Digital Holographic Microscopy

Unlike traditional microscopes that are limited by an objective’s depth of field, digital holographic microscopy (DHM) allows for examining microorganisms in volumes with significantly larger depths. By using a coherent light source for illumination, the phase and amplitude of a specimen’s optical field can be captured using a standard camera installed on an inverted microscope. Numerical algorithms are then used to reconstruct the experimental volume and find the depth location of organisms or particles. Once the locations of the organisms are found, they are linked together to form 3D trajectories such as the ones shown in the video. The volume here is roughly 1.5×1.5x2mm^3.

Probing dual gradients in-vitro: Nazca device

While the vast majority of studies of tactic behaviors in bacteria focused on single gradients, microorganisms live in highly structured microenvironments featuring possibly counteracting gradients of both liquid and gas species (e.g. aminoacids and oxygen/nitrogen). Investigating how such complex environments affect motility patterns in microorganisms plays a key role in allowing a deeper understanding of behavioral traits in microbial ecology. To this aim we developed the “Nazca device” a new microfluidic platform enabling the formation of steady opposing gradients of attractants in both liquid and gas phase. The device is composed of 5 channels, each 600 um wide (see Fig. 1).

CIMG0676_m2CIMG0672_m2Nazca device

The outer channels (red and blue, in Fig. 1 and 2) are physically separated from the other three by PDMS walls and are meant to host gases. When molecular oxygen and nitrogen are flown in the two channels a stable gradient is generated in the test channel (black in Fig. 1) where cells are localized. Diffusion of solutes between this channel and the flanking ones (i.e. “source” and “sink”, green and yellow in Fig. 1 and 2) is allowed by agarose walls (see Fig. 3) fabricated in-situ following a protocol developed in our lab. By flowing chemoattractants in the “source” channel and a buffer solution in the “sink” we can establish stable gradients (see gradient in the “test” channel, Fig. 2) cells in the “test” channel can be exposed to. This device is currently being used to inform modeling of behavioral traits of marine bacteria.

 

Construction of a 3-color fluorescent reporter system for real-time visualization of DMSP degradation gene expression (suported by Moore Fundation)

fig2 cherry Read the protocol by Cherry Gao

 

Microfluidic designs:

 

three-inlet-device

Three inlet device.  Files by Jeffrey Guasto.  This microfluidic device has been used to test chemotaxis in bacteria by initially filling one side with chemoattractant and the other with bacteria, then stopping the flow.  (see e.g. Garren et al 2014) PDF

 

three-channel-device

Three channel device.   Files by Kwangmin Son. This device is used to generate stable chemical gradients.  By placing a PDM device casted from this design on a hydrogel, the outer channels are used to generate a gradient for the inner channel with bacteria.  (See e.g. Yawata et al. 2016) PDF

 

round-sample-chamber

Round sample chamber.  Files by Jeffrey Guasto.  This design is used for examining the motility of bacteria or other organisms under stable uniform conditions. PDF