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.
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