Stimulated emission depletion microscopy

Patent metadata

Field	Value
Publication number	US-9575302-B2
Application number	US-201314423188-A
Country	US
Kind code	B2
Filing date	Aug 16, 2013
Priority date	Aug 23, 2012
Publication date	Feb 21, 2017
Grant date	Feb 21, 2017

Abstract

Aberrations in stimulated emission depletion microscopy are corrected using an adaptive optics approach using a metric which combines both image sharpness and brightness. Light modulators ( 22,32 ) are used to perform aberration correction in one or more of the depletion path ( 10 ), the excitation path ( 12 ), or the emission path from sample to detector.

First claim

The invention claimed is:

1. A method of stimulated emission depletion microscopy using adaptive optics comprising:
(a) capturing a fluorescence image from a stimulated emission depletion microscope having an excitation light path and a first light modulator in a depletion light path defining a pattern on the first light modulator, wherein the first light modulator is a spatial light modulator or a deformable mirror;
(b) calculating a measure of image sharpness of the fluorescence image,
calculating a measure of image brightness of the fluorescence image, and
calculating a metric combining the measure of image brightness and the measure of image sharpness;
wherein the metric M is defined by
M=S+σβBf ( S )  (2)

wherein M is the metric, S an image sharpness measure, B an image brightness measure, σ is constant that has a value −1 or 1 depending on whether the aberration mode being corrected produces a maximum or minimum brightness, β is a constant and f(S) is a function of S that has a larger absolute value for a value of S above a threshold value S T and a lower absolute value for a value of S below the threshold value S T :
(c) adjusting the pattern on the first light modulator; and
repeating (a), (b) and (c) to maximise or minimise the metric combining the measure of image brightness and the measure of image sharpness.

2. A method of stimulated emission depletion microscopy according to claim 1 wherein (a) capturing the fluorescence image comprises:
forming synchronised depletion and excitation beams, the depletion beam having a longer wavelength than the excitation beam;
directing the excitation beam along the excitation light path through an objective onto a sample and generating fluorescence;
directing the depletion beam along the depletion light path onto a depletion light modulator and directing the depletion beam from the depletion light modulator through the objective onto the sample to form a depletion beam with a point spread function having a minimum value at a center to de-excite the fluorescence away from the center;
capturing the fluorescence from the center;
moving the center relative to the sample to a plurality of positions to build up the fluorescence image.

3. A method according to claim 2 , wherein the method further comprises:
directing the excitation beam onto an excitation light modulator and directing the excitation beam from the excitation light modulator through the objective onto the sample to generate fluorescence; and
adapting the pattern on the excitation light modulator to reduce optical aberrations by maximising or minimising the image brightness measure B.

4. A method according to claim 2 , wherein the path from the sample to the detector is an emission light path, the method further comprising:
directing the light from the sample through the objective onto a second light modulator and then onto the detector; and
adapting the pattern on the second light modulator to reduce optical aberrations by maximising or minimising a further metric being a measure of image brightness.

5. A method according to claim 4 wherein the second light modulator is also in the excitation light path and wherein:
adapting the pattern on the second light modulator to reduce optical aberrations by maximising or minimising a further metric includes adapting the pattern on the second light modulator to reduce optical aberrations in both the excitation light path and the emission light path.

6. A method according to claim 5 wherein the second light modulator is in the depletion light path as well as the emission light path and the excitation light path and wherein:
adapting the pattern on the second light modulator to reduce optical aberrations by maximising or minimising a further metric includes adapting the pattern on the second light modulator to reduce optical aberrations in each of the depletion light path, the excitation light path and the emission light path.

7. A method according to claim 1 including correcting for a plurality of optical aberration modes.

8. A method according to claim 2 wherein the pattern on the depletion light modulator is a pattern arranged to direct a non-zero order diffraction peak onto a sample as the depletion beam with a point spread function.

9. A method according to claim 1 wherein the metric M is defined by

M
=

S
+

σβ
⁢

⁢

B
⁡

[

1

1
+

ⅇ

-

k
⁡

(

S
-

S
T

)

]

(
1
)

where k is a constant of proportionality.

10. A method according to claim 1 wherein the measure of image brightness B is calculated as the sum of the pixel values in the image.

11. A method according to claim 1 wherein the measure of image sharpness S is defined as the second moment of the image Fourier transform (FT) and calculated as

S
=

∑

n
,
m

⁢

μ

n
,
m

⁢

I
^

Assignees

• Isis Innovation
• Isis Innovation

Inventors

• Booth Martin
• Burke Daniel
• Bewersdorf Joerg
• Gould Travis J

Classifications

• G01N21/636

  using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties; (non-linear optics per se G02F1/35) · CPC title

• G01N21/6456

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title

• G02B26/06

  for controlling the phase of light (G02B26/08 takes precedence {, measuring optical phase difference G01J9/00}) · CPC title