Biophysics Tools and Techniques for the Physics of Life (Mark C. Leake) (1)

3.6 Basic Fluorescence Microscopy Illumination Modes

Worked Case Example 3.1: TIRF Microscopy

Video-rate fluorescence microscopy was performed using objective lens TIRF on single

living Saccharomyces cerevisiae cells (“budding yeast”), spherical cells of mean diam

eter 5 μm, immobilized onto a glass coverslip in a low-fluorescence water-based growth

medium. An orange dye was used to stain the cell membrane that aligns its electric dipole

axis parallel to the hydrophobic tail of phospholipid groups in the membrane. The dye was

excited into fluorescence using a 561 nm wavelength 20 mW laser with Gaussian beam

profile of full width at half maximum 1.0 mm as it comes out of the laser head, which was

then expanded by a factor of 3 prior to propagating through a neutral density filter of

ND = 0.6 then through a +200 mm focal length lens 200 mm downstream of the back focal

plane of an objective lens of effective focal length +2 mm and NA 1.49, in between which

was positioned a dichroic mirror that reflected 90% of the laser light onto the sample, with

net transmission power losses from all lenses between the laser and the sample being

~20%. Initially, the angle of incidence of the laser was set at 0° relative with the normal of

the coverslip–water interface and was purely p-polarized. The focal plane was set to the

coverslip surface and a cell positioned to the center of the camera field of view, with the

measured intensity values on the camera detector at the center of the camera during con

tinuous illumination being [112, 108, 111, 114, 112, 109, 111, 112, 107, 110, 113] counts

per pixel measured using 10 consecutive image frames from the start of laser illumin

ation. Similar values were obtained in a separate experiment performed in the absence

of any dye.

Calculate the approximate mean intensity of the laser at the sample in units of

W cm⁻². Comment on the results in regard to the dye and autofluorescence and esti

mate the camera readout noise.

The angle of incidence of the laser was then increased to the critical angle so that the

illumination was only just in TIRF mode. With the focal plane set again to the coverslip

surface and a new cell not previously exposed to the laser positioned to the center of

the camera field of view, the measured intensity values on the camera detector at the

center of the camera during continuous illumination were [411, 335, 274, 229, 199,

176, 154, 141, 133, 122, 118] counts per pixel. Comment on the difference with the

intensity data values of (a) and estimate the photobleach time of the dye under these

conditions.

The angle of incidence was then increased further to generate the thinnest evanes

cent field possible for TIRF excitation. Assuming the photon absorption of the dye is

not saturated, make a quantitative sketch of the variation in expected initial fluores

cence intensity per pixel if a 1D intensity profile is plotted parallel to the focal plane

through the center of the cell image.

Answers

The power incident on the sample after known attenuation and transmission

losses is given by

P =

−

(

) =

−

0 9

1 0 2

3 6

0 6.

. mW

The width w of the excitation field at the sample is given by

1 0

2 200

0 03

3 10 ³

×(

) =

−

For a rough approximation, the area of the excitation field is ~πw². Therefore, the

mean excitation intensity in units of W cm⁻² is

3 ²

4 5 10

3 10

130

≈

(

)

(

)

(

) ^≈

−

Wcm