μDish so that the bacteria are trapped between the agarose pad
and the coverslip bottom. Close and seal the lid of the μDish
using grease to minimize drying of the pad (see Note 17).
10. Place a drop of immersion oil on the 100Â objective and
mount the coverslip or the μDish on the microscope stage,
such that the glass side faces the objective.
11. Carry out snapshot imaging or setup a short-term time-lapse
microscopy experiment.
3.3 Preparation
of Semipermeable
Membranes
for Microfluidic
Microscopy
1. Cut a few pieces of dialysis membrane about 10 cm long with
scissors, which have been previously wiped with 70% ethanol.
2. Soak the membranes in 500 mL of sterile ultrapure water in a
1 L beaker for 1 h.
3. Use a magnetic stirrer to mix gently the water, change the
water once, and wash the membranes for 2 h (see Note 18).
4. Fish the pieces of membrane out of the water one at a time, and
cut out rectangles of 11 Â 50 mm size, using a ruler, a glass
sheet, and a scalpel blade (see Note 19).
5. Transfer the membrane rectangles into a sterile beaker containing sterile ultrapure water and allow them to soak for 1 h with
very gentle stirring.
6. Fish out the membrane rectangles from the water and remove
excess water by touching a filter paper.
7. Soak the membranes in 250 mL methanol overnight, in a
chemical fume hood, under very gentle stirring.
8. Remove the membranes from methanol one by one and place
them between sheets of filter paper, cut of the same size of a
glass slide.
9. Generate a stack, alternating one membrane to a piece of filter
paper, sandwich the stack between two glass slides, and clamp
the stack with a binder clip.
10. Place the assembled stack in a vacuum chamber under vacuum
for at least 2 days.
3.4 Microfluidic
Design
and Manufacturing
of Low-Resolution
Plastic Masks
1. Download a software suitable for designing microsystems (see
Notes 3 and 4).
2. Select a company that offers high-precision photolithography,
including laser photoplotting on film, to produce a
low-resolution plastic mask (see Note 5). The example presented here has microstructures in the order of hundreds of
μm, and therefore, a plastic mask is suitable (Fig. 1a).
3. First draw the outline of a single 5 Â 5 in. mask or mask array
(see Note 6). Inside each frame, draw a circle of 100 mm
diameter, representing the silicon wafer, and at the sides of
Microfluidic Microscopy of Mycobacteria
213
and the coverslip bottom. Close and seal the lid of the μDish
using grease to minimize drying of the pad (see Note 17).
10. Place a drop of immersion oil on the 100Â objective and
mount the coverslip or the μDish on the microscope stage,
such that the glass side faces the objective.
11. Carry out snapshot imaging or setup a short-term time-lapse
microscopy experiment.
3.3 Preparation
of Semipermeable
Membranes
for Microfluidic
Microscopy
1. Cut a few pieces of dialysis membrane about 10 cm long with
scissors, which have been previously wiped with 70% ethanol.
2. Soak the membranes in 500 mL of sterile ultrapure water in a
1 L beaker for 1 h.
3. Use a magnetic stirrer to mix gently the water, change the
water once, and wash the membranes for 2 h (see Note 18).
4. Fish the pieces of membrane out of the water one at a time, and
cut out rectangles of 11 Â 50 mm size, using a ruler, a glass
sheet, and a scalpel blade (see Note 19).
5. Transfer the membrane rectangles into a sterile beaker containing sterile ultrapure water and allow them to soak for 1 h with
very gentle stirring.
6. Fish out the membrane rectangles from the water and remove
excess water by touching a filter paper.
7. Soak the membranes in 250 mL methanol overnight, in a
chemical fume hood, under very gentle stirring.
8. Remove the membranes from methanol one by one and place
them between sheets of filter paper, cut of the same size of a
glass slide.
9. Generate a stack, alternating one membrane to a piece of filter
paper, sandwich the stack between two glass slides, and clamp
the stack with a binder clip.
10. Place the assembled stack in a vacuum chamber under vacuum
for at least 2 days.
3.4 Microfluidic
Design
and Manufacturing
of Low-Resolution
Plastic Masks
1. Download a software suitable for designing microsystems (see
Notes 3 and 4).
2. Select a company that offers high-precision photolithography,
including laser photoplotting on film, to produce a
low-resolution plastic mask (see Note 5). The example presented here has microstructures in the order of hundreds of
μm, and therefore, a plastic mask is suitable (Fig. 1a).
3. First draw the outline of a single 5 Â 5 in. mask or mask array
(see Note 6). Inside each frame, draw a circle of 100 mm
diameter, representing the silicon wafer, and at the sides of
Microfluidic Microscopy of Mycobacteria
213