Traditional microbiology approaches rely on measurements of
biological entities at the bulk population level. While this makes it
feasible to rapidly assess the population on the whole, it does not
capture the phenotypic diversity among individuals comprising this
population. Studying the mechanisms underlying phenotypic diversification and its functional relevance is of paramount importance
for understanding the bacterial cell biology. The approaches that
are being employed to capture phenotypic heterogeneity at the
single-cell level include—flow cytometry, time-lapse microscopy,
single-cell omics, mass spectrometry-based approaches, etc. [14–
19].
Considering the implications of phenotypic heterogeneity in
antibiotic persistence and the differing outcomes on host-pathogen
interactions, it is not surprising that there have been an increasing
number of studies addressing the physiology and behavior of mycobacteria at the single-cell level. Several recent studies have demonstrated the heterogeneity in mycobacterial populations not only in
bacteria growing in nutrient-rich medium, but also during growth
in the host or host-mimetic stress conditions [8, 20, 21]. These
include use of time-lapse imaging to study the fate of mycobacteria
when exposed to antibiotics [13, 22–25]; to study dynamic hostmicrobe interactions [26–29]; and to map cell cycle events [30–
33]. There have been significant advances in construction of
reporter strains and development of novel fluorescent probes for
aiding these single-cell studies [25, 30, 34–36]. Technology
advancements have also enabled combining time-lapse microscopy
with other techniques such as atomic force microscopy [37, 38] to
visualize single-cell features at the nanoscales, or with Raman imaging spectroscopy to unravel the metabolic activity of individual
bacteria [39].
Here, we focus specifically on the use of time-lapse microfluidic
microscopy to capture phenotypic heterogeneity. Since most of the
phenotypes observed among individuals in a population are
dynamic, we introduce an approach to study transient cellular
events through space and time. Microfluidic engineering techniques are useful for the study of different aspects of microbiology
[40, 41]. Here, we describe the construction, assembly, and use of a
microfluidic device consisting of micropatterned silicone polymer
and glass [42]. This microfluidic platform, which we refer to as
Hexa-device, is not only compatible with single-cell imaging of
mycobacterial growth over long periods of time, but also with the
ability to image six different bacterial strains at the same time,
increasing the experimental capacity compared to our firstgeneration device [43]. After the image sequences of bacteria
growing inside the microfluidic device have been collected by
time-lapse microscopy, the parameters related to individual cells
and lineages have to be systematically extracted and processed
[44, 45]. To this aim, a number of bioimage analysis platforms
206
Giulia Manina and Neeraj Dhar
biological entities at the bulk population level. While this makes it
feasible to rapidly assess the population on the whole, it does not
capture the phenotypic diversity among individuals comprising this
population. Studying the mechanisms underlying phenotypic diversification and its functional relevance is of paramount importance
for understanding the bacterial cell biology. The approaches that
are being employed to capture phenotypic heterogeneity at the
single-cell level include—flow cytometry, time-lapse microscopy,
single-cell omics, mass spectrometry-based approaches, etc. [14–
19].
Considering the implications of phenotypic heterogeneity in
antibiotic persistence and the differing outcomes on host-pathogen
interactions, it is not surprising that there have been an increasing
number of studies addressing the physiology and behavior of mycobacteria at the single-cell level. Several recent studies have demonstrated the heterogeneity in mycobacterial populations not only in
bacteria growing in nutrient-rich medium, but also during growth
in the host or host-mimetic stress conditions [8, 20, 21]. These
include use of time-lapse imaging to study the fate of mycobacteria
when exposed to antibiotics [13, 22–25]; to study dynamic hostmicrobe interactions [26–29]; and to map cell cycle events [30–
33]. There have been significant advances in construction of
reporter strains and development of novel fluorescent probes for
aiding these single-cell studies [25, 30, 34–36]. Technology
advancements have also enabled combining time-lapse microscopy
with other techniques such as atomic force microscopy [37, 38] to
visualize single-cell features at the nanoscales, or with Raman imaging spectroscopy to unravel the metabolic activity of individual
bacteria [39].
Here, we focus specifically on the use of time-lapse microfluidic
microscopy to capture phenotypic heterogeneity. Since most of the
phenotypes observed among individuals in a population are
dynamic, we introduce an approach to study transient cellular
events through space and time. Microfluidic engineering techniques are useful for the study of different aspects of microbiology
[40, 41]. Here, we describe the construction, assembly, and use of a
microfluidic device consisting of micropatterned silicone polymer
and glass [42]. This microfluidic platform, which we refer to as
Hexa-device, is not only compatible with single-cell imaging of
mycobacterial growth over long periods of time, but also with the
ability to image six different bacterial strains at the same time,
increasing the experimental capacity compared to our firstgeneration device [43]. After the image sequences of bacteria
growing inside the microfluidic device have been collected by
time-lapse microscopy, the parameters related to individual cells
and lineages have to be systematically extracted and processed
[44, 45]. To this aim, a number of bioimage analysis platforms
206
Giulia Manina and Neeraj Dhar