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org/journal/acsodf Review

Tools of the Trade: Image Analysis Programs for Confocal Laser-

Scanning Microscopy Studies of Biofilms and Considerations for
Their Use by Experimental Researchers
Shreeya Mhade and Karishma S Kaushik*

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ABSTRACT: Confocal laser-scanning microscopy (CLSM) is the

bedrock of the microscopic visualization of biofilms. Previous
applications of CLSM in biofilm studies have largely focused on
observations of bacterial or fungal elements of biofilms, often seen
as aggregates or mats of cells. However, the field of biofilm
research is moving beyond qualitative observations alone, toward
the quantitative analysis of the structural and functional features of
biofilms, across clinical, environmental, and laboratory conditions.
In recent times, several image analysis programs have been
developed to extract and quantify biofilm properties from confocal
micrographs. These tools not only vary in their scope and
relevance to the specific biofilm features under study but also with
respect to the user interface, compatibility with operating systems,
and raw image requirements. Understanding these considerations is important when selecting tools for quantitative biofilm analysis,
including at the initial experimental stages of image acquisition. In this review, we provide an overview of image analysis programs for
confocal micrographs of biofilms, with a focus on tool selection and image acquisition parameters that are relevant for experimental
researchers to ensure reliability and compatibility with downstream image processing.

■ INTRODUCTION
Biofilms are aggregates or clumps of microbial cells, most often
■ CONFOCAL LASER-SCANNING MICROSCOPY FOR
BIOFILM STUDIES
bacteria and fungi, attached to a surface or each other and Confocal laser-scanning microscopy (CLSM) has emerged as
embedded in a self-produced extracellular matrix.1 Since their the most widely used imaging technology for the microscopic
first description in 1978,2,3 biofilms have been increasingly visualization of biofilms.11,13,19,20 A versatile and powerful
technique, CLSM allows in situ, real-time, and nondestructive
observed across a range of healthcare and environmental
examination of living biofilms, along with 3D reconstruction of
settings. Clinical biofilm infections include pneumonia the entire sample.21 For this, CLSM excites fluorescence
(particularly in diseased lungs), eye, ear, skin, and wound signals from different planes within the sample, resulting in
infections, as well as life-threatening cardiac and endovascular multiple image acquisitions from across the depth of the
infections,4−6 and are notoriously tolerant to antibiotic biofilm. CLSM has made possible qualitative observations of
treatments. In the environment, biofilms play a role in clinical, environmental, and laboratory biofilms, which has led
bioremediation, biofiltration and wastewater management but to fundamental insights into the formation, development,
are also associated with biofouling and food contamination.7,8 morphology, structure, and architecture of biofilms under
different conditions.11,17,18,22−27 Further, with certain mod-
These clinical and environmental implications have prompted
ifications, CLSM has also been used to assess the viability and
the study of biofilms under native and laboratory conditions,
using an array of technologies across molecular and microbial
Received: November 11, 2022
scales.9−11 Notably, a large segment of biofilm research Accepted: May 11, 2023
includes microscopic visualization,12−14 which allows direct Published: May 26, 2023
observations of biofilm formation and development, structure
and organization, as well as viability and metabolic activity in
the presence of therapeutic agents.15−18
© 2023 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acsomega.2c07255
20163 ACS Omega 2023, 8, 20163−20177
 Table 1. Overview of Image Analysis Programs for Biofilm Confocal Micrographs with a Focus on Tool Scope and Selection of Relevance to Experimental Researchersa
operating
name of the image availability for down- system re- system require-
analysis program load quirements ments user interface scope of the tool capabilities limitations
ACS Omega

Bacterial Cell open-access; available Windows 10 requires MAT- MATLAB segmentation ability to study single bacterial cells in biofilm aggregates; uses deep cannot process images with a low signal-to-
Morphometry 3D at https://github. LAB, Jupyter and Python CNNs with mathematical image analysis to accurately segment background ratio such as images in which
(BCM3D)46 com/ Notebook scripts and classify single bacterial cells, including in mixed cell objects do not have well-defined structure
GahlmannLab/ scripts for based populations, from 3D fluorescent images or edges or high fluorescence; requires
BCM3D.git training data, understanding of programming languages
Cell Modeller such as Python and MATLAB for
and NiftyNet running scripts; requires a MATLAB
license, which is expensive
Biofilm Viability open-access, Macros Windows, requires Mor- uses the Im- segmentation; bio- can process multiple images in a folder at once; low computational runs only on TIFF files; can only process
Checker28 script available at Linux, and phoLibJ to run ageJ inter- mass calculation time; calculates biomass as percentage coverage of live and dead fluorescent images of single-species bio-
https://github. macOS the algorithm, face; the al- bacteria in each single-species (live/dead stained) biofilm image films stained with FilmTracer LIVE/
com/sophie- works on FIJI gorithm is a DEAD Biofilm Viability Kit; requires
mountcastle/ as a plugin macros understanding of Macros script editing to
Biofilm-Viability- script that is modify the code to fit the study data
Checker/ run as a FIJI
plugin
BIAM (Biofilm In- open-access, JAVA Windows, requires 4 GB GUI segmentation; fluo- provides intensity calculations for genetic reporters and fluorophores TIF files only; can only segment images
tensity and Archi- based Macros script Linux, and RAM and Im- rescence intensity in biofilm; allows intensity correction; uses three different with a single-labeled fluorophore, not
tecture Measure- available at https:// macOS ageJ version calculation; archi- segmentation strategies suitable for colocalization studies with
ment)47,48 github.com/ 1.51k or later tectural/structural two different fluorophores
BCinquin/Biofilm_ and Java7 to measurements
Analysis run the script such as area and
perimeter of bio-
film
BiofilmQ32 open-access, available Windows 10, minimal require- GUI segmentation using accepts input files in 8 different file formats; allows import of tool does not work on computers with

20164
for download at macOS X, ment is Intel i5 cube cytometry; presegmented images; provides spatial measurements in images lower specifications (lower version of
https://drescherlab. Linux with 16GB can measure fluo- where single-cell resolution is not possible processor or less RAM space); when
org/data/biofilmQ/ RAM; requires rescence, architec- there is heterogeneity in cell sizes, cube
MATLAB tural, and spatial segmentation does not work well; tool
R2017b or properties; data requires a MATLAB license to run
subsequent visualization advanced functionality; for batch pro-
versions cessing of multiple files, tool requires
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MATLAB script
BioFilmAnalyzer49 free, open-access, and Windows no specific re- GUI segmentation; enu- counts live and dead cells (can be used for viability counts); can be not available for other OS; Only provides
available at https:// quirements meration of cells; used for eukaryotic cells (example, host cells in clinical biofilms); cell count and not structural attributes of
bitbucket.org/ mentioned data visualization quantification of fluorescently labeled subpopulations from 2D cells or aggregates
rogex/ micrographs
biofilmanalyzer/
downloads/
CMEIAS v3.150,51 free, open-access col- Windows requires the GUI of segmentation; can can categorize microbial cells into 11 predominant bacterial runs only on the UTHSCSA ImageTool
lection of plugins UTHSCSA UTHSCSA analyze organiza- morphotype classes: cocci, spiral, curved rod, U-shaped rod,
formerly available at ImageTool for ImageTool tional and struc- regular rod, unbranched filament, ellipsoid, club, prosthecate,
http://cme.msu. running plu- tural properties of rudimentary branched rod and branched filaments
edu/cmeias gins microbial cells
CMEIAS Color free, open-access, for- Windows run as a module GUI segmentation can accurately define biomass object pixels uses TIFF format only
Segmentation52 merly available at under
http://cme.msu. CMEIAS, re-
edu/cmeias quires Image-
Tools to run
CMEIAS
CMEIAS JFrad53 free, open-access, for- Windows, requires JAVA 6 GUI measures spatial and built to support multiple file formats; user-defined thresholding; user-defined thresholding can introduce
merly available at Linux, and or higher to architectural bio- allows batch processing bias
http://cme.msu. macOS run the tool film properties
Review

edu/cmeias such as height,

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 Table 1. continued
operating
name of the image availability for down- system re- system require-
analysis program load quirements ments user interface scope of the tool capabilities limitations
ACS Omega

width, area, and

percentage surface
coverage using
fractal geometry
COMSTAT34 free, open-access, Windows, requires MAT- command segmentation; bio- user-defined thresholding; quantification of structural elements requires a MATLAB license, which is
available at www. macOS, LAB to run the line-based film features such expensive; command line-based interface;
comstat.dk Linux script interface as biovolume, area, can only process image formats compat-
thickness distribu- ible with MATLAB
tion, mean thick-
ness, identification
of microcolonies,
and area of micro-
colonies
COMSTAT254 free, open-access, Windows, runs as a plugin GUI based on segmentation; calcu- does not require MATLAB; properties calculated are similar to unable to calculate surface-to-volume ratio
available at www. macOS, on ImageJ/FIJI ImageJ-plu- lation of structural COMSTAT; can read multiple image formats; multiple thresh- like COMSTAT
comstat.dk Linux gin API properties olding methods
daime35 free, open-access Windows, requires 1 GB GUI segmentation; capa- no programming skills needed; can also analyze conventional input images should be in TIFF format only
available at https:// Linux RAM and at ble of noise re- epifluorescence, bright field, or phase contrast micrographs;
www.microbial- least a 512 MB duction, filtration, special tools for biofilm image analysis; allows quantification of
ecology.net/daime graphic card upscaling and fluorescence intensity, population abundance, spatial arrange-
downscaling of ments, morphometry, and object classification; visualization using
resolution; data interactive volume rendering, ray tracing, and 3D image clipping;
visualization special image analysis function for identifying correct stringency of
new rRNA-targeted probes for FISH; connectivity with R
ISA3D55,56 free, open-access no specific no specific de- MATLAB segmentation; calcu- offers automated thresholding; can calculate textural and volumetric no download link available

20165
details tails mentioned based script lation of structural parameters of biofilms
mentioned parameters
Iris57 free, open-access, Windows, requires ImageJ- GUI segmentation; calcu- ImageJ based so does not require additional software installation; quantification of kinetics data requires R;
available at http:// Linux, based API or lates colony mor- high-throughput and works well for colony arrays; can quantify programming packages to plot growth
critichu.github.io/ macOS plugin to run phology and ki- kinetics data for time series experiments curves; requires colony array to be in grid
Iris the tool netics data, phe- format
http://pubs.acs.org/journal/acsodf

notypic and
genotypic activity
PHLIP58 free, open-access, Windows, MATLAB tool- GUI segmentation; bio- allows batch processing; offers both manual and automated (Otsu’s MATLAB-based and requires MATLAB
available at https:// Linux, box-based film volume, mor- Method) thresholding; multichannel analysis; biofilm spatial and licensing; requires preprocessing steps
sourceforge.net/ macOS script phology, and sur- temporal calculations: biovolume, substratum coverage, thickness, such as image inversion and x-section
projects/phlip/ face characteriza- roughness and colocalization; offers a data conversion option resolution
tion (PHLIP-ML) to convert different image formats to input format;
has direct connectivity to PHLIP-ML (file convertor) for Leica
Systems
qBA algorithm59 free, open-access, Windows, no specific de- MATLAB segmentation; bacte- viability counts designed for counting bacterial cells; based
available at https:// Linux tails mentioned based script rial count enumer- on an adaptive algorithm (rather than
www. ation binary segmentation); no GUI; requires
infectognostics.de/ MATLAB and understanding of MAT-
forschung/ LAB scripts for troubleshooting
technologien/qba-
algorithm
R based method for free, open-access R Windows, R software pack- R program biofilm thickness calculates biofilm thickness using a statistical program only processes UTF-8 encoded import
biofilm thickness script Linux, ages, data. GUI) calculation data; requires histogram data of fluo-
by Frühauf et al.60 macOS table, reshape2, rescence intensity for each ROI imported
dplyr, ggplot2, from LAS X software; no automated
and cowplot image segmentation; can calculate only
one parameter (biofilm thickness)
Review

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 Table 1. continued
operating
name of the image availability for down- system re- system require-
analysis program load quirements ments user interface scope of the tool capabilities limitations
ACS Omega

ImageJ for biofilm free, open-access, Windows, requires JAVA GUI segmentation; calcu- processes multiple image formats; generate histograms and profile requires plug-ins to be separately installed;
analysis61,62 available at https:// Linux, and lates structural and plots; allows creation of customized Macros script for sharing it is a general image analysis tool and
imagej.nih.gov/ij macOS spatial attributes protocols; connectivity with many microscope customized does not offer advanced parameter
such as aggregate softwares, e.g., LAS X calculations or options specific for bio-
size, biomass dis- films
tribution, biomass
thickness, biomass
intensity, to name
a few
ParaView63 free, open-access, Windows, requires visual- GUI data visualization allows batch-processing; generates high-quality 3D rendering requires .vtk files; offers only visualization
available at https:// Linux, and ization toolkit and rendering
www.paraview.org/ macOS
Napari64 free, open-access, Windows, requires Python Python -based data visualization allows creation of customized plugins; generates high-quality 3D offers only visualization; requires under-
available at https:// Linux, and 3.7 or higher; script and and rendering rendering standing of Python for troubleshooting
napari.org/ macOS requires Py- GUI
thon libraries
and packages,
Qt library for
graphic user
interface, VisPy
for rendering
Three-Dimensional no details available Linux requires MAT- MATLAB image reconstruc- 3D image reconstruction for calculation of structural parameters requires MATLAB; runs only on Linux; no
Biofilm Image Re- LAB R2016a based script tion GUI
construction Algo-
rithm19

20166
Python based meth- Free, open-access, Windows requires Python Python based segmentation; mor- macrocolony calculation; fluorescence intensity calculations; can only Otsu’s thresholding method can be
od for measuring available at https:// 3 or higher; script phology and calculate growth kinetics and distance of macrocolony to used; morphological calculations done at
bacterial biofilm github.com/ requires Py- growth calcula- antimicrobial source the core of the colony
morphology and cohenoa/An-Open- thon libraries tions
growth calculation Source- and packages
by Gingichashvili Computational-
et al.65 Tool-for-
http://pubs.acs.org/journal/acsodf

Measuring-
Bacterial-Biofilm-
Morphology-and-
Growth-Kinetics
a
Abbreviations: CNN, convolutional neural network; GUI, graphic user interface; CMEIAS, Center for Microbial Ecology Image Analysis System; daime, digital image analysis in microbial ecology;
ISA3D, Image Structure Analyzer in 3D; PHLIP, phobia laser scanning microscopy imaging processor); qBA, quantitative biofilm analysis and bacteria generator.
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species diversity within biofilms.22,28 More recently, the displayed on the monitor enabling real-time visualization and
application of CLSM for biofilm studies is moving beyond microphotography of the sample.77 The micrographs are
descriptive observations alone, toward a more in-depth typically acquired as a series of optical sections (Z-stacks or Z-
characterization of biofilms from high-resolution image layers, that are perpendicular to the optical axis), which are
stacks.17,29,30 For this, CLSM is combined with image analysis stacked together and reconstructed to produce a 3D
tools, which can together provide quantitative insights into representation of the sample.78 For this, the object within
biofilm features under different conditions.31−33 These image the sample is built up pixel-by-pixel from the detected signal,
analysis programs include not only broadly applicable image with each pixel having four horizontal and vertical neighbors.
processing tools, such as ImageJ and IMARIS, but also The entire sample is displayed using mathematical operations
programs that are tailor-made to the study of biofilms, such as on the array, where two pixels with the same dimensions
COMSTAT,34 daime,35 and BiofilmQ,32 to name a few. This (either with coordinates that match or intensities that are the
shift in the field is reflected across several previous reviews, same) are paired together, leading to the final 3D image
which focus on microscopy applications and approaches, and construction.
image analysis tools and techniques for various biofilm Experimental Setup for Biofilm Confocal Microgra-
components, including single cells or small groups of bacterial phy. CLSM has been used for acquiring biofilm micrographs
cells, across different scales.17,33,36−45 In this review, we present from a range of experimental setups such as microtiter wells,
an account of the considerations related to confocal micro- colonies on agar, flow cells, microfluidic devices,22,65,79−81 as
graph acquisition and processing for biofilm experimental well as large volume receptacles.82 Irrespective of the setup,
research and provide an in-depth overview of image analysis biofilm experiments tend to show significant variability, even
programs for biofilm confocal micrographs, including their under nearly identical conditions.83,84 To account for this, it is
scope, user requirements, and limitations. While the present recommended that biofilm experiments are performed in at
suite of programs provides the biofilm researcher with an array least three biological replicates, where each biological replicate
of options, the specifications for each tool, including their is set up from a separate overnight bacterial or fungal culture.85
scope, suitability, and limitations, require significant consid- Further, for each biofilm replicate, images should be acquired
eration (Table 1). Further, given that biofilm experiments are from multiple fields of view, data from which can then be
time-, resource-, and labor-intensive, this overview will be consolidated for a single data point for that replicate. For time-
particularly important for biofilm researchers to ensure series studies, to account for the effect of prolonged laser
reliability and compatibility between the experimental setup exposure on the bacterial or fungal cells, the experiment may
and downstream image analysis. need to be set up with multiple technical replicates (the same

■ GENERAL CONSIDERATIONS FOR CONFOCAL

MICROGRAPH ACQUISITION AND PROCESSING
biofilm set up in different wells), each imaged at a different
time point.30 This setup will allow a technical replicate to be
exposed to the laser only for one time point, thereby
FOR BIOFILM STUDIES precluding the effects of photobleaching or phototoxicity of
The process of extraction of quantitative properties of biofilms the biofilm sample across multiple time points. In addition, for
from confocal micrographs is a multistep process, starting with comparisons across conditions such as different treatments or
the experimental setup and initial acquisition of the micro- time points, it would be important to ensure identical
graphs, followed by the subsequent export of images and parameters of laser intensity, object magnification, and image
visualization and analysis of confocal micrographs. While resolution, as far as possible. Finally, there are guidelines to
computational image analysis tools enable automated analysis address variability and ensure repeatability and reproducibility
of confocal biofilm micrographs, a basic understanding of the in the setup of biofilm experiments, including standards for
image acquisition process and parameters is important to reporting minimum information about a biofilm experiment
ensure that the experimental setup is compatible with the (MIABie) and the design of biofilm imaging experi-
subsequent image analysis. ments.83,86,87
Basics of Confocal Micrographs of Biofilms. Confocal Image Acquisition. With currently available commercial
laser scanning microscopy is based on the selective collection CLSM systems, there are only a few critical parameters that
of point-by-point fluorescence signals from optical sections need manual adjustments. While researchers are typically
within the sample.66 For confocal biofilm microscopy, bacterial familiar with adjustments related to object magnification and
or fungal cells are typically tagged with fluorophores or stained optical section thickness, prior acquaintance with additional
with fluorescent stains or probes.30,67−69 Following the photon relevant CLSM settings can significantly improve micrograph
excitation of fluorophores using a laser (gas lasers such as quality and reliability.
argon-ion, helium−neon, dye lasers, solid-state lasers,70 and Scan Speed and Pixel Dwell Time. An important initial
mercury and xenon arc lamps71), the detected electrical signal consideration during CLSM imaging is scan speed or data
is converted to gray scale values, which correspond to pixel acquisition time.88−91 Scan speed depends on the type of
intensity in that region (grayscale intensity values lie between 0 CLSM system used, as well as the optical magnification,
and 255, where 0 means black, 255 means white, and numbers numerical aperture, and lateral and axial resolution. Pixel dwell
in between represent shades of gray ranging from black to time is the amount of time that a laser beam is allowed to rest
white). Done at high speed by a digitizer at the back-end of the on a single pixel in the image.92 Typically, dwell times of 6−12
software, this enables real-time visualization and micro- μs are sufficient to get reasonable image parameters.93 Higher
photography of the sample. CLSM micrographs are 2D arrays resolution images typically require more dwell time per pixel to
or matrices, consisting of a fixed number of elements or collect more photons per pixel and therefore a slow scan speed.
“pixels”, each with a particular location (x and y coordinates, in However, setting a higher value for dwell time can increase the
the plane of the sample) and a fixed intensity value.72−76 The risk of photobleaching of the specimen.91 There is therefore a
image information is temporarily stored in the computer and trade-off between achieving high spatial and temporal
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resolution and scan speed, given that longer scanning and confocal microscopy image acquisition, is well predicted and
acquisition times could result in photodamage.94 governed by the Beer−Lambert Law, which explains how light
Laser Intensity. Given that CLSM micrograph acquisition is absorption exponentially decays as the laser is focused deeper
based on the intensity of fluorescent signals, the selection of into a specimen.105−108 This results in artificially low
the type and power of the laser are important considerations at fluorescence intensities in the biofilm’s deeper layers, which
the start of the image acquisition process. Typically, the is caused by spherical aberration of the objective lens.77,109,110
selection of the laser and wavelength depends on the excitation Because the configuration of confocal microscopy systems is
and emission spectra of the fluorophores in the experimental based on using the same optical lens for fluorescence excitation
setup.95−97 For this, it is important to understand the spectral and detection,77,100 the excited beam passing through the
and physical properties of fluorophores or fluorescent stains or outermost edge of the lens focuses at a different point on the
probes; Fluorescence SpectraViewer98 is an interactive tool axial plane than the beam passing through the center of the
that can be used to select probes and filters for fluorescence- lens. The emitted light suffers from the same spherical
based experimental design. Finally, while setting the laser aberration, which significantly reduces image resolution.105
intensity can vary across samples and experimental conditions, As a result, it is critical to consider that image resolution is
it is important to consider the effects of laser intensity on the compromised at depths greater than 50−80 μm.105,108 To
micrographs. At low laser intensities, valuable signal overcome this challenge to some extent, advanced confocal
information from the micrographs may be missed, whereas microscopy techniques such as multiphoton confocal micros-
higher laser intensities can lead to signal saturation and copy or advanced statistical methods during image analysis
photodamage. Notably, several acquisition software programs could be used.106,108,111
display autohistograms, which can help with laser intensity Zoom Settings. In general, the zoom control varies the size
adjustments.88,91,99 Finally, for time-series experiments, the of the area of the specimen scanned, and typically there is only
laser intensity may have to be adjusted across time points. This one optimal zoom setting for a particular combination of
is relevant in the context of biofilm growth over time, where wavelength, numerical aperture, and objective magnification.
bacteria grow to form large mats of biomass, in which case the This setting provides a pixel size that matches the Nyquist
fluorescence intensity of the sample is likely to increase, or criterion,88,112 which states that the pixel size of an image
with the effect of treatments, in which case the intensity could needs to be at least 2.3 times smaller than the object being
decrease. In this case, adjustments to the laser intensity might resolved, and can be calculated with an online Nyquist
be needed, and this should be considered when performing calculator.113 Notably, suboptimal zoom settings can result in
and interpreting downstream image analysis. For example, over-sampling or under-sampling of data.112 For example, the
when analyzing and comparing intensity values, biofilm time diffusion of certain dye molecules can cause a small variation in
points or conditions exposed to different laser intensities would fluorescence intensity, and in this case, choosing a higher
need to be normalized to ensure accurate interpretation of the spatial resolution could result in a large pixel-to-pixel variation.
values. For confocal micrographs of biofilm mats or aggregates, images
Pinhole Diameter. In CLSM systems, a portion of the of 512 × 512 or 1024 × 1024 pixels with a resolution of 300
emitted fluorescence is collected by the objective lens and dpi are widely used.59,88,112,114 While spatial resolution
focused into a pinhole in front of the detector.100 The amount corresponds to image quality, setting a higher resolution may
of fluorescent light that reaches the detector is determined by increase the image acquisition time and could also lead to
the detector pinhole. While the diameter of the pinhole is photobleaching or photodamage.87,115
usually set at ∼1 Airy unit, it is important to note that the Z-Thickness and Step Size. An important consideration
pinhole aperture determines the thickness of the optical during CLSM image acquisition is adjustments to the Z-
section and can therefore influence the resolution of the thickness of the acquired micrographs.91,116 Current CLSM
micrographs.88,91,99 A larger pinhole diameter will result in systems possess built-in features that enable scanning of the
more extrafocal intensity reaching the detector and could sample across the Z-axis in “live” mode.117 Following this
compromise the sharpness of the optical sections. However, for initial scanning, the lower and upper Z-layers are typically set
low fluorescence signals or fading fluorescence, the user may to include the lowermost and uppermost regions of the biofilm
need to open the pinhole to capture as much fluorescence as sample.30,31 Given that the number of Z-slices, and therefore
possible, which can compromise the Z-resolution of the the total Z-stack size, may vary across replicates, this may have
micrographs.101 For multicolor or multichannel images, for a to be set manually for each experimental replicate or condition.
consistent optical slice or Z-layer resolution, it is crucial that A step size of 1−2 μm is typically used to cover the entire
the pinhole size is adjusted depending on the wavelength of the biofilm sample.31,87,118 While the step size can be increased or
emitted light.102−104 decreased, this has to be considered in the context of the
Penetration Depth. Confocal laser scanning microscopy is experimental setup; decreasing the step size would result in
primarily based on capturing a portion of the nonscattered thinner optical sections for more accurate 3D reconstruction,
emitted fluorescence that originates at the focal plane of the while increasing the step size would result in faster image
objective lens.77 Absorption and scattering of laser light cause a acquisition.
significant reduction in image quality in thicker biofilm Taken together, image acquisition parameters for biofilm
samples, as the intensity of fluorescence in different sections confocal micrography can be adjusted using commercial
of the biofilm sample can differ due to laser attenuation. The acquisition software systems, depending on the type and
laser intensity in the lower section decreases as the penetration version of the CLSM system being used.38,118,119 For Leica
depth increases because a significant fraction of the excitation SP8 Confocal systems, an online tool is available to calculate
light is absorbed in the top layers of the sample, resulting in the zoom factor and Z-step size.120
less excitation light reaching the deeper layer of the biofilms. Additional CLSM Features for Image Acquisition. For
This laser attenuation property, a critical aspect during large specimens (such as entire organs, organisms, or
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microtiter wells), the mosaic function in CLSM systems can be microscope file types and conversion of these files into an
used for visualization at high resolution.121,122 This function internal format.32
combines multiple images into a single, large superimage of the Image Processing of Confocal Micrographs. Following
specimen. A tile scan, which records a set number of adjoining the export of confocal micrographs from the CLSM system, the
single images of the sample (the “tiles”), is required for such a imported images need to undergo initial processing for object
stitched superimage.66,79,84 While the sample is moved using identification and analysis.33,130 The general image analysis
the motorized stage to ensure precision, a small percentage pipeline for biofilm studies is divided into three sections: image
(2−10%) of overlapping pixels between single images is preprocessing, image segmentation, and feature extraction and
required to perfectly match the tiles.123 Certain CLSM systems analysis. Image preprocessing techniques such as median
also allow you to define two reference points (bounding box) filtering, background subtraction noise reduction, signal
at opposite corners of the specimen, and then the number and attenuation correction, sharpness, edge detection, and the
position of the fields to cover the specimen, including deconvolution algorithm can aid in the reduction or removal of
predefined overlapping regions, are calculated. The mosaic unwanted image artifacts. It is important to note, however, that
function is not limited to xy images (2D) but can also be used these preprocessing steps can be computationally expensive,
to generate large 3D images.39,124,125 Finally, high content and caution should be exercised when fine-tuning the
screening confocal laser microscopy (HCS-CLM) employs a preprocessing parameters to ensure that unwanted artifacts
special mode to improve raw data quality (higher resolution) are removed without losing the object of interest.131 Before
through the use of predefined and fully customizable one can extract morphological and functional measurements
automated analysis (unbiased) workflows (faster acquisi- from a micrograph, it is important that there is discrete
tion).126 Used to study 4D structural dynamics of biofilms in identification or labeling of voxels. Each pixel (or voxel,
96-well microplates, this computer aided microscopy (CAM) volumetric pixels in 3D micrographs) in a confocal micrograph
tool allows the user to create a scanning template or workflow has a distinct gray value that corresponds to the intensity of the
that is suitable for the experimental conditions. The raw data captured signal.
generated from this tool is usually in a platform-independent Image segmentation130−133 refers to the process of
OME-TIFF format, which is compatible with several image transitioning voxels from a conceptually simple array of point
analysis tools. measurements of fluorescence intensity to the more
Exporting Confocal Micrographs from CLSM Sys- informative concept of image objects. Regions and boundaries
tems. In addition to basic features for image acquisition and are two commonly used concepts in image segmentation.
analysis, commercial CLSM software systems include several Techniques for image segmentation can be divided into three
options for exporting and saving confocal micrographs.91 categories: bottom-up methods, top-down methods, and
Along with the image, the software allows additional data to be hybrid methods.131 Bottom-up methods rely on identifying
saved in manufacturer-customized or proprietary image regions that represent objects of interest and iteratively
formats, such as the image number, date of acquisition, merging voxels with similar measures. The top-down approach
microscope settings, exposure values, as well as the size and requires some prior knowledge about object shapes, object
scale details of the image (for example, .lif for Leica systems, models, and the number of regions; however, this requirement
.czi for Zeiss microscopes, .nd2 for Nikon, and .oib for for some level of knowledge tends to limit the applicability of
Olympus microscopes).127 Confocal micrographs are typically this segmentation approach. Finally, the hybrid method
saved as 8-bit images, with each pixel of the cross-section with combines both the bottom-up and top-down approaches and
intensity values in the gray value range of 0−255, though can be computationally demanding.
higher bit depths (12-bits) can also be generated with intensity The bottom-up method can be further divided into four
values in the gray value range of 0−4095.90 However, higher categories: intensity thresholding, region-based, boundary-
bit depths necessitate more file memory space, and certain based, and integrated segmentation. In the intensity-based
image analysis software programs cannot read 12-bit images. thresholding approach, a threshold is applied and all voxels
Several CLSM softwares also provide the option of batch- whose values lie within a certain range of fluorescence intensity
exporting and batch-processing of several exported images at belong to one class. This is done to separate the object of
once; a notable example is the Batch Tools Workspace in the interest (in case of biofilms, biomass) from the background.
ZEN Blue software (Zeiss).117,128 While CLSM systems allow This results in image binarization (1 for biomass and 0 for
users to save images in various formats, it is recommended that background).82,134 A voxel is considered a background voxel if
users use the manufacturer’s image format. These customized its shade intensity is less than the threshold value, else it is
image formats can then be converted into universal and considered a biomass voxel.133,135 Each voxel in the segmented
platform-independent formats such as .tiff, ome-tiff, .jpeg, or image contains information about the object class to which it
.png using the BioFormats plugin (available in ImageJ61 and belongs, with background voxels having a zero-voxel value.
FIJI129). It is important to note, however, that recently Using a single threshold value for an entire image allows for
developed image analysis tools obviate the need for a separate global thresholding of the image. Adaptive or local thresh-
conversion to .tiff (by incorporating a BioFormats plugin into olding occurs when the threshold value changes for different
their source code), and several customized image formats, such regions of the image. Local thresholding is applied under
as Nikon NIS-Elements ND2 files (.nd2), Zeuss CZI files specific conditions such as when the user needs to select an
(.czi), Zeiss LSM (laser scanning microscope) 510/710 files individual threshold for each pixel. For local thresholding, the
(.lsm), Leica LAS AF LIF (Leica Image File Format) files (.lif), image needs to be cropped into small sections and each section
and Olympus FluoView FV1000 files (.oif or .oib), can be is thresholded using the selected method.72,136 The region-
directly imported in formats compatible with image analysis based segmentation method assumes that neighboring voxels
programs.97 A notable example is the image analysis program within a specific region of an object are homogeneous. The
BiofilmQ, with file input and recognition features for standard general procedure in region-based segmentation is to compare
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each voxel to its neighbors. If the homogeneity criteria are met, subsequent processing steps, it is beneficial for the
the voxel is said to belong to the same class. However, the experimental researcher to understand the tools available and
performance of this approach is heavily reliant on the choice of determine the most suitable tool for the desired analysis. It is
homogeneity criteria. The boundary-based approach, on the important to note that the experimental study may require a
other hand, is heavily reliant on edge detection and the combination of several tools to analyze different biofilm
integration of boundary information for classification. These features. Recent reviews provide a list of comprehensive points
edges identify image discontinuities in gray levels, color, of consideration, which can be used as a starting point for tool
texture, and so on, and aid in image segmentation. Lastly, selection.17,33,36−45 In addition, based on our experience, we
integrated segmentation combines the characteristics of both suggest certain experimental factors which would be valuable
region and boundary criteria; a notable example is the to consider at the start of and during the biofilm confocal
watershed algorithm,137 which is based on an integrated micrograph acquisition and analysis process.
segmentation approach. In recent years, there has been an What Are the Biofilms and Other Biological Elements
increase in the development of novel segmentation approaches in the Biofilm Confocal Micrographs Expected to Look
based on convolutional neural networks and deep learning, Like? Depending on the bacteria and fungi under study,
with DeepCell 2.0138 and CellProfiler 3.0139 being notable biofilms typically grow from single cells or clumps of cells to
examples. However, CLSM biofilm image segmentation larger aggregates and dense mats of biomass.145−150 Growth
typically employs a traditional thresholding technique that conditions, including in vitro platforms and media conditions
selects a threshold value to segment voxels in the images based are known to influence biofilm properties.11,84,151,152 Taken
on fluorescence or grayscale intensity. together, a combination of biofilm features, including the
Thresholding in biofilm studies can be performed either spatiotemporal distribution, biomass intensity, and biofilm
manually or automatically and can be applied to individual 2D components (bacterial or fungal cells, matrix) and morphology
images or applied together for a stack of confocal micrographs. (single cells, aggregates, dense mats, pellicles), will constitute
Manual thresholding relies on the visual determination of the raw data set of the confocal micrographs. In the case of
thresholds140 and is therefore prone to bias in the measure- clinical and in vivo biofilms, host tissue and cellular factors may
ment of biofilm features, where setting a low threshold can also be imaged.153−156 In the case of environmental biofilms,
falsely capture the presence of biomass when there is none and plant structures and additional biological elements (diatoms,
a threshold that is too high can result in missing the algae) may also be present in the sample.157−160 Identifying
measurements of low-intensity biomass.31,55,82,130 Automated and analyzing these biofilm and non-biofilm features may
thresholding methods can be global or local, depending on the require special consideration at the time of image acquisition
specificity and sensitivity needed for the detection of the (laser intensity, zoom factor, Z-slice thickness, presence of
regions of interest. Otsu’s thresholding approach, which is used autofluorescence) to ensure appropriate image processing and
in software such as COMSTAT and BiofilmQ, is a widely used tool selection.
global method based on traditional segmentation that selects What Are the Analysis Parameters Needed for the
an optimum threshold that can provide maximum variation in Study? At the stage of image acquisition, it would be
shade intensity and class separability between the biomass and important to consider the downstream processing and analyses
background.141 Other global methods include iterative needed for the goals of the experiment. This is relevant not
selection142 in software like BiofilmQ and daime, robust only for tool selection but also for ensuring that the
automatic threshold selection (RATS)143 in software like experimental conditions and image acquisition parameters
daime, and objective threshold selection (OTS)144 in software meet these requirements. For example, if biomass thickness is
like PHLIP. Notably, several recently available tools for biofilm expected to be a parameter under study, it would be optimum
analysis are based on Otsu thresholding and iterative to select multiple regions of the biofilm for imaging for each
selection.31,55 replicate, which can then be pooled to obtain biomass
Under certain experimental conditions, it could be difficult thickness for that replicate. This would account for variability
to distinguish individual microbial cells in the image, for in thickness across different regions of the replicate (for
example, in confocal micrographs of dense biofilm aggregates example, in a 96-well).83,84 Another example would be
or mats. In this case, the pixel location of the biofilm edge may quantifying biofilm aggregate dimensions, which can be done
not be uniquely defined, given that the biofilm edge consists of using area or volume, using growth rate as a proxy
a fluorescence signal gradient that can span several cell measure.30,161,162 For the size of the aggregates, a Z-stack
diameters.31,55 As a result, different segmentation algorithms projection would provide details about area and mean gray
will identify the biofilm edge in slightly different locations. value for each aggregate cluster, which can be computed using
Because images of microbial communities without single-cell several available programs.32,34,35,48,53,54,58,59,61 Quantification
resolution are frequently analyzed in biofilm research, the need of aggregate volume would require high-resolution images and
for accurate semantic segmentation has resulted in a range of user familiarity with advanced tools such as BiofilmQ,32
different algorithmic solutions for biofilm analysis such as COMSTAT,34 COMSTAT2,54 Iris,57 ISA3D,56 PHLIP,58 qBA
Bacterial Cell Morphometry 3D (BCM3D),46 BiofilmQ,32 algorithm,59 with features to quantify the biovolume of each
daime,35 and qBA algorithm.59 aggregate. Finally, an aggregate can also be quantified using

■ SPECIFIC EXPERIMENTAL CONSIDERATIONS FOR

IMAGE ACQUISITION AND ANALYSIS FOR
growth rate as a proxy measure, using biomass measurements
as a function of time.162
In the Case of Time-Lapse and Treatment Experi-
BIOFILM CONFOCAL MICROGRAPHS ments, How Is the Image Acquisition and Analysis
The first step in the analysis of biofilm confocal micrographs is Likely to Change Across the Experiment? The growth of
the selection of an image analysis program that is best suited to biofilms over time and the effects of treatments can cause
the parameters under study. Because this decision affects all marked shifts in fluorescence intensity from the sample, which
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Figure 1. General workflow for analysis of confocal micrographs of biofilms can be divided into (A) experimental setup and image acquisition, (B)
image processing, visualization and analysis, and (C) analysis of quantitative data and representation of results. Understanding the confocal
micrograph acquisition process and parameters as well as the selection and scope of the image analysis programs is important to ensure
compatibility between the experimental setup and subsequent image analysis.

will require modifications in image acquisition settings.30,163,164 example, when analyzing the spatial organization of biomass in
For example, the formation of large, dense biomass mats could a given condition, the location of the upper and lower bounds
increase the fluorescence intensity from the biofilm, prompting of the biofilm can vary across replicates. The biofilm replicates
a decrease in the laser intensity. On the other hand, reduction may not be attached all the way to the top or start at the same
in biomass following antibiotic or antimicrobial treatments location at the base of the well or extend across the same
could require an increase in laser intensity to visualize the height in the well. In this case, representing the data for each
remnant biomass.165 This can also be the case when studying replicate separately could be considered.
spatial and temporal biofilm features, such as biofilm Are There Possible Artifacts in the Image Acquisition
morphology and viability, which could demonstrate hetero- Process That Should Be Considered at the Time of
geneity across the experimental time points.164 Importantly, Analysis? During the image acquisition process, it is
these changes in settings will need to be accounted for during important to make a visual note of the biofilm features
the image analysis, to prevent erroneous conclusions from the observed and check for possible inconsistencies, which could
experiments. While the considerations will vary based on result in the misrepresentation of results from downstream
experimental conditions, this can be possibly overcome by image analysis.84 Given that image analysis programs will
preliminary determination of a “sweet spot” of CLSM settings analyze biofilm features irrespective of whether they are native
that works across time points and conditions. Alternatively, the phenomena or artifacts, it is for the experimental researcher to
differences in CLSM settings can be accounted for by determine if the observations are real. For example, when
normalizing the data or representing the data as ratios across measuring biomass distribution, the dispersal of biomass due
time points. For example, the intensity of the biomass at a to technical errors (such as perturbation of the biofilm in a 96-
subsequent time point can be normalized to the biomass well-plate due to physical movement or pipetting) could
intensity at the initial (“zero” time point30,166). erroneously increase the vertical biomass distribution width.
How Does the Experimental Researcher Plan to This would also influence the size and properties of biofilm
Represent the Data for a Given Biofilm Feature or aggregates and therefore be an artifact in the analysis. Another
Parameter? While the graphical representation of data related example of an artifact that could result during aggregate
to biofilm features and parameters will likely be finalized only analysis is the presence of dense aggregates, as can occur
after the image processing and analysis, it is worthwhile to during long periods of biofilm growth.30 In this case, the
consider how the experimental conditions could influence the inability to distinguish between closely placed aggregates might
final representation of data. Given the nature of biofilm result in larger aggregate dimensions as readouts. Therefore,
experiments, including the substantial variation observed while a particle size analysis tool (such as ImageJ) will provide
across replicates, data representation is particularly important a distribution of aggregate dimensions regardless of the
in the context of spatial studies of biofilms.30,147,148 For biomass density, the experimenter should consider the results
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of the analysis in conjunction with the visual biofilm features at scripts, if any) and purchase of licensed software, are also
the time of image acquisition. important. Taken together, the current tools of the trade are
Taken together, once a raw set of images have been acquired well-poised to further quantitative biofilm research and, along
and a suitable tool for an image analysis task has been with recent advances in single-cell analysis and AI/ML based
identified, new users could use a series of test images to methods, lend themselves well to a fine-tuned analysis of
familiarize themselves with the user interface, tool features and biofilm features from confocal micrographs.
options, and analysis sensitivity and results. Further, exper-
imental researchers can also reach out to tool developers for
technical challenges, and several tools also have community
■ AUTHOR INFORMATION
Corresponding Author
forums for troubleshooting.
Karishma S Kaushik − Department of Biotechnology,
Finally, it is vital for the experimental biofilm researcher to
Savitribai Phule Pune University, Pune 411007, India;
document in detail the CLSM settings used for image
orcid.org/0000-0001-5131-1798;
acquisition and image processing tools and analysis parameters
Email: [email protected]
employed (in the methods section of a manuscript, for
example) to ensure reproducibility and reliability of the Author
experimental results. It is evident that several factors in the Shreeya Mhade − Department of Biotechnology, Savitribai
CLSM image analysis workflow, including the experimental Phule Pune University, Pune 411007, India; orcid.org/
setup, microscopy settings, image segmentation and thresh- 0000-0002-3050-802X
olding, and image analysis parameters (Figure 1), would be
important for subsequent researchers to build upon or adapt Complete contact information is available at:
the biofilm study conditions. https://pubs.acs.org/10.1021/acsomega.2c07255

■ OVERVIEW OF IMAGE ANALYSIS PROGRAMS FOR

ANALYSIS OF BIOFILM CONFOCAL
Funding
K.S.K.’s appointment is supported by the Ramalingaswami Re-
entry Fellowship (BT/HRD/16/2006), Department of Bio-
MICROGRAPHS technology, Government of India. This work was supported by
In this section, we present an overview of the tools available for the Har Gobind Khorana-Innovative Young Biotechnologist
biofilm confocal micrograph segmentation and postsegmenta- Award (IYBA) to K.S.K (BT/12/IYBA/2019/05).
tion quantitative image analysis. In particular, we focus on the Notes
aspects of tool selection relevant to experimental researchers The authors declare no competing financial interest.
such as the user requirements, scope, capabilities, and
limitations of the program (Table 1). It is important to note
that this review focuses on biofilm image analysis at a
community level (aggregates, mats of bacterial or fungal cells)
■ ACKNOWLEDGMENTS
The authors’ thank Radhika Dhekane for helpful discussions
and does not extensively cover single-cell biofilm analysis. on confocal microscopy settings and image acquisition for
Further, we have largely focused on conventional image biofilm experiments.
analysis tools, which are widely used and have well-established
protocols and technical support. Together, albeit not all
inclusive, this will serve as a primary set of image analysis
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