
    DEFINITIONS
    BACTERIA
    are single-celled organisms with a unique
    internal structure. Humans and other
    multicellular organisms are eukaryotes, which
    means our cells have distinct nuclei bound
    with a membrane. Bacteria are prokaryotes,
    meaning they don't have organized nuclei or
    any other membrane-bound organelles.
    01 THE BEGINNINGS OF IN VIVO FLOURESCENCE
    IMAGING IN MICROBIOLOGY

    In microbiology, in vivo fluorescence imaging is an imaging


    technique that uses fluorescence to image cells within live
    organisms. This type of microscopy provides highly accurate
    results and allows for the observation of the development of
    certain processes.
    When it comes to laboratory strain sizes in
    microbiology, they are typically on par with
    the resolution limit of optical microscopy
    between 200 and 300 nm.s. Due to the
    frequently extreme growth conditions of
    archaeal organisms, which can include
    extremely high or low temperature, high
    salinity, acidic, alkaline, or anaerobic
    environments, as well as the ability of
    colorful pigments to cause autofluorescence,
    standard fluorescence microscopy is still a
    rarely used tool for archaeal studies.
    02 SUBCELLULAR IMAGING IN
    BACTERIAL CELLS
    Tracking single copies of allowed for the first
    identification of a single FP-labeled protein in live
    bacteria a membrane protein because protein diffusion
    in the membrane moves much more slowly than it does
    in the cytosol, which makes things simpler. This simple
    genetic tagging method is obviously not directly
    transferable to other cellular components like
    nucleotides, lipids, or carbohydrates. Furthermore, for
    the majority of these subcellular measurements,
    proteins of interests were genetically tagged by FPs,
    using the bright GFP-variant eYFP.
    MAINTAINING AND
    MANIPULATING BACTERIAL
    CELL PHYSIOLOGY ON THE
    MICROSCOPE STAGE
    The single-molecule microscopy in bacterial cells is
    utilizing unique photophysical characteristics of
    fluorescent molecules, single molecule localization
    microscopy overcomes this diffraction limit (or
    fluorophores). These can be seen because they are linked
    to proteins of interest that are present inside of cells.
    This is accomplished through fluorescence blinking for
    single-molecule super-resolution microscopy techniques.
    The density of fluorescent spots in a single image can
    therefore be kept at a low, single-spot level by
    controlling the number of fluorescent emitters that are
    simultaneously active.and by capturing an imaging
    sequence, the single-molecule signals can be read out by
    sensitive detectors over time.
    OPEN CHALLENGES IN SINGLE-
    05 MOLECULE MICROBIOLOGY
    the open challenges in single-molecule microbiology is we
    have outlined some of the key issues we think will soon be
    present in the field of these tiny molecular machines, but
    there is still much to learn about them. Combining the two
    essential single-molecule strategies of fluorescence and
    optical trapping can solve many of these problems. The
    limited number of compatible fluorophores and labeling
    options that satisfy the stringent requirements for high-
    quality single-molecule microscopy, yielding satisfactory
    labeling specificity and efficiency, and being read-out at low
    channel background/cross-talk and at highest
    spatiotemporal resolution, currently dictate the majority of
    experimental designs that are feasible. Also, new imaging
    techniques promise drastic improvements in temporal
    resolution and long-term imaging without increased
    phototoxicity.
    CONCLUSION
    Significant progress has been made in understanding the
    biological processes at work within individual bacterial
    cells as well as their functions within the complex
    environments of living cultures. Promising developments
    included enhanced fluorescent probes and labels, cutting-
    edge imaging methods like single-molecule microscopy,
    and microfluidic systems to preserve bacterial cell
    physiology. While some issues remain open, such as
    adapting single-molecule imaging for non-model systems
    or creating suitable multi-target and correlative
    strategies.
    THANK YOU!
    Any questions? Don't hesitate to
    ask for our help

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